Search

Marker genes for the monocyte-macrophage-DC subpopulations identified in Supplementary Figure 5H. Only genes with adjusted p-value < 0.01 and average log fold-change > 0.3 were included. gene, gene name; p_val, p-value calculated based on Wilcoxon test; avg_logFC, log fold-change of the average expression between the given cluster and all other clusters; pct.1, fraction of gene-expressing cells in the given cluster; pct.2, fraction of gene-expressing cells in all other clusters; pct.diff, Difference between the percentage of gene-expressing cells in the given cluster and all other clusters; p_val_adj, Bonferroni adjusted p-value; cluster, cluster annotation based on the gene markers identified; resolution, clustering resolution parameter used in Seurat.

Pharmacodynamics of STAT3 ASO in CT26 STK11 KO model. (A) Percentage of CD45+ immune cells among live cells from untreated or drug-treated STK11 KO CT26 tumors as indicated. (B) Percentage of Ly6G+ granulocytic cells among live CD45+ cells. (C) Percentage of intratumoral CD103+ cDC1 dendritic cells among CD45+ immune cells. (D) CD86 MFI of CD103+ cDC1s. (E) Percentage of migratory DC among CD45+ cells in TDLN of tumor engrafted mice. (F) CD86 MFI of these migratory DC in TDLNs. (G) Flow cytometry plots of CD86 levels on TDLN migratory DC. (H) Percentage of CD64+ CD11b+ myeloid cells among live intratumoral CD45+ cells. (I) Flow cytometry plots of CD86 on these CD64+ CD11b+ myeloid cells. (J) Percentages of MHCII+ CD206+ intratumoral myeloid cells. (K) Percentage of MHCII+ CD163+ intratumoral myeloid cells.

Supplementary Appendix

Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non–small cell lung cancer (NSCLC) enrolled in three phase I/II trials. STK11 mutations were associated with resistance to the anti–PD-L1 antibody durvalumab (alone/with the anti-CTLA4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood, and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e., CXCL2, IL6), Th17 contexture (i.e., IL17A), and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy.Significance:Patients with nonsquamous STK11-mutant (STK11mut) NSCLC are less likely than STK11 wild-type (STK11wt) patients to respond to anti–PD-L1 ± anti-CTLA4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC.This article is highlighted in the In This Issue feature, p. 2659

Purpose:Antibody–drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance.Experimental Design:The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR.Results:Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs.Conclusions:Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.

Supplementary methods and materials, and Supplemental Figures

Figure S1. Expression of tumor-associated antigens in mouse tumor cell lines; Table S1. Number of complete responses in CT26 tumor-bearing mice that were treated with EphA2-Tub or EphA2-PBD; Figure S2. Activity of EphA2-Tub and EphA2-PBD in the Renca tumor model; Fig. S3. Control IgG-ADCs are minimally active in CT26, MCA205, 4T1, and RENCA tumor models, demonstrating target specificity for EphA2-ADC; Figure S4. CD8+ T cells are important for the efficacy of ADCs in the CT26 model; Table S2. Summary of results from combining ADCs with checkpoint inhibitors or TNFR agonists; Figure S5. Blood lymphocyte count of non-tumor bearing BALB/c and C57BL/6 mice following dosing with EphA2-Tub or EphA2-PBD; Figure S6. Combination of EphA2-ADCs with anti-PD1 or PD-L1; Figure S7. Combination of EphA2-ADCs with GITRL FP or OX40L FP; Figure S8. Activity of ADCs in the MCA205 model; Figure S9. Antitumor activity studies in the Renca model; Figure S10. Normalized CD45+ cells in the Renca model; Fig. S11. Detection of EphA2 in syngeneic mouse models via immunohistochemistry; Figure S12. Immunophenotyping of myeloid cells in the Renca model

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody–drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. Cancer Res; 77(10); 2686–98. ©2017 AACR.

Supplementary table showing the relative expression of 43 target genes in a panel of EphA2-positive cell lines.

Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non–small cell lung cancer (NSCLC) enrolled in three phase I/II trials. STK11 mutations were associated with resistance to the anti–PD-L1 antibody durvalumab (alone/with the anti-CTLA4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood, and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e., CXCL2, IL6), Th17 contexture (i.e., IL17A), and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy. Significance: Patients with nonsquamous STK11-mutant (STK11mut) NSCLC are less likely than STK11 wild-type (STK11wt) patients to respond to anti–PD-L1 ± anti-CTLA4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC. This article is highlighted in the In This Issue feature, p. 2659

Prostate cancer is traditionally considered an immunologically “cold” tumor type rendering patients insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent anti tumor immune response to “heat up” the tumor microenvironment. However, many antigens expressed on prostate tumors are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a sub-optimal therapeutic index. Our target discovery and validation efforts identified STEAP2 as a superior prostate antigen for therapeutic targeting. Importantly, STEAP2 is a highly prevalent prostate cancer antigen displaying high, homogeneous cell surface expression across all stages of disease. A novel lead generation approach facilitated the development of a potent and specific armored STEAP2 CAR-T therapeutic candidate, AZD0754. This second generation CAR-T product is armored with a dominant-negative TGFβRII, thereby bolstering activity in the TGFβ-rich immunosuppressive environment of prostate cancer. Armored STEAP2 CAR-T cells demonstrate favorable in vitro properties, robust dose dependent in vivo efficacy in STEAP2 expressing cell line- and patient derived- mouse xenograft models and encouraging preclinical safety. Taken together, this data builds confidence in the specificity and potency of this potential first in class STEAP2 targeted CAR-T therapy and supports future clinical development. Citation Format: Dewald van Dyk, Peter Zanvit, Christine Fazenbaker, Kelly McGlinchey, Weichuan Luo, Jessica Pezold, John Meekin, Chien-ying Chang, Benjamin Clark, Philip L. Martin, Clare Hoover, Eric Tu, Ryan Gilbreth, Mark Cobbold, Gordon Moody, Emily E. Bosco. Antitumor activity of AZD0754, a dnTGFbRII armored STEAP2 targeted CAR-T therapy, in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB085.

Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.

Mutations in the

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors.

Purpose: Antibody–drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. Experimental Design: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. Results: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. Conclusions: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody–drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. Cancer Res; 77(10); 2686–98. ©2017 AACR.

Antibody drug conjugates (ADCs) combine a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer. ADCs bearing non-cleavable drug-linkers such as the pyrrolobenzodiazepine (PBD) SG3376 or the maytansinoid DM1 rely upon lysosomal degradation of the antibody for release of the cytotoxic warheads, which must then escape the lysosome and bind to their intracellular targets. We sought to identify the mechanism of lysosomal transport of these membrane-insoluble warheads and have recently reported that the lysosomal transporter SLC46A3 is required for cytotoxic activity of ADCs bearing SG3376 and DM1, and that loss of SLC46A3 is a mechanism of acquired resistance to Trastuzumab-DM1 (T-DM1). Here we explore the potential translational relevance of SLC46A3. Analysis of SLC46A3 mRNA expression across several tumors showed that breast cancer cell lines and primary tumor samples have relatively high levels of SLC46A3 compared to samples derived from multiple myeloma (MM), esophageal, and upper aerodigestive tract malignancies, all of which are being targeted in clinical trials with non-cleavable DM1 ADCs. Deeper analysis indicates that levels of SLC46A3 are decreased in MM patients compared to healthy donors, and levels are further decreased in the relapsed/refractory setting compared to samples taken at diagnosis. We measured the expression of SLC46A3 in a panel of MM cell lines by RT-PCR and found that 5 of 9 (56%) had undetectable levels of SLC46A3. In vitro cytotoxicity assays demonstrate that SLC46A3-negative cells are resistant to B-cell maturation antigen (BCMA)-targeted ADCs prepared with non-cleavable DM1 and SG3376 warheads, but remain sensitive to the cleavable payload PBD SG3249 (Tesirine). Taken together, our results suggest that SLC46A3 may be a predictive response marker for ADCs bearing the non-cleavable warheads SG3376 and DM1, and this marker may be particularly useful in trials involving MM. Citation Format: Krista Kinneer, Yu-Tzu Tai, Sriram Sridhar, John Meekin, Sandrina Phipps, Ben Ruddle, Ryan Fleming, Nazzareno Dimasi, Ronald Herbst, Arnaud Tiberghien, Luke Masterson, Philip Howard, Kenneth Anderson, David Tice. The lysosomal transporter SLC46A3 is a potential predictive biomarker for antibody-drug conjugates bearing the non-cleavable warheads SG3376 and DM1 [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 567.

Antibody-drug conjugates (ADCs) combine a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer. ADCs bearing non-cleavable warheads such as the pyrrolobenzodiazepine (PBD) SG3376 rely upon lysosomal degradation of the antibody for release of the cytotoxic warhead. Here we sought to identify the mechanism of lysosomal membrane transport following release of these membrane-insoluble warheads. We used a multiplexed RT-PCR screen to compare the expression of 43 lysosomal membrane-specific genes in cell lines with inherent sensitivity or resistance to various SG3376-based ADCs. We discovered that only the expression of SLC46A3 correlated with sensitivity to the ADCs. Functional validation through gene knockout or forced gene expression in vitro revealed that loss or gain of SLC46A3 altered the potency of SG3376- and DM1-ADCs but had no effect on ADCs carrying the non-cleavable warhead MMAF or the cleavable PBD payload SG3249. To explore a role in acquired resistance, we interrogated trastuzumab-DM1 (T-DM1) sensitive and resistant cell line pairs and found that resistant lines had lost expression of SLC46A3. Sensitivity to both SG3376- and DM1-ADCs was restored upon reintroduction of SLC46A3. Furthermore, we examined a patient-derived xenograft (PDX) breast cancer model of acquired T-DM1 resistance and found that expression of SLC46A3 was decreased by 92% compared to the parental, T-DM1 sensitive model. Taken together, our results establish SLC46A3 as a transporter of both SG3376 and DM1 catabolites from the lysosome, and suggest that SLC46A3 may be a predictive response marker for ADCs bearing these non-cleavable warheads. Citation Format: Krista Kinneer, John Meekin, Sandrina Phipps, Binyam Bezabeh, Ryan Fleming, Christine Kiefer, Marlon Rebelatto, Nazzareno Dimasi, Michael J. Wick, Alyssa Moriarty, Megan Groves, Ronald Herbst, Arnaud Tiberghien, Luke Masterson, Philip Howard, David Tice. Loss of the lysosomal transporter SLC46A3 is a mechanism for innate and acquired resistance to non-cleavable maytansine and pyrrolobenzodiazepine-based antibody-drug conjugates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A138.

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether ADCs conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induced ICD, modulated the immune microenvironment, and could combine with IO drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent vs. immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. These ADCs also induced immunological memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of these ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in the antitumor activity. Combinations of ADCs in different tumor models with IO drugs including PD-1 or PD-L1 antibodies, or OX40 ligand or GITR ligand fusion proteins produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADCs and ADC/IO combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADCs and IO drugs that may increase clinical responses. Citation Format: Jonathan Rios-Doria, Jay Harper, Raymond Rothstein, Leslie Wetzel, Jon Chesebrough, Allison Marrero, Cui Chen, Patrick Strout, Kathy Mulgrew, Kelly McGlinchey, Ryan Fleming, Binyam Bezabeh, John Meekin, David Stewart, Maureen Kennedy, Philip Martin, Andrew Buchanan, Nazzareno Dimasi, Emil Michelotti, Robert Hollingsworth. Antibody-drug conjugates bearing pyrrolobenzodiazepine or tubulysin payloads alter the tumor immune microenvironment and synergize with multiple immunotherapies [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 4596. doi:10.1158/1538-7445.AM2017-4596

Lung cancer is the most common type of cancer, with approximately 222,000 new cases expected each year in the United States. Approximately 72% of the individuals diagnosed with lung cancer will fall victim to the disease. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, but current treatments will not cure the disease for most patients. In an effort to identify novel therapeutics to address this unmet need, a phenotypic screening campaign was initiated. This type of target identification approach allows the discovery of novel drug targets by selection of antibodies based on their ability to elicit a specific desired phenotype. Once antibodies with the desired function are isolated, the targets to which the “hit” antibodies bind can be identified using a variety of approaches including IP-mass spec. Although phenotypic screens have been performed previously, the current effort has important advantages over those described previously. Specifically, this included the use of several unique screening assays run in parallel to allow the evaluation of multiple phenotypes simultaneously. Also unique was the use of primary material from NSCLC patients for both selection and functional screening. Specifically, we demonstrate the use of primary tumor cells in high throughput screening assays. We show the utility of incorporating novel screening assays including anoikis and spheroids with multiple endpoint readouts. Finally we provide data describing a series of “hit” antibodies isolated in the screen which had a variety of activities across the screening assays. These studies show the utility of function-first screening in the identification of unique potential therapeutic targets for oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 974. doi:1538-7445.AM2012-974