Abstract Chimeric Antigen Receptor (CAR) T cell therapy has transformed outcomes for hematologic malignancies, yet optimizing manufacturing conditions remains critical to improving potency and consistency. We previously demonstrated that culturing CAR-T cells under physiological oxygen and pressurized conditions improved persistence and efficacy in a disseminated NALM6 human ALL model.In this study, we investigated whether these optimized manufacturing conditions similarly improve antitumor activity in a subcutaneous NALM6 xenograft model. CD19-targeted CAR-T cells were expanded under three conditions: standard CO2 incubation (21% O2 + 0 PSI), mild hypoxia with hyperbaric pressure (15% O2 + 5 PSI), reflecting arterial and peripheral blood environments, and hypoxia with hyperbaric pressure (5% O2 + 5 PSI), modeling the solid tumor and bone marrow microenvironments. After a 10-day tumor stage, a dose of 2.5e6 CAR+ T cells achieved complete tumor clearance in all conditions at 20 days post T cell dose, with faster tumor regression under 5% O2 + 5 PSI conditions (7 days post T cell dose) compared to normoxia or mild hypoxia (10 days post T cell dose). These in vivo outcomes were consistent with enhanced in vitro functional activity, characterized by greater cytoxicity at CAR-T-to-effector ratios under 1:30 and increased cytokine production. This consistency mirrors our previous findings in the disseminated model, where CAR-T cells cultured under physiologically relevant conditions demonstrated the in vitro characteristics predictive of in vivo efficacy. Preliminary findings show enhanced potency and persistence under 5% O2 + 5 PSI conditions. To determine whether these manufacturing advantages are sustained at lower CAR-T doses, a dose titration study is currently in progress, with complete results forthcoming.These data highlight the potential of physiological manufacturing with the GMP AVATAR Foundry to enhance CAR-T function and lower effective doses, supporting scalable next-generation cell therapies. Citation Format: Candy Garcia, Anita J. Zaitouna, Abriel Czachorowski, Lauren Kucharczyk, Natalie Czeryba, Philip Edward Lapinski, Andrea Hodgins-Davis, Thomas Sullivan, Yelena Bronevetsky, NINGCHUN LIU, Sheri Barnes, Shannon Eaker, Scott Wise, James Lim. Environmental conditioning during CD19-CAR T cell manufacturing impacts antitumor potency in a subcutaneous NALM6 tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4273.

Antibody-drug conjugates (ADCs) represent groundbreaking advancement in the cancer treatment landscape. ADCs combine the specific binding capacity of a monoclonal antibody with a cytotoxic payload that can kill target and bystander cells. The antibody moiety of ADCs confers payloads with the ability to target cancer cells with minimal damage to healthy tissues. A key consideration in defining appropriate breast cancer treatment regimen is the expression level of human epidermal growth factor receptor 2 (HER-2), targeted with the antibody Trastuzumab. Breast cancers are classified as high, low, or negative for HER-2, which dictates the therapeutic approach. Trastuzumab alone may not be an effective treatment because tumors can either be initially resistant or develop resistance over time. Around 70% of patients experience resistance within the first year of treatment. To overcome this, ADCs such as Trastuzumab-Emtansine (DM1) or Trastuzumab-Deruxtecan (Dxd) can be used at lower doses, reducing the risk of resistance and potentially enhancing treatment efficacy. Assessment of Trastuzumab ADCs in multiple murine breast cancer models can be costly, but in vitro evaluation of various cancer histotypes facilitates the selective identification of the best murine model. Having a robust screening tool is an important asset. To assess Trastuzumab ADCs, we screened five breast cancer cells (BT-474, Hs 578T, MDA-MB-231, MDA-MB-361, SK-BR-3) with varying levels of HER-2 expression. They were evaluated for binding efficiency, competitive inhibition, and cytotoxicity of ADCs. Additionally, the ability of the ADC to kill bystander cells (HER-2 low or negative cells) was assessed using two bystander assay methods. The half maximal effective concentration (EC50) values for Trastuzumab and Trastuzumab-Dxd were measured on two cell lines. The HER-2 high cell line had a tenfold higher EC50 value. Competitive inhibition of Trastuzumab-ADC binding was tested with a PE labeled Trastuzumab on two cell lines. The Trastuzumab-ADC was able to outcompete Trastuzumab on HER-2 high cells but not on HER-2 low/negative cells. Proliferation assays on multiple cell lines were used to assess the ability of Trastuzumab-DM1 to inhibit cell growth in comparison to DM1 (payload alone) or Trastuzumab (antibody alone). Overall, Trastuzumab-DM1 was better able to inhibit growth of HER-2 positive cells compared to payload or antibody alone. Two ADC bystander assays were developed and tested. The first involved flow cytometry and the second a luciferase assay. In both assays, bystander cells were more effectively targeted only in the presence of HER-2 positive cells. The ability of ADCs to target bystander cells suggest that even in heterogenous HER-2 expressing tumors, these therapies may still be effective. Our cutting-edge approaches provide precise insights into the activity and efficacy of Trastuzumab ADCs. Michael Steffey, Stacey Roys, Anita J. Zaitouna, Philip Lapinski, Kerry-Ann Bright, Sheri Barnes, David Draper, Scott Wise. Multi-modal assessment of an anti-HER2 (trastuzumab) antibody-drug conjugate screening assay for breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6746.

Nearly one million people worldwide are diagnosed with gastric cancer, with approximately 660,000 people succumbing to the disease every year. Sixty-four percent of gastric cancer cases occur in Asia, and approximately 3% in the United States. Primary therapies for the treatment of gastric cancer include surgery, chemotherapy, immunotherapy, and targeted therapies such as blocking antibodies and antibody-drug conjugates (ADCs). Human Epidermal growth factor Receptor 2 (HER2) is a protein overexpressed in a variety of cancer types of epithelial origin, including gastric cancer. The anti-HER2 monoclonal antibody trastuzumab is the standard of care for certain types of HER2+ cancers. Trastuzumab has a multi-faceted mechanism of action, including Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), which is primarily mediated by Natural Killer (NK) cells in the clinic. However, in the absence of NK cells, neutrophil- and macrophage-dependent ADCC and Antibody Dependent Cellular Phagocytosis (ADCP) may contribute to the mechanism of action of monoclonal antibodies in immunocompromised mice (such as NSG mice). The gastric carcinoma tumor cell line NCI-N87 overexpresses HER2, and thus this model was chosen to evaluate the effect of multiple standard of care agents, including trastuzumab and the ADC trastuzumab-DXd, in the presence and absence of human NK cells. NCI-N87 cells were evaluated for HER2 expression by flow cytometry and were found to be approximately 99% HER2 positive. FcγRI and FcγRIIa-H mediated ADCP and FcγRIIIa mediated ADCC of trastuzumab and ADCs trastuzumab-DXd, trastuzumab-DM1, and trastuzumab-MMAE, were measured in vitro using reporter assays. Similar activity between trastuzumab and ADCs was observed, although it is presumed the antibody-payload linker may be interfering with binding to either the NCI-N87 cells or the Fc receptor, causing some variability in fold induction. For in vivo experiments, NCI-N87 cells were mixed with 50% extracellular matrix, and subcutaneously implanted in female NSG mice. Mice with established tumors were treated with osimertinib mesylate, 5-fluorouracil, lapatinib, trastuzumab, and trastuzumab-DXd to assess anti-tumor response. Additionally, a cohort of mice was humanized with peripheral blood mononuclear cells (PBMC) to evaluate the impact of human immune cells on the efficacy of trastuzumab and trastuzumab-DXd. All standard of care agents had significant anti-tumor activity, with trastuzumab-DXd being highly effective. Humanization did not appear to provide additional efficacy. In summary, a well-characterized HER2+ model of gastric cancer has been established. Future studies include testing the activity of a T-cell-engaging HER2-targeting bispecific antibody in the humanized NCI-N87 xenograft model. Hillary J. Millar Quinn, Kyla Emerson, Sean Shukait, Natalie Czeryba, Lauren Kucharczyk, Anita J. Zaitouna, Sheri Barnes, Scott Wise. Development, characterization, and humanization of NCI-N87 human gastric carcinoma xenograft model in NSG mice [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B089.

Abstract Amid the growing arsenal of cancer treatments, antibody-dependent cellular cytotoxicity (ADCC) assays have emerged as a significant contributor to the quest for more effective therapies. The ADCC-based antibody therapy often exploits Natural Killer (NK) cells as effectors. This immunotherapeutic approach harnesses the innate cytotoxic potential of NK cells, offering a promising solution for the precision targeting of malignant cells. In this study, we employed two methods that can be used to measure ADCC potency and specificity during preclinical drug discovery. The first was a flow cytometry-based approach that focused on enhancing biological relevance by utilizing primary human peripheral blood mononuclear cells (PBMCs) as a source for NK effector cells. To this end, target cells were fluorescently labeled with CFSE and cultured with freshly isolated PBMCs at 3 effector to target (E:T) ratios, in the presence of ADCC antibody. After 16h incubation, a viability dye was added, and percent cytotoxicity was measured in CFSE+ targets cells by flow cytometry. The second method offered a high-throughput plate reader-based approach (ADCC Reporter Bioassay; Promega), that utilized an ADCC reporter effector cell line harboring a luciferase cassette driven by an NFAT promoter, which is activated when effector and targets cells are bridged by a therapeutic antibody. Cytotoxicity was read out by measuring light output on a Cytation 3 plate imaging reader (Agilent Technologies) after a 6h cell culture incubation using the recommended 6:1 E:T ratio. Preclinical application for both assays was demonstrated using an anti-HER2 therapeutic antibody (trastuzumab) against BT-474, which is a HER2 positive cell line. To demonstrate specificity, an anti-VEGF antibody (bevacizumab) and Hs 578T cells (HER2 low) were used as controls. Our results revealed trastuzumab induced a strong dose-dependent increase in cytotoxicity in the BT-474 cells but not the Hs 578T cell line (range: 40-70%) while bevacizumab failed to induce any cytotoxicity above background levels at any concentration. Furthermore, the flow cytometry and plate reader methodologies produced similar trends. In summary, both applications can be used to produce reliable ADCC drug screening assessments. Additional considerations include investigational needs surrounding high throughput capability versus biological relevance. Unlike traditional models using immortalized cell lines, primary human PBMCs more faithfully recapitulate the physiological interactions between effector cells and target cells. On the other hand, the more streamlined reporter cell line approach not only accelerates assay throughput but also ensures consistent and reliable results in an attractive option for large-scale screening and drug development efforts. Citation Format: David William Draper, Yewei Xing, Scott Wise. NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 83.

Abstract Cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapy, have revolutionized the treatment of hematological malignancies, but their use in solid tumors remains a significant challenge for the field. This is due, in part, to immunosuppressive mechanisms in the tumor microenvironment (TME), which include reduced oxygen tension, high interstitial pressure, and an abundance of immunosuppressive proteins, resulting in mitochondrial dysfunction, exhaustion, and depletion of adoptively transferred cells. Metabolic manipulation of therapeutic cells could overcome these barriers to enable cell therapy success in solid tumors. To this end, we have employed AVATAR technology (Xcellbio, San Francisco, CA), an incubation system that enables precise control of oxygen tension and hyperbaric pressure on cells in culture to improve the manufacture of adoptive T cell therapies. Using a CD19 CAR T model system, we have shown that transduction and expansion of CAR T cells under reduced oxygen and hyperbaric pressure conditions yield greater percentages and total numbers of lentiviral-transduced cells. While cells transduced in a standard CO2 incubator yielded 10-20% CD19 CAR+ cells, transduction in the AVATAR system with increased pressure yielded 15-40% CD19 CAR+ cells. Similarly, T cell cultures performed under pressurized AVATAR conditions generated ~2X more viable cells after 10 days. Furthermore, these metabolically reprogrammed cells exhibit enhanced potency, with improved anti-tumor cytotoxic activity in vitro. We have extended these studies to measure the ability of AVATAR-expanded CD19 CAR T cells to control the growth of CD19-expressing NALM6-Luc tumor cells in vivo compared to CAR T cells grown in a conventional CO2 incubator. Mice (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories, Bar Harbor, ME) dosed with the AVATAR-expanded cells exhibited good tumor control and increased persistence of CAR T cells in relevant organs, along with alterations in cell trafficking to the bone marrow and spleen. The transferred cells maintained a less differentiated phenotype, with central memory and effector memory populations, as measured by expression of the markers CD45RA and CD62L, dominating the T cell compartment. This work showcases the benefits of metabolic reprogramming to improve the yield and functional potency of CAR T cells, which has direct applications to reducing the cost and improving the efficacy of these lifesaving treatments. The AVATAR technology is currently being translated into a closed system bioreactor for use in GMP cell therapy manufacturing. Citation Format: Yewei Xing, Shannon Eaker, Yelena Bronevetsky, Candy Garcia, Hadia Lemar, Evan Massi, Albert Wong, Sheri Barnes, Derrik Germain, Scott Wise, James Lim. Metabolic reprogramming enhances expansion and potency of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6334.

Abstract Traditional cancer cell lines used as surrogate models in vitro and in vivo have made significant contributions to cancer research and drug discovery. While cell culture and animal studies are critical steps in determining the efficacy, pharmacodynamics, and mechanism of action of novel anti-cancer drugs, it is well-known that cells grown in 2D culture systems have modified growth characteristics and different responses to chemotherapeutic drugs compared to tumors in vivo as a result of differing structural and environmental conditions that fail to replicate in vivo tumor microenvironments. AVATAR bioreactors (Xcell Biosciences, San Francisco, CA) were invented to address these limitations by providing discrete control over oxygen and hyperbaric pressure levels to better mimic the in vivo environment and allow cultured cells to be grown in conditions which mimic the human body. To better serve preclinical research needs, we generated new tumor cell models by long-term acclimating (LTA) existing cancer cells in the AVATAR incubator system using 1% O2 and 2 PSI conditions similar to the conditions expected in the tumor microenvironment (TME). Six different cancer cell lines of diverse origins including pancreas (MIA PaCa-2), breast (BT-474), colon (HCT116), prostate (LNCaP, PC-3) and melanoma (A-375) were adapted for 2-3 months with phenotypic changes recorded. Key marker changes were commonly observed in LTA cells compared to parental lines. For example, in HCT116 colorectal cancer cells, TME culture conditions induced a higher expression level of E-cadherin in both protein and mRNA levels (2.2-fold change), indicating a mesenchymal-epithelial transition of the cell after adaptation. This coincides with the observation that LTA adapted HCT116 cell bodies had a more angular cell morphology as compared to parental cells grown under conventional normoxic culture conditions. In-depth gene-expression analysis using a Nanostring tumor signaling pathway panel identified several common and tissue-specific gene and pathway changes. These included VEGFA, EGF receptor signaling pathway (EGFR, E-Cadherin, GRB7, c-Fos), TGF pathway (IGFBP3, TGFB1, TGFB2, SMAD7), and extracellular matrix signaling (LAMB3, ITGA5, ITGB3, ITGB8, PDGFA, CCND2). Current studies are focusing on drug response testing in 2D and 3D cultures based on above-mentioned gene and signaling pathway changes. Cell proliferation and migration capacity of xenograft LTA cells will be evaluated for the purposes of creating novel murine models for drug screening applications. Citation Format: Yewei Xing, Abriel Czachorowski, James Lim, Scott Wise. Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6005.

Syngeneic tumor models for ovarian cancer are limited in preclinical research. One of these models, ID8-luc, is established intraperitoneally with disease progression resulting in the accumulation of ascites fluid. Treatment efficacy can be determined by non-invasive bioluminescence imaging (BLI), but solid ID8-luc tumors do not form in the peritoneal cavity, making assessment of immunomodulation challenging. To this end, we have developed the ID8-luc model as a tool to investigate immuno-modulatory drug candidates by utilizing tissues in the peritoneal cavity of diseased animals for ex vivo analyses. Specifically, flow cytometry of diseased ovaries was employed to examine the effects of checkpoint inhibition on T cell phenotypes and NK cell infiltration. To identify tumor deposits in organs from mice bearing ID8-luc disease for analysis tumor infiltrate, the model was established in untreated C57BL/6 albino mice, and ovaries, liver, peritoneal wall, pancreas, and spleen were evaluated pathologically. Progressive establishment of tumor deposits were seen in the ovaries, pancreas, and peritoneal wall of untreated mice, but not in naïve mice. Further, immune infiltration in ovaries were markedly higher compared to naïve mice. To assess immunomodulation, disease was established in a second cohort of mice, and mice were treated with anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4. Disease progression was monitored by BLI, and following treatment, ovaries containing tumor deposits were collected and lymphoid infiltrate was analyzed by flow cytometry. Checkpoint blockade did not result in efficacy under the conditions tested. However, differences in T cell infiltration were notable with treatment of anti-PD-1 and anti-mPD-L1. Particularly, a marked increase of five to ten fold CD8+ and CD4+ T cells by absolute counts was seen with anti-mPD-1 and anti-mPD-L1 treatment compared to control. Further, the percentage of CD8+ T cells expressing the activation markers CD69 and ICOS as well as PD-1, TIM-3, and LAG-3 checkpoint proteins increased with anti-mPD-1 treatment. Less prominent yet similar trends in this biomarker expression were seen with anti-mPD-L1 treatment. NK and NKT infiltration also increased five to eight fold in ovaries following anti-mPD-1 treatment. Interestingly, anti-mCTLA-4 treatment did not result in any remarkable changes in infiltration or activation marker dynamics. Taken together, these findings are suggestive of a mounting immune response following PD-1 and PD-L1 blockade, but not CTLA-4 blockade, characterized by changes in the lymphocyte compartment in the ovaries. This response is seen in the absence of efficacy, suggesting the potential that this immunomodulation can be exploited in rational combination strategies with checkpoint blockade for immune-oncology drug candidates using the ID8-luc ovarian tumor model. Citation Format: Sheri Barnes, Anita Zaitouna, Lauren Kucharczyk, Scott Wise. Differential immunomodulation following checkpoint blockade in the orthotopic ID8-luc ovarian model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4654.

Supplementary Materials and Methods. Supplementary table 1: XRay crystallography parameters for DP-4157/MET complex. Supplementary table 2: Source and description of kinases. Supplementary table 3: Selectivity of profile of altiratinib inhibition from Reaction Biology 295 kinase panel (fold selectivity versus IC50 value of 2.7 nM MET inhibition). Supplementary table 4: A. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (30 mg/kg); B. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (10 mg/kg). Supplementary figure 1: Co-crystal structures of altiratinib analog DP-4157. Supplementary figure 2: Comparison of altiratinib with other clinical stage MET inhibitors regarding potency versus activation loop mutant forms D1228H, D1228N, Y1230D, Y1230C, and Y1230H. Supplementary figure 3: Determination of off-rate, residency time, and tight-binding inhibitor constant Kd for altiratinib binding to the MET kinase domain. Supplementary figure 4: Michaelis-Menton analysis of altiratinib inhibition of MET with respect to ATP. Supplementary figure 5: Kinome tree interaction profile for altiratinib vs 295 human kinases. Altiratinib was evaluated using Kinase HotspotSM platform from Reaction Biology (Malvern, PA). Supplementary figure 6: Inhibitory potencies for inhibiting HUVEC cell signaling activated by HGF/MET (black), VEGF-A/VEGFR2 (melon), and ANG2/TIE2 (tan). Comparative potencies are shown for altiratinib, E-7050, cabozantinib, and MGCD-265. Supplementary figure 7: Upper panel: inhibition of capillary tube formation by various concentrations of altiratinib. Tube formation was initiated by addition of 200 ng/mL of angiopoietin 2 (ANG2). Lower table: titrated IC50 values for inhibition of capillary tube formation induced by ANG2, HGF, and VEGF-A. Supplementary figure 8: Inhibition of proliferation in the mutant B-RAF melanoma SK-MEL-28 cell line in the absence or presence of HGF or MRC-5 fibroblast conditioned medium. Supplementary figure 9: Ratio of brain: plasma concentrations of altiratinib through 24 hr after administration of a single dose of 5 mg/kg IV to C57/Black mice.

Chimeric antigen receptor (CAR) T cell therapy holds great promise for the treatment of various cancers, including solid tumors. However, attempts to model the behavior and effectiveness of CAR-T cell therapies for blood cancers and solid tumors have been challenging due to the unique tumor microenvironments in which these cancer cells are found. The tumor microenvironment (TME) is often characterized by hypoxia, increased acidity, and high interstitial fluid pressures, allowing cancer cells to effectively evade immune surveillance. This immunosuppressive TME also contributes to CAR-T cell exhaustion, thereby limiting its antitumor activity and function. To address these concerns, we have developed a proprietary cell-based assay to measure CAR-T cell potency and cytotoxic function in three-dimensional (3D) in vitro cell culture system, human acute B cell lymphoblastic leukemia mouse model, and immunosuppressive tumor microenvironments. Utilizing the AVATAR system, we replicated the oxygen and interstitial fluid pressures found in the vasculature, the bone marrow and solid tumor microenvironments. Tumor cytolysis assays were conducted in these environments to measure cell exhaustion as analyzed by flow cytometry and electrical impedance. In addition, serial tumor challenge assays were performed to examine CAR-T potency and effectiveness in TME. Proof-of-concept experiments were performed using ROR1 CAR-T cells targeting the ovarian adenocarcinoma cell line, SKOV3. CD19 CAR-T were also used targeting the acute lymphoblastic leukemia cell line, NALM6. Defined ratios of effector T cells to tumor cells was assessed to model CAR-T potency in vitro and elevated CD19 CAR-T mediated cytotoxicity was confirmed with the increased ratio of effector T cells to tumor cells in both 2D and 3D culture system. CD19 CAR-T cells also exhibited in vivo dose dependent efficacy against the systemic NALM6-Luc acute lymphoblastic leukemia (ALL) mouse model, quantified by bioluminescence (BLI) image monitoring method. Multiple pressure and oxygen settings were examined to model the cross-section of the bone marrow and solid tumor microenvironments (0 PSI to 5 PSI, 1% to 10% O2). Initial results from these screening experiments show significant decline in ROR1 CAR-T mediated cytotoxicity when performed under TME conditions. However, CD19 CAR-T showed effective cell killing under TME conditions. Interestingly, acclimating and expanding ROR1 CAR-T cells to high pressure and decreased oxygen culture conditions improved potency levels and warrants further investigation. In summary, we observe CAR-T cells comprise the tumor cell killing ability in both in vitro 3D and in vivo animal models. We also describe a physiologically relevant potency assay that incorporates hyperbaric and hypoxic incubation technology to predict the behavior of cell therapies in immunosuppressive tumor microenvironments. Citation Format: Yelena Bronevetsky, Evan Massi, Candy Garcia, Ningchun Liu, Yewei Xing, Natalie Czeryba, Scott Wise, James Lim. Functional potency assay predicts CAR-T effectiveness in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1782.

As it is rarely discovered early, the prognosis for people with pancreatic cancer is poor, with an average 5-year survival rate of 10% and only 3% for those with metastatic illness. Pancreatic cancer patients have few therapeutic options and innovative therapies are sorely needed to improve treatment of the disease. This is mainly due to the late diagnosis and partially due to the biology of the disease. Capturing the components of the tumor microenvironment, which serve as both a physical barrier and a source of stromal-driven resistance to therapeutics, is one of the issues faced by drug developers searching for agents to combat pancreatic cancer. We have created a 3D model of the pancreatic tumor microenvironment, the Reconstructed Pancreas (r-Pancreas), using Mia PaCa-2, PANC-1, and BxPC-3 pancreatic tumor cell lines embedded in an extracellular matrix (ECM) that was formulated to recapitulate the typical ECM of pancreatic tumors. We have shown that gemcitabine was effective against pancreatic tumor cells cultured in r-Pancreas (IC50=0.4-0.8µM), while 5-fluorouracil was ineffective (IC50 not reached), mimicking clinical response. In nude mice, subcutaneous PANC-1 tumors exhibited similar responses to gemcitabine and 5-fluorocurcil. Treatment with gemcitabine resulted in a Day 63 median ΔT/ΔC of 37% while 5-fluorouracil resulted in a Day 63 median delta ΔT/ΔC of 102%. Incorporating a collagen-rich capsule increases the physiological relevance of the r-Pancreas model because it has been shown that human pancreatic tumors have an outer layer of stiff ECM that functions as a physical barrier and prevents drugs from penetrating the tumor. Recently, we showed that, at least in the Mia PaCa-2 and BxPC-3 tumor cell lines, introducing such a collagen capsule dramatically reduced sensitivity to gemcitabine. Disrupting the collagen capsule with MMP-9 restored the sensitivity of the tumor cells to gemcitabine demonstrating that the capsule provides a physical barrier to drug entry. Additionally, creating a co-culture of primary activated pancreatic stromal cells and pancreatic tumor cell lines produces a more complete 3D model that permits the testing of potential therapeutic agents inside the pancreatic tumor microenvironment. Hence, we have created a 3D model where pancreatic tumor cells were co-cultured with human pancreatic fibroblasts. According to our results, the IC50 values for pancreatic tumor cell lines treated with gemcitabine increased by twofold when co-cultured with activated pancreatic fibroblasts compared to control sets of tumor cells alone (IC50=1-3µM). Together, these data imply that, to be clinically relevant, in vitro models of pancreatic cancer must integrate tumor-specific components of the microenvironment, including the collagen capsule and activated fibroblasts. Citation Format: Arnat Balabyev, Justin D. Phillips, Michael Steffey, Eleanore J. Kirshner, Aayushi Ahlawat, Arlette H. Uihlein, Daniel Saims, Scott Wise, Julia Kirshner. 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4561.

Lung cancer remains a devastating cancer diagnosis around the world, resulting in 18% of all cancer-related deaths. Lung cancer often metastasizes to the brain, significantly reducing life expectancy. Up to 7% of patients with non-small cell lung cancer (NSCLC) already have brain metastasis when they are first diagnosed, and 20%-40% of patients with NSCLC will develop this complication during disease progression. Currently there are no targeted therapies specific for brain metastases, and the blood-brain barrier can pose a physiologic impediment to many cytotoxic drugs and antibody-based therapies. Here, we will discuss the development and use of xenograft models to address metastatic brain disease via a direct intracranial implant coupled with a subcutaneous “primary” tumor to effectively allow evaluation of the response to treatment at both locations. Treatment with targeted therapies could have significantly different penetration and absorption rates, which will alter pharmacokinetic and/or pharmacodynamic (PK/PD) assessment across either tissue. The dual implant technique can be a powerful tool to assess the simultaneous impact of treatment on established metastatic disease and primary tumor. We have characterized the dual disease induction parameters for two human NSCLC cell lines: NCI-H1975-Luc and PC-9-Luc. Both cell lines have been transfected with luciferase to allow for bioluminescence imaging (BLI) to monitor the intracranial disease progression. Both NCI-H1975-Luc and PC-9-Luc are of interest to the research community due to their unique mutational EGFR T790M status. Consistent with the historical and published data, the H1975-luc and PC-9-luc models behaved as expected, producing reliable tumor progression with minimal intragroup variability. In each case, subcutaneous tumors (primary site) reached an evaluation size of ~1000 mm3 in ~20 days, and intracranial tumors (metastatic site) exhibited a total flux doubling time of ~2 days by BLI. Treatment with Osimertinib, an approved first-line treatment for EGFR+ NSCLC patients, was effective against both the subcutaneous and intracranial tumors. Treatment with Osimertinib at 25 mg/kg, given orally, produced 100% complete regressions (CR) against the subcutaneous tumors, which never re-grew prior to study termination on Day 26 post implant and 100% partial regressions (PR) against the established intracranial tumors, which re-grew following the conclusion of treatment at the same rate as the control brain tumors. These data show a difference between the responsive nature of subcutaneous and intracranial NSCLC tumors representing the difference in responsiveness of primary tumors and brain metastases. Utilizing this in vivo dual implant technique can be effective in comparing and assessing the ability of targeted therapies to inhibit both primary and metastatic diseases. Citation Format: Erin Trachet, Scott Wise. A novel technique to preclinically assess the ability of targeted therapies to inhibit both primary and metastatic non small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 37.

Preclinical in vivo models are used to profile or refine CAR T therapies before advancing to human studies. Establishing long-term CAR T persistence and efficacy continues to challenge progress in the CAR T space, thus development of robust platforms that can provide longitudinal assessments of CAR T persistence and functionality is paramount. To this end, we used the NALM6-Luc ALL model to develop a flow cytometry platform that provides quantitative analysis of CAR T cells over time as well as surface markers that are documented to correlate with sustained T cell persistence and activation in vivo. Tumored NSG mice were enrolled into treatment groups based on tumor burden calculated from bioluminescence imaging (BLI) data. T cells transduced to express a CD19 CAR (or left untransduced (UTD)) were injected intravenously at 1.0E+07 cells/mouse. Tumor burden was monitored by BLI and flow cytometry was performed weekly on survival bleeds to measure CAR T persistence and examine phenotype. Treatment with CD19 CAR T cells delayed tumor growth resulting in an increase in time to progression of 107.1% compared to UTD controls. However, no regressions were observed. On day 1 post-transfer, CD3+ T cells were detectable in mice that received both CAR T and UTD cells (140+/-26 and 162+/-56 cells/uL blood respectively). T cells declined to near undetectable levels by Day 20, a point when all animals in the UTD treatment group had reached euthanasia criteria. After Day 20, T cells expanded in circulation in the CD19 CAR T treatment group reaching 695+/-327 cells/uL blood by day 40. T cell expansion coincided with exponential tumor outgrowth in the treatment group. To assess T cell functionality, flow cytometry was used to measure the expression of biomarkers for T cell activation (CD25, 4-1BB, and ICOS) and exhaustion markers (TIM-3, PD-1, and LAG-3). CD25, 4-1BB, and ICOS expression did not exceed positivity on more than 15% of CD8+ T cells and peaked by day 30 before downregulation was observed. Notably, PD-1 and LAG-3 expression levels continued to increase throughout the study, suggesting T cells were taking on an exhausted phenotype. Similar trends were observed on CD4+ T cells. To investigate whether the late phase T cell expansion was a graft vs. host response, CAR T cell measurements were compared to non-tumored animals. Expansion as well as PD-1/LAG-3 expression was only observed in tumor-bearing mice indicating the responses were tumor-specific. Taken together, these data demonstrate that CD19 CAR T cells can inhibit NALM6-Luc tumor growth in vivo and expand in circulation in an antigen-specific manner. Furthermore, CAR T cell failure to control tumor growth may be due to onset of an exhausted phenotype. Finally, we demonstrate that flow cytometry can be used to characterize T cell persistence and functionality in murine xenograft tumor models. Citation Format: David W. Draper, Derrik Germain, Stacey Roys, Olivia Nelson, Scott Wise. Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2749.

1030 Background: Immune evasion is one of the mechanisms by which cancer cells gain the ability to metastasize from the primary tumor to distant sites. In triple-negative breast cancer (TNBC), metastatic tumors are shown to be more immunologically silent than primary tumors. As a result, there are varying degrees of responses to immunotherapy between early-stage and metastatic tumors. Positive clinical responses to immune checkpoint inhibitors (ICIs) are seen in patients with early-stage TNBC regardless of PD-L1 expression. In this study, we investigated the differences in the immune signatures of primary and metastatic breast cancer in a real-world patient population. Methods: We analyzed retrospective comprehensive genomic and immune profiling (CGIP) results from 529 breast tumors tested in the real-world clinical setting. Tumor specimens were classified as primary breast (PB), any lymph nodes (LN; regional and non-regional) or metastatic visceral (MV) sites. LN samples were chosen as positive controls due to expected elevated inflammatory signaling. PD-L1 (CPS positive ≥10) and TMB (high ≥10 Mut/Mb) were determined using IHC and DNA sequencing, respectively. mRNA expression signatures of tumor inflammation (TIGS, strong/moderate/weak) and expression of LAG3, TIGIT and TIM3 were determined by RNA-sequencing from a 395-gene panel. LAG3, TIGIT and TIM3 were selected due to emerging clinical trials in solid tumors. We used over-representation and proportion analysis using chi-squared test to determine the association of specimen sites to various genomic and immune correlates. Results: Among 529 cases, the median patient age was 63.2 years (25.5-93.5). The majority of patients were female (519, 98%), and the most common tumor histology was invasive ductal carcinoma (287, 54%). A total of 224 (42%), 72 (14%), and 232 (44%) of patients had specimens from PB, LN, MV, respectively, while 1 (0.19%) patient had an unknown source. Samples of PB harbored greater degree of immune infiltration, demonstrating higher TIGS score than MV (p=0.014). Primary lesions also demonstrated a greater proportion of PD-L1 positive than metastatic lesions (44% vs 21%, p < 0.001) and higher expressions of immune checkpoints such as TIGIT (p<0.001), LAG3 (p=0.037) and TIM3 (p<0.001). Conclusions: We found that non-lymph node breast cancer metastases harbor a less active immune response than primary breast lesions, showing a lower degree of immune cell infiltration and decreased expression of immune checkpoint markers. These findings support the notion that the immune microenvironment of breast cancer metastases is immunosuppressive and may exhibit a tempered response to ICIs. Therefore, combination treatments of ICIs with chemotherapy, targeted therapies or cancer vaccines may be promising therapeutic approaches to enhance the immune responses and potentially overcome resistance to ICIs in metastatic breast cancer.

Supplementary Material and Methods

Tumor microenvironments (TME) are rich in cells that potentially inhibit T cell function, such as regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs). Many TMEs are dominated by myeloid cells, making targeting these cells an area of interest. The purpose of this study was to develop an in vitro assay system to assess the ability of novel drugs to overcome MDSC-mediated immune suppression by monitoring changes to T cell function. Arginase1 (ARG1), an enzyme that catalyzes L-arginine into L-ornithine and urea, is highly expressed in MDSCs. An ARG1 target is currently in clinical trials (INCB001158, CB-1158 from Calithera Biosciences) and was used as a target of interest in this study. MDSC generation from bone marrow cells (BM) was compared using GM-CSF and IL-6 or GM-CSF, IFNγ, and LPS. α-Difluormethylornithine (DFMO), a 2nd generation ARG1 inhibitor that irreversibly binds ARG1, which leads to increased availability of L-arginine, known to modulate T cell activity, was tested during MDSC generation. BM were harvested and characterized with a myeloid flow cytometry panel. GM-CSF, IFNγ, and LPS generated more reproducible MDSCs that express ARG1. M-MDSC-derived BM expressed 2 times more ARG1 as compared to DFMO cultured MDSC-derived BM. The cytokines and other growth factors produced by MDSC-derived BM have the potential to alter the activation state of CD8+ T cells; therefore, the effect of a suppressive TME on CD8+ T cells was monitored by co-culturing CD8+ T cells with MDSCs in vitro. MDSC-derived BM were co-cultured for 4 days with CD8+ T cells (purified from C57BL/6 splenocytes) and activated with anti-CD3/anti-CD28 beads. CD8+ T cells were characterized for proliferation using CellTraceTM violet, surface activation markers (CD69 and PD-1), and intracellular cytokines (IFNγ, TNFα, and IL-2). CD8+T cells co-cultured with DFMO-inhibited MDSC-derived BM were more proliferative, had greater surface activation, and greater intracellular cytokine of CD8+ T cells as compared to when co-cultured with MDSC-derived BM. CD8+ T cell proliferation was measured at multiple ratios of CD8+ T cells and MDSC-derived BM with a proliferation range of 88-7% when CD8+ T cells were co-cultured with MDSC-derived BM and 91-30% when co-cultured with DFMO-inhibited MDSC-derived BM. Similar trends were observed with CD69, PD-1, IFN-y, TNF-a and IL-2 expression in CD8+ T cells. Overall, the ability to monitor changes in T cell activation following MDSC has broad applications in in vitro screening of novel drug compounds that alter the TME as well as sets the experimental framework for ex vivo analysis of cells isolated from TME following treatment. Citation Format: Anita J. Zaitouna, Philip Lapinski, Amber Rowse, David Draper, Scott Wise. Inhibition of murine myeloid suppressor cells increases CD8+ T cell activation in Vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2762.

Supplementary Tables 1-4, Figure 1 from The ABL Switch Control Inhibitor DCC-2036 Is Active against the Chronic Myeloid Leukemia Mutant BCR-ABLT315I and Exhibits a Narrow Resistance Profile

6506 Background: CGP concurrently tests tumor tissue for guideline-recommended predictive biomarkers for optimal therapy selection and identification of clinical trials in NSCLC patients. A common practice in the community oncology setting is to first order SGT, then consider CGP when SGT results are negative or after treatment failure. Given the limited tissue available and the scope of alterations not tested by SGT, we sought to characterize the effects of prior SGT on subsequent CGP testing success, and therapeutic opportunities identified by CGP beyond SGT for NSCLC patients in the community setting. Methods: Reference laboratory information systems were used to prospectively contact clinicians across 80 community practices who ordered at least 1 SGT for guideline-recommended genomic variants in NSCLC. CGP was offered for their patients prior to SGT or upon receipt of negative SGT results. SGT included individual assays for BRAF, EGFR, KRAS, MET exon 14 skipping mutations; ALK, RET, and ROS1 rearrangements; and PD-L1 IHC . CGP included DNA-seq for mutations, copy number variants in 523 genes including guideline-recommended genomic variants, MSI, and tumor mutational burden; RNA-seq for rearrangements/fusions/splice variants; and PD-L1 IHC. Results: Among a total of 580 NSCLC patients with CGP ordered (May 2021-December 2022), 168 (29%) had ≥1 SGT ordered prior to CGP (median=5). No patients had all SGT performed, with untested cases ranging from 10% for ALK to 90% for MET exon 14. The same FFPE tissue block was used for CGP in 150/168 (89%) of cases with prior negative SGT. Compared to CGP-only cases, CGP for cases with prior negative SGT was canceled twice as often at tissue review (16% vs 7%; p=.001), had higher DNA extraction failures (13% vs 8%; p=.09), and lower DNA sequencing success (70% vs 83%, p<.001). CGP detected guideline-recommended variants in 51% of all cases. Among prior negative SGT cases, frequencies were higher in genes where SGT was performed less often and is less sensitive than CGP RNA-seq ( RET fusions , MET exon 14 skipping). CGP also identified guideline-recommended variants in genes with no SGT offered during the study period ( ERBB2 mutations , NTRK2/3 fusions), as well as variants with emerging evidence for FDA expedited program-designated clinical trial therapies in 28% of patients, including NRG1 fusions and BRAF non-V600E , KEAP1, KRAS non-G12C, NFE2L2, STK11, and TP53 Y220C mutations. Conclusions: Prior negative SGT doubled subsequent CGP test cancellations for NSCLC due to tissue insufficiency and increased CGP DNA extraction failures. SGT practice in the community oncology setting does not meet practice guideline recommendations and negatively impacts the potential benefit of subsequent CGP for NSCLC patients.

1029 Background: Preclinical evidence suggests that breast cancer with features of genomic instability may upregulate the host antitumor immune response by producing neoantigens through DNA damage and increasing interferon production through the stimulator of interferon genes (STING) pathway. In this study, we evaluated the association between features of genomic instability and immune response in a real-world breast cancer population. Methods: We analyzed retrospective comprehensive genomic and immune profiling (CGIP) results from 529 breast tumors tested in the real-world clinical setting. We defined the HRD phenotype as tumor with any single nucleotide variants (SNV), indels, copy number variations (CNV) or fusions in the following genes: ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CHEK1/2, FANCA, MRE11A, NBN, PALB2, RAD50 and RAD51. Otherwise, they were considered HR-proficient (HRP). PD-L1 expression (CPS positive ≥10) and TMB (high ≥10 Mut/Mb) were determined using IHC and DNA sequencing, respectively. mRNA expression signatures of tumor inflammation (TIGS, strong/moderate/weak), cell proliferation (CP, high/moderate/poor) and cancer testis antigen burden (CTAB, high/low) were determined by RNA-sequencing from a 395-gene panel. We used over-representation and proportion analysis using chi-squared test to determine association of HRD to immune correlates. Results: Among 529 cases, the median patient age was 63.2 years (25.5-93.5). The majority of patients were female (519, 98%), and the most common tumor histology was invasive ductal carcinoma (287, 54%). A total of 405 (77%) and 124 (23%) of patients had HRD and HRP phenotypes, respectively. A greater proportion of HRD tumors (16%) had higher TMB compared to HRP (5.6%, p=0.003). HRP tumors showed a relatively low CP index, whereas HRD tumors were associated with a moderate CP index score (p = 0.007). HRD phenotype was associated with a significantly higher proportion of weakly inflamed tumors, as represented by lower TIGS scores (p=0.007). No significant difference in PD-L1 or CTAB was found between HRD and HRP phenotypes. Conclusions: Breast tumors with mutations in the HR genes demonstrated greater TMB and moderate cellular proliferation index of both tumor and immune cells, which suggest susceptibility to immune checkpoint inhibitors. Interestingly, this cohort lacked elevated markers of immune infiltration (TIGS), indicating a mechanism of potential tumor immune evasion. These results suggest that CGIP may allow for more informed treatment decisions in HRD breast cancers. Furthermore, assessment of HR status and immunotherapy susceptibility may support clinical trial selections for therapies targeting the complex interplay of genomic and immune components of breast cancer (e.g., a combination of PARP inhibitor and immunotherapy).