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Despite dramatic improvements in the treatment of primary breast cancers, there currently are no effective targeted therapeutics for women diagnosed with metastatic triple negative breast cancer (mTNBC), resulting in an overall survival of just ~13 months in these patients. Drug-loaded nanoparticles (NPs) offer the potential to improve the therapeutic efficacy and pharmacokinetic profile of drugs through use of passive and active targeting to metastatic tumors. However, effective and active targeting of nanodrug formulations to tumor cells is complicated by the adsorption of proteins to NP surfaces upon exposure to the systemic circulation. Termed ‘protein coronas’, this protein coat can drastically reduce the blood circulation time and targeting capability of NPs in vivo. We have developed paclitaxel (PTX)-loaded NPs that are engineered for decreased non-specific adhesivity and receptor-targeting (‘DART’) characteristics, which balance minimal recognition by circulating immune cells and low non-specific binding to tumor extracellular matrix proteins with maximal targeting to tumor tissues. These DARTs selectively bind the fibroblast growth factor-inducible 14 (Fn14) cell surface receptor, which is overexpressed in over a dozen solid cancers and their metastases, including mTNBC tumors. We recently demonstrated the enhanced therapeutic efficacy of Fn14-targeted DARTs in comparison to a non-targeted nanoformulation and Abraxane, an FDA-approved nanoformulation for mTNBC, in xenograft models of primary and intracranial TNBC. In addition, we found that these DARTs retain targeting capability and traffic to Fn14+ tumors in the presence of an endogenous protein corona in vitro and in vivo. This encouraged further investigation into the specific mechanisms of DART NP uptake in tumor cells in both the presence and absence of protein coronas using surface plasmon resonance, flow cytometry, total internal reflection fluorescence and confocal microscopy, and cytotoxicity assays. Understanding the role of protein coronas on this drug delivery platform is crucial for its clinical development and these results provide valuable new information pertaining to the optimization of NP surface properties for minimizing the impact of protein coronas and improving mTNBC tumor targeting in vivo. Citation Format: Christine P Carney, Anshika Kapur, Nikhil Pandey, Jimena G Dancy, Aniket S Wadajkar, Graeme F Woodworth, Jeffrey A Winkles, Anthony J Kim. Impact of protein corona formation on Fn14-targeted DART nanoparticle selectivity, uptake, and cytotoxicity on TNBC cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-24.

MEN1611 is a PI3K inhibitor currently in clinical development targeting the p110 α, β and γ isoforms, while sparing the δ subunit. In preclinical models, MEN1611 has demonstrated a long lasting antitumor activity when combined with trastuzumab in HER2+/PiK3CA mutated breast cancer. Aim of this work is to characterize the effects of MEN1611 on the PI3Kγ isoform, highly expressed in tumor-associated macrophages (TAMs), in order to understand whether the anti-tumor activity of MEN1611 might be also mediated by the inflammatory microenvironment. Previous evidences have shown that selective targeting of PI3Kγ by IPI-549 can reshape the inflammatory cells infiltrating tumors towards a less immunosuppressive phenotype and promote cytotoxic T cell-mediated tumor regression. In order to evaluate the effect of MEN1611 on TAMs, we established an in vitro model by differentiating murine and human macrophages from bone marrow-derived monocytes or buffy coats-isolated monocytes respectively. Pro-inflammatory M1 macrophages have been differentiated by treatment with Lipopolysaccharide (LPS) and interferon γ (IFNγ), while pro-tumoral M2 macrophages by interleukin 4 (IL-4) stimulation. The cells, thus obtained, have been incubated with MEN1611 or IPI-549 (positive control) and the effects on their phenotype, gene and protein expression, evaluated through confocal microscopy, RNASeq and flow cytometry respectively. A syngeneic xenograft model of breast cancer based on 4T1 cells has been also established in order to investigate in vivo the activity of MEN1611 on the inflammatory environment (TAMs and T cells). In vitro data showed MEN1611 and IPI-549 ability to repolarize murine and human macrophages towards a pro-inflammatory phenotype. Confocal analysis revealed a shape remodeling from an elongated M2-like towards a round M1-like; gene and protein expression analysis revealed a significant increase of immuno-stimulating factors mRNA and M1 chemokines and cytokines secretion, respectively. A modulation of the inflammatory infiltrate was also observed in vivo where RNASeq and flow cytometry analysis on dissociated treated tumors highlighted an increase of pro-inflammatory markers. The in-silico ingenuity pathway analysis (IPA) performed on genes modulated by MEN1611 revealed the enhancement of some processes related to an immune activating switch, such as the recruitment of myeloid cells or the cytotoxicity of lymphocytes and natural killer cells. In conclusion, we demonstrated in a cellular context that MEN1611 activity on PI3Kγ isoform is responsible for macrophages reprogramming from an immune-suppressive to an immune-activating phenotype. Moreover, we observed that in 4T1 murine breast cancer model, tumor regression induced by MEN1611 was also sustained by a modulation of the inflammatory microenvironment, characterized by an increased recruitment and cytotoxicity of T cells. Citation Format: Stefania Capano, Alessandro Paoli, Alessio Fiascarelli, Andrea Massimiliano Tomirotti, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Massimiliano Salerno, Monica Binaschi. Men1611 promotes immune activating myeloid cell polarization [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-26.

Purpose: Diagnosis of bone metastasis (BM) in breast cancer relies on structural changes of bone. We investigated whether circulating osteocalcin-positivecells (cOC) could detect the incipient and/or progressive BM earlier than image-based diagnostics. Experimental design: Metastatic breast cancer patients with or without bone metastasis (designated BM+ or BM-) were enrolled and cOC were quantified at baseline by flow cytometry. The progression of BM was evaluated after 18 months. Murine BM models were established by intra-tibial or -cardiac injection of MDA-MB-231 or 4T1 breast cancer cells. Results: In the baseline analysis, cOC was significantly higher in BM+ than BM- group. In the 18-month follow-up study of BM+ patients (n=63), baseline cOC was significantly associated with BM disease progression. Among BM- patients (n=33), three patients developed new bone metastasis within 18 months and were found to have had higher cOC at baseline. The patients with higher cOC showed shortened BM progression-free survival, compared with those with lower cOC (cutoff=0.045%; P Conclusions: cOC is increased in the early phase of breast cancer BM and predict the disease progression. cOC can be a novel biomarker for early diagnosis and progression of BM. Citation Format: Sun Wook W Cho, Kyung-Hun Lee, Tae-Yong Kim, Serk In Park, Seock-Ah Im. Circulating osteocalcin-positive cells as a novel diagnostic biomarker for bone metastasis in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-19-02.

Purpose/Objective(s): To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines. Materials/Methods: Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using CellTiter-Glo® assays and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival fractions were determined by cell survival after 5 population doubling times. Results: The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity, both effects are strictly additive. Conclusion: Although intrinsic HER2 expression is a factor of radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion. Citation Format: Fabien Mignot, Youlia Kirova, Pierre Verrelle, Marie-Paule Teulade-Fichou, Frédérique Megnin-Chanet. Trastuzumab Emtansine (T-DM1): No radiosensitizing effect in vitro on HER2-positive breast cancer cells (study in vitro) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-24.

Background: Triple-negative breast cancer (TNBC) still lacks defined molecular biomarkers. Thus, targeting of specific gene/protein molecular signature of tumors emerged among the best anticancer strategies. Recently, increased expression of the cell surface receptor Sortilin was reported in tumors from patients with TNBC. Given Sortilin’s functions in protein internalization and trafficking; we developed a new Sortilin-targeted peptide-anticancer drug conjugate to treat Sortilin-positive breast cancer. The conjugate consists of 2 molecules of Docetaxel linked to a 17 amino acids peptide (TH-1902) which enables specific binding to Sortilin and subsequent internalization of TH-1902. Methods: MDA-MB-231 breast cancer cells were used as a TNBC model for in vitro and in vivo xenograft assays. Female CD-1 nude mice (Crl:NU-Foxn1nu) were used for xenograft tumor models, female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were used in hematotoxicology and female Crl: CD-1 mice were used for pharmacokinetic evaluation. In vitro cell migration was assessed using the xCELLigence real-time system, whereas MTT assay was used for cell proliferation analysis. Apoptosis biomarkers expression was assessed by immunoblotting. The microtubule polymerization and depolymerization assay was performed by spectrofluorimetry, whereas fluorescent TH-peptide uptake was assessed by flow cytometry. Tissue microarrays included 9 normal adjacent tissues, 35 infiltrating ductal carcinomas (IDC), and 10 lymph nodes metastatic (LNM) carcinomas. Hematotoxicity assay allowed for blood neutrophil counts analysis in plasma samples. Results: Fluorescent Alexa488-labeled TH-peptide was found to be internalized in MDA-MB-231 cells and reduced by 72% upon transient SORT1 gene silencing. Alexa488-labeled TH-peptide uptake was inhibited by 65% in the presence of an excess of unlabeled TH-peptide and by the Sortilin ligands Neurotensin and Progranulin. TH-1902 exerted potent anti-proliferative and anti-migratory activities in vitro. TH-1902, like Docetaxel alone, triggered cell death mechanisms. The apoptotic and anti-migratory effects were reversed upon siRNA-mediated gene silencing of SORT1. Conjugation of Docetaxel to TH-peptide did not affect its capacity to alter microtubule stabilization, once internalized and cleaved from the TH-peptide within the cells. This further triggered the down-regulation of IL-6, Bcl-xL and mutant p53 pro-survival biomarkers. Sortilin immunolabeling showed very low levels in normal adjacent tissues, whereas high levels of Sortilin labeling were scored in IDC and LNM. In vivo, TH-1902 exhibited a greater tumor regression capacity with a prolonged survival in a murine MDA-MB-231 xenograft tumor model than did Docetaxel. Hematotoxic assays revealed that neutrophils count decreased significantly in Docetaxel-treated mice at MTD after 3 cycles, whereas they remained within normal limits in TH-1902-treated mice at an equivalent dose of Docetaxel even after six injections of TH-1902. Preliminary assessment of TH-1902 in normal CD-1 mice revealed that most Docetaxel remained associated with the peptide over the time course analyzed after a single IV bolus injection at 50 mg/kg. Absence of neutropenia in the presence of TH-1902 may be explained by the low levels of free Docetaxel in mouse plasma. Conclusions: The results demonstrate that Sortilin can be used as a target for treatment of TNBC. In addition, TH-1902 specifically internalized Docetaxel through a receptor-mediated mechanism exploiting Sortilin’s functions and significantly decreased the concentration of Docetaxel in normal cells. Overall, TH-1902 improved the efficacy and safety profiles compared to Docetaxel, which makes it a promising novel therapy for the treatment of TNBC. Citation Format: Michel Demeule, Cyndia Charfi, Jean-Christophe C Currie, Alain Larocque, Alain Zgheib, Christian Marsolais, Richard Béliveau, Borhane Annabi. Receptor-mediated chemotherapy using a new Docetaxel-peptide conjugate for Sortilin-positive triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-07.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent–targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry–based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D-driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. Significance: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Elucidation of the underlying molecular mechanisms of immune evasion in cancer is critical for the development of immunotherapies aimed to restore and stimulate effective antitumor immunity. Here, we evaluate the role of the actin cytoskeleton in breast cancer cell resistance to cytotoxic natural killer (NK) cells. A significant fraction of breast cancer cells responded to NK-cell attack via a surprisingly rapid and massive accumulation of F-actin near the immunologic synapse, a process we termed “actin response.” Live-cell imaging provided direct evidence that the actin response is associated with tumor cell resistance to NK-cell–mediated cell death. High-throughput imaging flow cytometry analyses showed that breast cancer cell lines highly resistant to NK cells were significantly enriched in actin response-competent cells as compared with susceptible cell lines. The actin response was not associated with a defect in NK-cell activation but correlated with reduced intracellular levels of the cytotoxic protease granzyme B and a lower rate of apoptosis in target cells. Inhibition of the actin response by knocking down CDC42 or N-WASP led to a significant increase in granzyme B levels in target cells and was sufficient to convert resistant breast cancer cell lines into a highly susceptible phenotype. The actin response and its protective effects were fully recapitulated using donor-derived primary NK cells as effector cells. Together, these findings establish the pivotal role of actin remodeling in breast cancer cell resistance to NK-cell–mediated killing. Significance: These findings establish the pivotal role of the actin cytoskeleton in driving breast cancer cell resistance to natural killer cells, a subset of cytotoxic lymphocytes with important roles in innate antitumor immunity. Cancer Res; 78(19); 5631–43. ©2018 AACR.

Background: The identification of new strategies aimed to optimize the treatment of breast cancer and its metastases represents a great technical and medical challenge. Target-specific therapies, such as Trastuzumab (TZ), have revolutionized the clinical scenario in certain subsets of cancer. However, the huge variability in response to therapy and the frequent onset of drug resistance in patients still hamper the therapeutic success. Antibody-conjugated nanoparticles may combine specific recognition of tumor cells with the capability to act as innovative reservoir of active drugs. Here, multivalent TZ-conjugated colloidal nanoparticles were developed as target-specific and biologically active nanosystem to enhance the therapeutic potential toward HER2+ breast cancer. Methods: Iron oxide nanoparticles conjugated with multiple half chains of TZ have been developed and tested in different HER2+ breast cancer cell lines, in comparison to free TZ or untargeted nanoparticles. Active targeting and specificity toward HER2 receptor was assessed by flow cytometry and confocal microscopy. Cellular uptake of nanoparticles and HER2 endocytosis were followed by electron or confocal microscopy. Direct anticancer efficacy was assessed by incubation of free or nanoformulated TZ on sensitive breast cancer cells, and analysis of cell viability, cell cycle, and expression of p27kip1. Finally, nanoparticles were tested on TZ-resistant breast cancer cell lines for capability of re-sensitization. Results: TZ-conjugated nanoparticles showed specific targeting of HER2, with induction of site-specific phosphorylation in the catalytic domain of the receptor and cellular uptake by endocytosis. Treatment with TZ-conjugated nanoparticles dramatically decreased cancer cell viability, by significantly improving the antitumor activity of TZ. This effect was independent from the ADCC mechanism, and associated with marked induction of p27kip1 expression and cell cycle arrest in G1 phase in TZ-sensitive SKBR-3 cells. TZ-conjugated nanoparticles also affected viability of breast cancer cells insensitive to TZ, further confirming enhanced potential of the nanoformulation and suggesting interference with some mechanisms of resistance. Conclusions: Our results provide evidence that multivalent exposure of TZ half chain on iron oxide nanoparticles affords enhanced antitumor potential and target-specific activity in HER2+ breast cancer cells. Powerful inhibition of HER2 signaling by TZ-conjugated nanoparticles could favor responsiveness of drug resistant cells, thus suggesting novel therapeutic strategies to overcome resistance. Citation Format: Truffi M, Monieri M, Sorrentino L, Mazzucchelli S, Colombo M, Pandolfi L, Prosperi D, Corsi F. Multivalent exposure of trastuzumab on iron oxide nanoparticles enhances antitumor activity and weakens drug resistance in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-11.

CD47 is a cell surface transmembrane protein that binds to signal regulatory protein alpha (SIRPα) on macrophages and results in a “don't eat me” signal that inhibits phagocytosis. Breast cancer cells, both primary and metastatic, frequently overexpress CD47 and exploit this pathway to evade macrophage-mediated destruction. Anti-CD47 monoclonal antibodies (mAbs) block the CD47/SIRPα interaction and promote tumor cell destruction via phagocytosis. Anti-CD47 mAbs also contribute to an anti-tumor T-cell response in immune-competent mice. Therefore, anti-CD47 antibodies represent a new class of immune checkpoint inhibitors that modulate both the innate and adaptive immune systems. CD47 is expressed on multiple cell types, including tumor cells and normal cells. Many anti-CD47 mAbs block the CD47/SIRPα interaction and cause phagocytosis of tumor cells, but do not directly induce the death of human tumor cells. Tioma has created new humanized anti-CD47 mAbs with novel and differentiated functional profiles to enhance functional heterogeneity. Ti-104, Ti-176 and Ti-108 block the binding of SIRPα to CD47 and increase phagocytosis of human tumor cells. They also induce cell death of human hematological and solid tumor cell lines (including breast cancer lines) in a cell autonomous manner. Cell death was determined by an increase in phosphatidylserine-positive/7AAD-positive tumor cells assessed by flow cytometry following incubation in media containing anti-CD47 mAb or a negative control immunoglobulin. In vitro, these mAbs bind to human tumor cell lines with apparent binding affinities ranging from low pM to low nM, depending on the cell line and method of analysis (solid-phase or cell-based ELISA, flow cytometry or surface plasmon resonance). In vitro, Ti-104 and Ti-108 bind to human RBCs, whereas Ti-176 has markedly reduced binding to human and cynomolgus monkey RBCs. In a four-week (once-weekly dosing) exploratory safety study in cynomolgus monkeys with Ti-176 and Ti-108, no dose-limiting toxicity or gross pathological or microscopic findings were identified after an initial dose of 5 mg/kg (Week 1) followed by doses of 50 mg/kg (Weeks 2, 3, and 4). Ti-176 treatment resulted in minimal decrease in red cell mass, hemoglobin and hematocrit, which corresponded in vitro to markedly reduced binding to cynomolgus monkey RBCs. Ti-108 caused transient reduction of RBC parameters comparable to some previously reported anti-CD47 mAbs. Ti-104, Ti-176 and Ti-108 showed potent, dose-dependent efficacy in multiple mouse tumor models, including in the MDA-MB-231 triple-negative breast cancer orthotopic model. These data provide the preclinical rationale for further evaluation of Ti-104, Ti-176 and Ti-108 as breast cancer treatments. Citation Format: Karr RW, Liu K, Hiebsch R, Capoccia B, Donio M, Crowley K, Puro R, Chanda S, Manning P. Development of humanized anti-CD47 monoclonal antibodies with differentiated functional profiles [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-15.

Background: PolyADP-ribose polymerase (PARP) inhibitors are a novel promising strategy toward triple-negative breast cancer (TNBC), which often shows genomic instability or BRCA mutations. However, clinical results are controversial, and no benefits were demonstrated in case of wild type BRCA, possibly due to poor bioavailability, and inadequate nuclear delivery. Nanotechnology could overcome these major limitations. The aim of this study was to assess the anticancer efficacy of H-Ferritin nanoformulated Olaparib (HOla) vs. free Olaparib (Ola) on BRCA-mutated and non mutated TNBC cells. Methods: BRCA-mutated HCC1937 cells and BRCA-wild type MDA MB-231 and MDA MB-468 cells were treated with HOla or free Ola in vitro. Active targeting and binding capability of HOla toward transferrin receptor 1 (TfR-1), over-expressed on TNBC cells, was assessed by flow cytometry. Internalization and intracellular localization of Ola and HOla was assessed by confocal microscopy. Anticancer efficacy was assessed by administration of increasing doses of HOla or Ola, comparing cell viability, cell cycle, cell death, PARP-1 cleavage and DNA damage. Finally, anti-PARP efficacy and proportion of drug in the nuclear compartment were compared between treatments. Results: All TNBC cell lines over-expressed TfR-1 and were succesfully recognized by HOla. Confocal microscopy showed a fast internalization of nanoparticles into cells, with intracellular persistence up to 48h. A marked increase in nuclear concentration of drug was observed with HOla compared to Ola, due to a strongly improved nuclear delivery by H-Ferritin mediated by a self-triggered mechanism. No significant antiproliferative effect was demonstrated with Ola at 10 nM, 50 nM or 100 nM. Conversely, HOla at 50 nM and 100 nM showed a 1000-fold higher anticancer activity in all TNBC cell lines. A possible contribution in cytotoxicity by H-Ferritin nanovector itself was excluded treating cells with void nanoparticles. Proportions of cell cycle arrest in G2/M, cell death, cleaved PARP-1 and DNA damage in terms of phosphorylated histone H2A.X were higher in HOla treated samples than in ones treated with free Ola. Conclusions:Our findings suggest that nanoformulation of Ola strongly enhances cytotoxic efficacy of PARP inhibition as a stand-alone therapy, on both BRCA-mutated and wild type TNBCs allowing a targeted delivery into TNBC cells and a prompt homing into the nuclear compartment. Citation Format: Mazzucchelli S, Truffi M, Sorrentino L, Bellini M, Rizzuto MA, Ottria R, Ciuffreda P, Prosperi D, Corsi F. Olaparib nanoformulation in H-ferritin as a promising option for both BRCA-mutated and sporadic triple negative breast cancer: An in vitro study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-13.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and scourging soft tissue tumor worldwide. Its current incidence is 13 per 100,000 US population with a very low 5-years survival rate (5%) and estimated to be the second leading cause of cancer-related death by 2030. Most diagnoses are made at advanced disease states warranting combination chemotherapy. Fluorouracil (5-FU) is an effective drug in treating PDAC; however, rapid degradation and systemic instability remain a drawback to its efficacy. The objective of this study was to chemically modify 5-FU to 1,3 bistetrahydrofuran-2yl-5-FU (MFU) to prolong its systemic stability and enhance the anticancer activity of 5-FU. Method: 5-FU was chemically modified to MFU using tetrahydrofuran-2-yl acetate and 1,8-Diazabicyclo (5.4. 0)undec-7-ene (DBU) in Dimethyl Formamide (DMF). MFU was characterized using nuclear magnetic resonance (NMR) to determine new bond formation, mass spectrometer (MS) for molecular weight determination, high-performance liquid chromatography (HPLC) to determine percent purity, and micro-elemental analysis used to ascertain the presence of elemental composition and purity. Percent conversion of MFU to 5FU was determined using PDAC cells. In vitro anticancer activity of MFU was tested using 2D and 3D MiaPaCa-2 and PANC-1 PDAC cultures and cell viability study performed using the alamar blue assay. Apoptotic and cell cycle studies were performed on MFU treated MiaPaCa-2 and PANC-1 cells using flow cytometry. Results: Molecular weight of synthesized MFU (C12H15FN2O4) was determined to be 270.02 using the Electrospray Ionization (ESI) (MS (M+H)+ = 270.08). While percent purity of MFU was found to be greater than 99.6%, melting point was determined to be 115±2 °C and retention time (RT) was 1.85 minutes. For MFU conversion to 5-FU studies, 4.1% of 25µM and 7.6% of 50µM of MFU incubated MiaPaCa-2 cells was converted to 5-FU over a period of 24 hr showing an incremental production of 5-FU from MFU based on concentration. For in vitro study, half-maximal inhibitory concentration (IC50) of 5-FU treated 2D MiaPaCa-2 culture was 3.4 ±1.1µM while that of MFU treated 2D MiaPaCa-2 culture was 2.1± 0.2 µM. The IC50 value of 5-FU treated 3D MiaPaCa-2 culture was found to be (8.5±1.2µM), while IC50 value for MFU treated 3D MiaPaCa-2 culture was (7.2±1.1µM). Put together, IC50 value for MFU for either 2D or 3D was significantly lower compared with IC50 value 5-FU (P=0.021 (MFU vs 5-FU) for 2D and P=0.04 (MFU vs 5-FU) for 3D). A similar trend was noted when PANC-1 cells were used. Apoptotic and cell cycle studies were similar to that of the 5-FU with most of the cells (54%) in late apoptosis after 48 h of treatment and 64% vs 33% of cells arrested at the G1 and S phase of the cell cycle respectively. Conclusion: The studies demonstrated that MFU may be an alternate approach to improve the delivery, systemic stability, and efficacy of 5-FU in the treatment of PDAC tumors. Citation Format: Nkafu Bechem Ndemazie, Andriana Inkoom, Xue Y. Zhu, Edward Agyare. Optimizing the efficacy of 5-FU as a chemotherapeutic agent in advanced pancreatic ductal adenocarcinoma (PDAC) using MIAPaCa-2 and PANC-1 cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-047.

Background: HPD has been described in 14-26% of NSCLC pts upon single agent ICI and correlates with poor survival. Currently, HPD occurrence has not been explored upon ICI-PCT and biomarkers of HPD are lacking. Although high circulating neutrophils (Ns) correlated with HPD in NSCLC pts, the role of specific Ns subsets is unknown. Methods: NSCLC pts treated with 1st line ICI as single agent or in combination with PCT were assessed for HPD and circulating Ns' phenotype. Tumor response was evaluated by RECIST 1.1. HPD required 3 assessment (2 before ICI, 1 upon ICI) and was defined as delta tumor growth rate (TGR) (TGR upon ICI - TGR before ICI) >50% and/or TGR ratio (TGR upon ICI/TGR before ICI) ≥2. Circulating low density Ns (LDNs) subtypes were assessed by flow cytometry (FC) on peripheral blood mononuclear cells (PBMCs). LDNs were defined as CD66b+CD15+ cells among CD11b+ PBMCs. Immature subtypes were defined as CD10- LDNs. Fatty acids (FA) (bodipy-palmitate) uptake was assessed by FC. T-cells of healthy donors were isolated and activated in vitro with CD3/CD28 beads in presence or not of pts' Ns to characterize their interplay. Results: Of 63 NSCLC pts, 22% had PD-L1 on tumor cells ≥50%. In the ICI single agent cohort (N=46), PD and HPD occurred in 21 (41%) and 4 (9%) pts, respectively. Before ICI start, HPD pts had significantly higher median percentage (%) of circulating immature CD10- LDNs [43.5 (min 29.5; max 82.6) vs 10.3 (min 0.1; max 79.4), p=0.01] compared to PD pts. In the ICI-PCT cohort (N=17), PD occurred in 4 (23%) pts, no HPD was reported. 5 pts had baseline CD10- LDNs ≥ 43.5% (median % of CD10- LDNs in HPD pts upon single agent ICI), 2 of them had SD and 3 PD upon ICI-PCT. In these 5 pts, CD10- LDNs significantly decreased during ICI-PCT compared to what observed in HPD pts during single agent ICI [median variation -43.4 (min -67.6, max -31.6) vs +6.9 (min -33, max +44), p= 0.03]. CD10- LDNs did not impair T-cells proliferation and IFNγ production, measured after 7 days of coculture but significantly reduced T-cell survival compared to CD10+LDNs. Baseline metabolic profile assessment showed that immature CD10- LDNs had a predominant lipid metabolism with a higher FA uptake [bodipy-palmitate mean florescence intensity (MFI) ± standard deviation (SD): 46754±11562] compared to lymphocytes [bodipy-palmitate MFI ±SD: 11542±405]. Conclusion: Higher baseline immature CD10- LDNs is a circulating biomarker of HPD upon single agent ICI. The addition of PCT prevents HPD reducing the immature Ns subset. The FA metabolism of immature LDNs does not affect T-cells proliferation/activation but reduces T-cells survival, suggesting that these two processes are differently regulated and that immature Ns reduce T-cells life span by metabolic competition. Citation Format: Roberto Ferrara, Elena Jachetti, Giuseppina Calareso, Marta Brambilla, Giuseppe Lo Russo, Claudia Proto, Arsela Prelaj, Diego Signorelli, Giulia Galli, Alessandro De Toma, Mario Occhipinti, Sara Manglaviti, Alice Labianca, Monica Ganzinelli, Sestina Maria Spanò, Giuliano Molino, Antonia Martinetti, Francesca Gabriella Greco, Marta Bini, Teresa Beninato, Filippo de Braud, Mario Paolo Colombo, Marina Chiara Garassino, Sabrina Sangaletti. Immunometabolism of circulating neutrophils in hyperprogressive disease (HPD) upon first-line PD-1/PD-L1 inhibitors (ICI) alone or in combination with platinum-based chemotherapy (PCT) in non-small cell lung cancer (NSCLC) patients (pts) [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 1669.

Background: NSCLC cells carrying EGFR mutations can gain resistance to cognate TKIs through amplification of Chr22q11.2 (Chr22amp), a chromosome segment containing CRKL. This also specifically associates with exquisite sensitivity to inhibitors of Aurora Kinase B (AZD2811), potentially mediated by other Chr22 genes. Furthermore, a phenotypic rewiring occurs in the response to AZD2811, from a senescent polyploidy in wildtype (WT) cells to apoptosis in Chr22amp cells. Here, we aimed to elucidate the underlying signaling alterations in this background by phosphoproteomic pathway analysis. Methods: The EGFR mutant cell line PC9 and 8 TKI resistant derivatives were profiled (4 Chr22amp and 4 WT). Kinetics of response to AZD2811 (100nM) and osimertinib (160 nM) were identified by flow cytometry. Samples (n=3) were prepared for phosphoproteomics, after 6, 24, and 48 h AZD2811 and 1 h osimertinib, with time matched controls. Cells were washed and lysed in urea, then digested with trypsin. Phosphorylated peptides were enriched with TiO2 and analyzed by Orbitrap LC-MS/MS. Computational analyses quantified peptides across samples. KScanTM bioinformatics identified differential phosphopeptides between Chr22amp and WT to determine kinase substrate profiles by KSEA, putative downstream targets (PDT) and differential compound target activity markers (CTAM). Results: Single cell time-course analysis of phenotypic response to AZD2811 in Chr22amp cells showed that >60% of cells become Annexin V+ by 48 h post-treatment. We took earlier timepoints of 6, 24 and 48 h post treatment. We focused the phosphoproteomic analysis on three comparisons of Chr22amp amplified cells to: 1) the basal signaling state compared to WT; 2) the signaling response to osimertinib in parental PC9; and 3) the altered kinetics of signaling in response to AZD2811 compared to WT. At the basal level, Chr22amp had CK1e, CDK2, p38a substrates differentially enriched, and MTOR inhibitor and Aurora B inhibitor modulated sites (p Conclusions: Here, we identified significant pathway deregulation in Chr22amp cells that subverted EGFR inhibition and enhanced sensitivity to AZD2811. Intriguingly, we detected enhanced Aurora B activity in Chr22amp cells at basal levels, and surprising impact of AZD2811 on the EGFR pathway. Citation Format: Arran Dokal, Jordi Bertran-Alamillo, Edmund Wilkes, Hilary Lewis, Ana Gimenez-Capitan, Calum Greenhalgh, Ruth Osuntola, Maruan Higazi-Vega, Shona Ellison, Vinothini Rajeeve, Giulia Fabbri, Urszula Polanska, J. Elizabeth Pease, Pedro Rodriguez-Cutillas, Jelena Urosevic, Miguel Angel Molina-Vila, David Britton, Jon Travers. Precision phosphoproteomic analysis in Chr22q11.2 amplified NSCLC cells reveals distinct signaling corruption and response to Aurora kinase B inhibition [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 1107.

Introduction: Toxin-conjugated agents targeting cell-surface antigens (e.g. antibody-drug conjugates) show promising clinical activity for some solid tumors, but none yet for OS. There is an urgent need to identify high-confidence OS cell-surface antigens and evaluate the antitumor activity of toxin conjugates that can deliver cytotoxic payloads to OS tumors expressing these targets. Methods: To identify cell-surface proteins that are highly expressed in OS but not in normal tissues, we prepared surface protein extracts from 4 standard, 4 patient-derived OS cell lines, and 10 OS PDX tissues. Quantitative mass spectrometry (MS) was used to profile the surface proteins. We included the proteomics data of OS and 39 types of normal human tissues via a public MS database (ProteomicsDB). We compared RNA-sequencing data from 101 OS patient samples via the NCI TARGET project with the RNA-sequencing data of 54 types of normal human tissue (GTEx, 17382 samples). We integrated the MS and RNA sequencing datasets to prioritize candidate surface targets by differential and absolute expression at both protein and mRNA levels. The cell-surface expression of the targets was further validated by IHC and flow cytometry with OS cell lines and tumor tissue microarray. BT1769, which consists of a bicyclic peptide targeting the tumor antigen MT1-MMP, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker was tested in 6 OS PDX models using standard PPTC methods at 3 mg/kg given IV weekly x 4. Ewing sarcoma (EWS) models lacking MT1-MMP expression were also tested. Results: MS identified 1156 surface proteins as highly expressed in OS cell lines/PDX tissues. 129 proteins overexpressed in OS were identified from the public MS database. RNA sequencing data identified 195 overexpressed plasma membrane-associated genes in OS compared to normal tissues. The targets identified by both proteomics and transcriptomic data analysis were cross-referenced with existing toxin-conjugated agents. Three potential targets, MT1-MMP, CD276, and MRC2 were validated and MT1-MMP was prioritized because it had the highest absolute expression level in OS. BT1769 significantly inhibited tumor growth in 6/6 OS PDX models when compared to vehicle, with complete response maintained at 6 weeks in 3/6 models. Minimal activity was observed in 2/2 EWS models. Correlation analysis between the MT1-MMP gene expression level, IHC staining intensity and drug response are underway and will be presented. Conclusion: Integrated proteomic and transcriptomic OS surfaceome profiling identified high-confidence OS cell-surface antigens as therapeutic targets. MT1-MMP was highly expressed in most OS patient samples and PDX models, and BT1769 was highly active against select OS PDX models. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana N. Lazcano Segura, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Eric J. Earley, Stephen W. Erickson, Edward A. Kolb, Malcolm A. Smith, Peter Houghton, Raushan Kurmasheva, Tara Gelb, Philip Huxley, Johanna Lahdenranta, Gemma Mudd, Richard Gorlick. Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC) [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 LB008.

Background: Cancer progression and response to therapy are governed by complex cellular interactions in the tumor microenvironment (TME). A better understanding of TME and the immune microenvironment requires robust experimental systems modeling patient specific immune interactions. Conventional 2D cultures lack in recapitulating 3D morphological structures and complex immune biology of native human tumors. A vastly heterogenous TME impacts patient treatment response. There is a dearth of 2D or 3D in vitro models that mimic in vivo diversity of TME, including infiltrating immune populations. 3D organoid cultures typically contain neoplastic epithelium and fail to retain syngeneic tumor-infiltrating lymphocytes (TILs) interactions vital for exhibiting a clinically significant drug response. Here we describe patient-derived xenograft (PDX) derived 3D organoid (PDXO) cultures with autologous TILs to avoid alloreactivity and simulate tumor specific immune response. The described model allows the evaluation of TIL infiltration and high throughput screening of immune-modulatory therapies. Methods: Patient-derived xenografts from mice were enzymatically digested to establish PDXO. PDXO were characterized for expression of cancer specific cell-surface markers. TILs were expanded in culture from PBMCs from matching patients and characterized using flow cytometry. Organoids and TILs from the same patient were fluorescently labeled and cocultured for four days, and tumor infiltration and drug cytotoxicity with and without immune-therapeutics was evaluated. CellInsight CX7 high content imaging platform was used to track TILs, and cancer cells, and TIL infiltration was confirmed via immunofluorescence. Luminex technology was used to quantify changes in the cytokine profile upon drug treatment. Results: PDXO were established to mimic in vivo tumor biology. Tumor-specific TILs were expanded and characterized for expression of memory, inhibitory, proliferation, and regulatory T cell markers. TIL infiltration in organoids was observed from day one in culture and maintained over four days. Immunofluorescence for T cell marker CD3 (cluster of differentiation 3) within PDXO confirmed TIL infiltration. Infiltrating TILs and tumor cell killing evaluation using fluorescent dyes via high throughput imaging platform showed disease and patient-specific response. PDXO treatment with immune-modulatory therapeutics led to changes in the secreted levels of cytokines IL2 (interleukin 2), IFNγ (interferon gamma), and TNFα (tumor necrotic factor alpha). Conclusion: Our patient autologous TILs - PDXO culture platform can be used for high throughput drug screening to model immune-therapeutic response with the tumor specific immune microenvironment. Citation Format: Garima Kaushik, Padma Kamineny, Amy Wesa. Unique 3D organoids autologous TIL coculture platform enables high throughput immune-oncology drug response studies [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 2978.

Cancer immunotherapies have the potential to induce durable anti-tumor immune responses; however the efficacy of these treatments varies among patients and partially depends on the complex interplay between malignant cells and non-malignant cells within the tumor microenvironment. In the tumor microenvironment, T cells play a crucial role in eliminating tumor cells and orchestrating anti-tumor immune responses; in contrast immunosuppressive cell types such as myeloid-derived suppressor cells can support tumor progression. Here we harnessed recent advances in single-cell multiomics that allow high-resolution functional characterization of intratumoral immune cells to improve our understanding of their dynamic relationships. Specifically, we utilized a One Solution Workflow to dissociate a solid tumor followed by in-depth dissection of the immune composition of the tumor microenvironment using both flow cytometry and the BD Rhapsody™ Single-Cell Analysis System. We characterized the cellular composition of tumor microenvironment using flow cytometry analysis. Importantly, by examining the expression of mRNA and surface protein markers at single-cell resolution, we unraveled the complexities of the tumor microenvironment, elucidated cellular heterogeneity and identified cell-type specific gene signatures. We propose this One Solution Workflow can be adopted to understand the transcriptional and proteomic phenotypes of tumor infiltrating leukocytes for development of novel immune therapeutic strategies for cancer. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product.BD-23279 (v1.0) 1120BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved. Citation Format: Xiaoshan Shi, Margaret Nakamoto, Aaron Middlebrook, Wei Huang, Evelyn Lo, Patrick T. Neuhoefer, Scott Bornheimer, Smita Ghanekar. Deep characterization of tumor infiltrating leukocytes using a combination of flow cytometry and single-cell multiomics [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 2788.

Introduction: Molecular nanomachines (MNMs) have been studied for their anti-tumor efficacy in a number of solid malignancies. They consist of small (1 nm) organic molecule-based motors that change conformation upon light activation and mechanically drill holes in the cell membrane resulting in cellular necrosis, representing a novel form of molecular mechanical therapy. Recently, we generated a blue light spectrum (395-405 nm)-activated MNMs which showed promising results in pancreatic cancer cell lines. Herein, we evaluate the in vitro efficacy in urothelial carcinoma, a disease characterized by high rates of recurrence, progression, and therapeutic resistance. Methods: MNMs activated by the blue light spectrum were synthesized. In vitro efficacy of the activated MNMs (8 µM in 0.1% DMSO) was investigated in TCCSUP and UC-3 human bladder cancer cell lines. Cells were incubated with MNMs for 30 minutes, then treatment groups were subjected to 300 mW/cm2 blue light (comparable to Blue Light Cystoscope) for 4 minutes. Control groups included MNMs without light, 0.1% DMSO (required for solubility of the MNMs) with light, and 0.1% DMSO without light. Cell viability was quantitatively evaluated utilizing propidium iodide and flow cytometry assay with each datapoint representing triplicates. Additionally, cells were grown and subjectively tested for cell adhesion to the culture dish at 1d following light and MNM treatment. In addition, we utilized the wound healing assay to detect changes in cell motility following MNM treatment. Results: TCCSUP cells exhibited almost 100% loss in viability when treated with light-activated MNMs. Solution only (0.1% DMSO) and unactivated MNMs exhibited less than 5% change in cell viability, whereas, solution and light exhibited 20% reduction in cell viability suggesting some light sensitivity of TCCSUP cells. Cell adhesion assay 1d post-treatment confirmed 100% cell death for the MNMs + light treated cells, while showing survival of cells with light only treatment. In addition, results from wound healing assay conducted on UC-3 cells demonstrated at 72 h, there was significant inhibition of cell motility in the treatment group (MNMs + light) compared to control groups. Conclusions: Light-activated MNM exhibit in vitro efficacy in multiple human bladder cancer cells lines. MNMs are a promising novel therapeutic modality for the intravesical treatment of bladder cancer, and there are no known resistance mechanisms. Further preclinical characterization of antitumor efficacy and tolerability are necessary in in vivo models. Citation Format: Supriya Nagaraju, Alexis van Venrooy, Patrick J. Hensley, Kelly K. Bree, Ciceron Ayala-Orozco, Nathan A. Brooks, David Izhaky, James M. Tour, Ashish M. Kamat. Light-activated molecular nanomachines kill bladder cancer cells [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 267.

Tissue-resident memory CD8+ T cells (TRMs), marked by expression of CD103, are considered to play a key role as anti-tumor immune response in breast cancer. However, prognostic value of TRMs and heterogeneity in CD103+ TRMs of breast cancer in terms of tumor-specificity needs further understanding to establish immuno-therapeutic implications. Here, we isolated tumor-infiltrating CD103+TRMs in breast cancer samples and matched peripheral blood mononuclear cells (PBMCs) which were prospectively collected from 127 patients who underwent surgical resection between July 2016 and May 2020 at Severance Hospital, Seoul, Republic of Korea. Multi-color flow cytometry analyses were performed.CD103+TRMs consist of both tumor-antigen specific CD8+ T cells and bystander CD8+ T cells, such as viral antigen-specific CD8+ T cells. Dissection of CD103+TRMs with CD39 expression reveals a clonally-distinct subpopulation exhibiting tumor-antigen specificity and reactivity with transcriptionally and phenotypically exhaustive features. CD39+CD103+ TRMs were enriched in HER2 enriched and triple-negative breast cancer(TNBC) subtypes, but not in Hormone receptor(HR) positive/HER2 not enriched subtypes, implying that the role of tumor-infiltrating CD8+ T cells in eliciting anti-tumor immune response differ among molecular subtypes. In line with early-exhaustive features of CD39+CD103+TRMs, inhibition of either PD-1 alone or both PD-1 and CTLA-4 reinvigorate the proliferation and the cytokine production of CD8+ TILs in HER2 and TNBC subtypes. Our findings indicate that different immuno-therapeutic strategies should be considered to different subtypes of breast cancer. Citation Format: Jee Ye Kim, Yong Joon Lee, Seung hyuck Jeon, Heejin Nam, Jae Hyung Jung, Seung Il Kim, Eui-Cheol Shin. Tumor-specificity of tumor-infiltrating CD8+ t cells determines the potential of immune checkpoint inhibitors in breast 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 1666.

Tumor-specific monoclonal antibodies (mAb) which induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer. Evaluating the potency of a new, tumor-specific mAb requires testing with multiple sources of donor cells since the degree of NK cell-mediated ADCC is known to be variable between patients. Thus, in addition to evaluating whether a new tumor-specific mAb can effectively induce NK cell-mediated ADCC, it is also important to characterize the donor NK cells utilized in order to provide insight into potential treatment efficacy. In the current study, we investigated the application of a novel, high throughput assay in assessing the ADCC potency of different anti-hCD20 mAbs against a Burkitt's lymphoma cell line using effector cells obtained from multiple donors. This multiplexed assay incorporates a cells and beads format to enable the simultaneous measurement of target cell killing, along with enumeration and phenotyping of the NK cells, and quantification of up to 8 secreted effector proteins and cytokines. The assay requires a sample volume of just 10 µl and can be performed in a single well of a 96 or 384 well plate using the iQue® platform for advanced flow cytometry. Data analysis and visualization was done using the integrated iQue Forecyt® software package. The results showed a mAb concentration-dependent increase in tumor cell killing, along with corresponding increases in secretion of Granzyme B and CD178 (Fas Ligand), and production of IFNγ, TNF, and RANTES. The degree of NK cell-mediated ADCC and effector protein secretion levels were dependent on both the anti-hCD20 mAb and donor cells used, with higher levels of ADCC and effector protein secretion observed using a non-fucosylated anti-hCD20 mAb as compared to a conventional, fucosylated anti-hCD20 mAb. This method enabled the rapid assessment of the potency of different mAbs in ADCC mediated by NK cells, and also effectively distinguished potential differences in mAb efficacy between donors. Citation Format: Julie Lovchik, Mark Carter. Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry [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 1856.

Background: Identification of ALK rearrangements in NSCLC patients has important implications as it significantly expands their therapeutic opportunities. However, the availability of tumors biopsies is sometimes limited in the clinical setting. The analysis of liquid biopsies can potentially overcome such limitation. Nevertheless, the identification of ALK rearrangements using liquid biopsies remains challenging as fusion transcripts are rapidly degraded by RNAses present in the blood. Nonetheless, encapsulated RNA inside extracellular vesicles (EVs) is protected from RNAses. The main objective of this study is to explore the potential value of exosomes for non-invasive ALK testing. Methods: H3122 and H2228 cell lines, derived from NSCLC patients with EML4-ALK variant 1 (E13;A20) and variant 3 (E6a/b;A20) respectively, were used to isolate exosomes by the sequential centrifugation of cell culture supernatants. Briefly, supernatants were centrifuged twice for 10 min at 200xg, twice for 500xg for 10 min, once for 30 min at 10,000xg and a final ultracentrifugation at 100,000×g for 2 h at 4 °C. Purified exosomes were verified in terms of morphology and size by transmission electron microscopy (TEM). In addition, specific EVs markers, namely CD63, CD81 and CD9, were measured by flow cytometry and Western-Blot. EML4-ALK variants were analyzed at the protein level by Western blot using 5A4 antibody, and RNA level by digital PCR (dPCR), using a QuantStudio® 3D Digital PCR System and specific TaqMan® assays for EML4-ALK variant 1 and 3. Samples were also submitted to NGS analysis using the Oncomine™ Pan-Cancer Cell-Free Assay kit. Results: TEM showed spherical vesicles around 100 nm in both cell lines. Flow cytometry and Western blot confirmed expression of the exosomal specific markers CD63, CD81 and CD9 tetraspanins. Western blot also showed EML4-ALK variant 1 (120 kDa) and variant 3 (87 kDa) in H3122 and H2228 purified exosomes, respectively. Isolated exosome RNA profile was composed of small RNA (< 200 nt), although 18S and 28S rRNA peaks were also present. dPCR and NGS confirmed EML4-ALK rearrangements in exosome derived from both cell lines. Conclusions: EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients. Citation Format: Estela Sánchez-Herrero, Carmen Campos-Silva, Yaiza Cáceres Martell, Sandra Sanz-Moreno, Roberto Serna-Blasco, Alejandro Rodríguez Festa, Miguel Ángel Molina-Vila, Mariano Provencio, Mar Valés-Gómez, Atocha Romero. ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing [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 561.

Obesity creates a chronic inflammatory environment that can lead to an exhausted phenotype among T cells. Since exhausted T cells gradually lose their effector functions, obese patients may be at higher risk of developing different disorders, including infectious diseases and cancer. Using the BD FACSymphonyTM A5 Cell Analyzer, we applied a 21-color high-parameter flow cytometry panel to assess various immune cell types in a mouse model of obesity. Also, a flow-based functional assay provided insights into obesity-associated T cell dysfunction. To address progressive changes that might have been caused by a high-fat diet over time, C57BL/6 mice were fed with a high-fat diet for a period of 4 or 16 weeks. Cell populations were examined in blood, spleen and epididymal fat tissue with the latter being known as a location that harbors local inflammation in obese mice. Computational tools for high-dimensional data analysis allowed a thorough characterization of tissue-specific cell phenotypes and their relationship with obesity. Notably, the frequency of effector/memory CD8 T cells and TCRγδ T cells were increased in the fat of obese mice. However, assessment of T cell effector functions in vitro revealed an impairment in cell proliferation, suggesting that fat CD8 T cells from obese mice may not effectively respond to stimulation. Though further studies are required, these results support previous published evidence that low-grade chronic inflammation associated with obesity may impact T cell responses. The data also represent an efficient stepwise approach for staining of surface and intracellular markers and for evaluation of T cell function in vitro and demonstrates the utility of computational tools available in FlowJo™ v10 Software. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product.BD-23275 (v1.0) 1120BD, the BD Logo, FACSymphony and FlowJo are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved. Citation Format: Gisele V. Baracho, Stephanie Widmann, Aaron Tyznik. Defining cell population diversity and T cell dysfunction in a mouse model of obesity [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 1815.

In colon cancer, the subset of cells fueling the clonal growth of the tumor have been shown to also possess enhanced chemoresistance abilities. These cells, known as cancer stem cells (CSC), are controlled by molecules and cells within the tumor microenvironment. In the latter reside enteric glial cells (EGC) that heavily regulate intestinal epithelial barrier function in a healthy colon. However, EGC influence on colon carcinogenesis remains poorly understood. Recent work from our lab shows that EGC promote CSC-driven tumorigenesis via the release of prostaglandin E2. Here, we investigate the impact of EGC on CSC chemoresistance. CD24Hi-CD44Hi CSC were FACS-isolated from human colon cancer primary tumors or cell lines and 3D-grown in the presence of murine or human EGC seeded on Transwell filters in vitro in the presence of the chemotherapeutic drug 5-Fluorouracil (5-FU) or injected subcutaneously with(out) EGC in vivo in 5-FU treated-immunodeficient mice. EGC impact was assessed on 5-FU-induced restriction of tumor formation and growth in vitro and in vivo. EGC-conditioned medium (CM) impact on 5-FU-induced apoptosis in CSC was measured by flow cytometry. EGC impact on expression of 48 chemoresistance-related genes in CSC was assessed by high throughput RT-qPCR. Impact of inhibition of ATM activity and ATM activation by the Mre11-Rad50-Nbs1 (MRN) complex in CSC was tested using 0.1µM KU-55933 and 12µM mirin. To understand the impact of 5-FU on EGC, levels of senescence markers p16, p19, p21 and H3K9me3 and β-gal activity were assessed. Proteomic analyses of EGC-CM were conducted by mass spectrometry. EGC promoted growth of CSC-derived tumors in the presence of 5-FU in vivo. EGC increased the number and size of tumor-organoids grown from CSC isolated from cell lines (HT29, HCT116, HCT15) and primary tumors in the presence of 5-FU in vitro. EGC-CM reduced apoptosis in 5-FU-treated CSC. Out of all genes tested, only ATM mRNA was significantly enriched in 5-FU-treated CSC cultured with EGC vs. alone. Co-culture studies using CSC derived from the ATM deficient SW1222 cell line or using the ATM inhibitor KU-55933 reduced EGC pro-chemoresistance effects. Furthermore, inhibition of ATM activation by the upstream DNA damage sensor MRN complex using mirin abolished EGC protective effects on CSC resistance to 5-FU. 5-FU-treated EGC showed increased levels of senescence markers and protein secretion, indicative of a senescence-associated secretory phenotype (SASP). Moreover, CM from 5-FU-treated EGC further reduced 5-FU-induced apoptosis in CSC as compared to CM from untreated EGC. Mass spectrometric analyses revealed that IGFBP7, a putative ATM activator, was >100 times more abundant in the CM of 5-FU-treated EGC vs. untreated EGC. Our data strongly indicate that EGC promote CSC chemoresistance via increased ATM signaling. Future studies will investigate the implication of IGFBP7 and downstream targets of ATM involved in DNA repair. Citation Format: Gregory Bacola, Simon Vales, Alice Prigent, Kelsie A. Dougherty, Deanna M. Peperno, Shaian Lashani, Bradley A. Wieland, Melissa Touvron, Lisa Oliver, François M. Vallette, Michel Neunlist, Laurianne Van Landeghem. Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells [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 119.

Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [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 1044.

T cell immunity is vital for the control of viral infections. Cytotoxic CD8 T cells play a crucial role in eliminating virus-infected cells while CD4 helper T cells mediate host immune responses and promote B cells to secrete antibodies. For both, T cell recognition of viral antigens in the form of short peptides presented by HLA complexes is a prerequisite for T cell activation. Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become a global pandemic that has disrupted economies and resulted in an enormous burden on health care systems. Despite the worldwide intensive efforts on development of COVID-19 therapeutics, a safe and effective COVID-19 treatment or vaccine is not yet available. In this study, we established an in vitro stimulation system in which peripheral blood mononuclear cells were treated with putative SARS-CoV-2 immunogenic peptides. T cell immune responses were measured using different quantitative flow cytometry panels that were designed for evaluating cytokine production or identifying T cell subsets. Evaluation of host immune responses with a suite of flow cytometric panels may prove to be useful in the study of COVID-19. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product.BD-23276 (v1.0) 1120BD and the BD Logo are trademarks of Becton, Dickinson and Company. © 2020 BD. All rights reserved. Citation Format: Xiaoshan Shi, Scott J. Bornheimer, Suraj Saksena, Stephanie Widmann, Aaron Tyznik. Flow cytometry analysis of immune response to COVID-19 vaccine candidates using an in vitro stimulation model [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 715.

Objectives: Cellular kinetic (CK) measurement of CAR-T cell expansion in-vivo by quantitative polymerase chain reaction (qPCR) has been measured in units of transgene copy number/μg of DNA. We propose a formula to convert the qPCR, and flow cytometry (FC) measurements to interpretable numbers of CAR T-cells/μL blood. Further, after the conversion CK parameters were correlated with efficacy/safety endpoints. Methods: CK data measured using both qPCR and FC assays were utilized from the ELIANA trial in pediatric and young adult patients with relapsed and refractory acute lymphoblastic leukemia (pALL). qPCR measures the presence of CAR transgene in cells with units of copies of CAR-DNA/μg of genomic DNA, while FC quantifies surface expression of CAR T-cells as the % of either T-cells or white blood cells (WBCs) that express CAR. Neither measurement accounts for the typical significant increase in WBCs following CAR-T infusion. We propose equations for converting FC (Eq1) or qPCR (Eq2) into concentration of CAR-cells/μL of blood, using WBC counts from the complete blood count. For qPCR, the equation relies on 3 additional parameters M·F/N: amount of DNA/WBC (M), average number of copies of CAR-DNA/CTL019 cell (N), and fraction of cells with CAR-DNA that express CAR receptor (F). To estimate M·F/N, we performed regression of the cells/μL estimate from FC vs qPCR. We fit the CK model [1] to obtain the model parameters. Finally, CK parameters with safety/efficacy were correlated to compare the converted estimates to the native CK units. • (CD3+CAR+cells)/μl blood= WBC/μl blood × (CD3+CAR+cells)/WBC (1) • (CAR+cells)/μl blood=WBC/μl blood × CAR DNA copies/μg DNA × M μg DNA/WBC × 1 CAR cell/N CAR DNA copies×F (2) Results: There was high correlation between FC and qPCR estimates of CAR-T in the blood of pALL patients (r2=0.775). The M·F/N value derived based on FC and qPCR results was estimated as 2.68e-6μg DNA/CAR copies. Assuming M=6.6e-6μg DNA/WBC [2] and F=1, this predicts that N=2.46 CAR copies/CAR-T cell. There was also high correlation between the copies/μg and cells/μL estimates using qPCR (r2=0.752). The relationship between CK parameters and safety/efficacy endpoints was not improved when cells/μL was used. This can be attributed to high correlation between these metrics. Using the CK model, 11x greater fold expansion was predicted using the cells/μL estimate compared to copies/μg. This is because cells/μL estimate accounts the expansion of both CAR-T and WBC numbers following lymphodepletion. Conclusions: The conversion of CK into cells/μL allows for a physiological interpretation of CK data with a high correlation between the cells/μL and copies/μg indicating either metric can be used to predict safety/efficacy. References: 1 Stein M et al. CPT: Pharmacometrics & Systems Pharmacology(2019) 2 Gillooly JF et al. Cold Spring Harbor Perspectives in Biology vol.7,7 a019091(2015) Citation Format: Anwesha Chaudhury, Andrew Stein, Stephan Grupp, John Levine, Michael Pulsipher, G Doug Myers, Edward Waldron, Xu Zhu, Fraser McBlane, Rakesh Awasthi, Edmund K. Waller. Conversion of cellular kinetic data for chimeric antigen receptor T-cell therapy (CAR-T) into interpretable units [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 509.

Background: Tyrosine kinase inhibitors (TKIs) have been developed against EGFR. However, in NSCLC (Non-small Cell Lung Cancer) and some other cancers, the occurrence of activating mutations in EGFR leads to acquired resistance. Anomalous cMET signaling additionally contributes to developing resistance to targeted therapies, including ones directed at EGFR. A number of bispecific antibodies targeting EGFR and cMET are in clinical trials and JNJ-61186372 (Amivantamab) from Janssen labs, has been given fast track approval by the FDA. In this study we created bispecific and trispecific antibody formats which target both cMET and EGFR. Trispecific (TsAb), unlike the bispecific (BsAb), binds to two different epitopes on cMET and one on EGFR resulting in enhanced internalization of the receptors. This study demonstrates that our antibodies exhibit enhanced capabilities than the benchmark at clinical stage in cell and non-cell based in vitro assays. Methods: The two cMET binding arms of TsAb were designed to block the cMET-HGF (ligand) interaction and enhance the internalization of the receptors. Cell based assays were performed using a NSCLC cell line NCI-H1975 (ATCC CRL-5908) and HGF-expressing NCI-H1975. Receptor binding and receptor/ligand blocking (cMET/HGF and EGFR/EGF) assays were performed using both ELISA and flow cytometry. Phosphorylation assays were performed using ELISA and Western blotting. Receptor internalization studies were executed using the pHrodo labelling kit and were further assessed through fluorescence microscopy and quantified through flow cytometry. ADCC assays (Antibody dependent cellular cytotoxicity) were performed through the Crystal violet method. Results: BsAb was more effective than benchmark in terms of its binding capabilities to cMET and EGFR receptors and also in blocking the interaction of cMET and EGFR with their ligands. Moreover, functional assays revealed that the new BsAb is in most cases better than benchmark in successfully inhibiting the phosphorylation of not only the cMET and EGFR receptors but also the downstream signaling pathways which are involved in cancer cell proliferation, survival and obstructing the apoptotic pathways. Furthermore, the new TsAb was found to be more effective than BsAb and benchmark in cMET/EGFR receptor internalization which may lead to additional extenuation of the downstream pathways for these receptors. Cancer cell killing assays for ABT BsAb show ADCC comparable to the benchmark. Conclusion: Comparing to BsAbs, TsAb induces enhanced internalization of the cMET/EGFR receptors leading to further extenuation of the downstream oncogenic signaling pathways. These data suggest the development of ABT BsAb and specifically TsAb as a new drug target for patients with lung cancer and NSCLC in particular. Citation Format: Mohd Zaman, Cai Huang, Sachith Gallolu Kankanamalage, Amit K. Chaudhary, Jianbo Dong, Yue Liu. Development of cMET/cMET/EGFR Trispecific antibody as therapeutic modality for Non-small Cell Lung 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 LB069.

Lung cancer is the leading cause of cancer-associated deaths worldwide. SCLC entails ∼15% of cases1 and is considered one of the most aggressive forms associated a dismal prognosis. Unfortunately, the treatment options for SCLC are practically unchanged since the 80s. In SCLC 20% of patients show amplification or transcriptional upregulation of Myc family members. Myc is also amplified in up to 33% of NSCLC patients and is a prognostic marker of early-stage tumors2,3. Thus c-MYC represents an attractive therapeutic target in lung cancer. However, the intra-nuclear localization and intrinsically disordered nature of the protein renders successful small molecule or antibody drug development extremely problematic. Consequent to these challenges, no direct inhibitor of c-MYC has yet advanced into the clinic.4Here, we present the mechanism of action, in vivo lung cancer efficacy and the drug-like properties of IDP-121, a stapled peptide specifically designed to directly target the c-MYC protein (see Abstract 1). IDP-121 penetrates tumor cells at nanomolar concentrations by a translocation mechanism, avoiding endosomal trapping and presenting a prolonged intra-nuclear half-life. IDP-121 leads to c-MYC/MAX complex disruption as demonstrated by immunoprecipitation, promotes c-MYC degradation and a decrease in DNA-binding activity to its consensus-binding site by ELISA-based assays. A reduction of c-MYC protein binding at promoters of Myc-occupied gene is observed in IDP-121 treated cells through the genome by ChIP-seq. Furthermore, c-Myc gene expression levels remain unaffected, consistent with a direct c-MYC inhibitor. Flow cytometry shows increase in apoptosis by Annexin V staining. In vivo, IDP-121 shows significant reduction of tumor growth in syngenic and orthotopic SCLC models administered i.v. and s.c. The HPLC-MS and IHC analyses of tumor tissue after treatment confirm effective tumoral tissue distribution and target engagement, with significant reduction of c-MYC protein and cell proliferation in vivo. IDP-121 is highly stable in plasma, is not affected by hepatic metabolism and it is highly bound to plasma proteins. Thus, evidence suggests that we have identified a first-in-class stapled peptide direct inhibitor of MYC that shows efficacy in lung cancers at low exposure levels with no systemic toxicity. The efficacy in hematological tumors of IDP-121 have been also demonstrated in vivo (see Abstract 2). In addition, IDP-121 has progressed through GLP toxicology studies without evidence of major systemic toxicity, allowing for the first viable Myc-targeted therapy to enter Phase 1 clinical trials in 2021. 1Bragelmann J. et al. Cell Cycle 2017;16:1489-14982Georige J. et al. Nature. 524(7563), 47–53 (2015) 3Mollaoglu G. et al. Cancer Cell. 31(2), 270–285 (2017)4 Whitfield J. R. et al. Front. Cell Dev. Biol. 2017 Citation Format: Haritz Moreno, Borja Ruiz Fernández-de-Córdoba, Carolina Zandueta, Susana Martínez-Canarias, Miguel Moreno, Joan J. Albertí, Laura Nevola, Santiago Esteban, Silvestre Vicent, Fernando Lecanda. A first-in-class c-MYC inhibitor shows in vivo efficacy in lung cancer models and has appropriate drug-like characteristics [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 1264.

Background: Cell surface plectin (CSP) is an abundant bioavailable target exclusively found on the surface of ovarian epithelial cancers while remaining entirely intracellular in pre-malignant tissue. Under current treatment options, 80% of women, even if remission is achieved, will experience recurrence of disease demonstrating an urgent need for more effective therapeutics. Herein, we investigate the anticancer effects of a CSP-targeting monoclonal antibody (ZB131) in in-vitro and in-vivo models of ovarian cancer (OC) as well as elucidate key signaling pathways it modulates. We demonstrate that treatment with ZB131 is efficacious both as a monotherapy and in combination with chemotherapy. Methods: A first-in-class CSP-targeting monoclonal antibody, ZB131, was developed and characterized for specificity and affinity via ELISA and kinetic binding assays. In-vitro efficacy of ZB131 was determined by Cell Titer-Glo assay and flow cytometry, impact on cell-cycle by flow cytometry, and on migration by scratch assay using multiple OC cell lines. ROS accumulation was evaluated by DCFDA assay. Phosphorylation changes of signaling kinases were measured by LiCor. In-vivo validation was performed in an OC xenograft model. The expression of proliferative markers was evaluated by IHC staining of tumor sections. Treatment toxicity was assessed from serum chemistry tests and histological evaluation of major organs. Similar in-vivo and in-vitro techniques were used to characterize ZB131's effect in combination with cisplatin. Results: ZB131 demonstrates high affinity (0.4±0.1nM) and specificity for both murine and human CSP. Treatment with ZB131 decreased cell proliferation, migration, induced G0/G1 growth arrest, and increased ROS accumulation followed by cell death in OC cell lines. ZB131 induced two coordinated events, decreased phosphorylation of cell proliferation cascades (AKT, PRAS40, ERK1/2, and GSK3alpha/beta) and increased phosphorylation of stress signaling pathways (JNK, c-Jun, p38, MSK1/2, and AMPK). In vivo, ZB131 therapy caused a 50% tumor growth reduction as well as decreased expression of cyclin D and Ki67 with no toxic side effects. Impressively, ZB131 synergizes with cisplatin resulting in a greater than 50-fold decrease in IC50 in-vitro and showed sustained tumor growth reduction with greater than 85% tumor necrosis over the course of treatment in-vivo. Conclusion: CSP is a novel highly abundant therapeutic target exclusively localized to the cell surface of ovarian, colorectal, lung, and pancreatic cancers which collectively account for more than half of all estimated cancer deaths. Our findings illustrate that CSP-targeting with ZB131 induces cytotoxicity by activating stress pathways and dampening proliferation and survival pathways. This study serves as the basis for informing a novel treatment strategy for CSP-positive tumors. Citation Format: Samantha Melinda Perez, Julien Dimastromatteo, Kimberly A. Kelly. A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models [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 946.

CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+ B cell malignancies. All the FDA-approved CART19, including tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel use anti-CD19 single-chain variable fragments (scFv) that bind to the same CD19 epitope. This epitope is located at the membrane-distal portion of CD19. We aimed to develop an anti-CD19 CAR that binds to a membrane-proximal domain. To this goal, we screened a chicken immune scFv library against the CD19 extracellular domain. We selected an scFv clone (1218) that was not competing with FMC63 for CD19 binding in competition enzyme immunoassay and real-time interaction analysis. Subsequent mutagenesis assay by replacing several residues with monkey residues showed that, as compared to FMC63, the epitope of the 1218 scFv is localized in a membrane-proximal location at three-dimensional modeling (Teplyakov A., Proteins, 2018). The chicken 1218 scFv was humanized by CDR-grafting to human germline genes and backmutations (h1218 scFv) and was confirmed to be specific for CD19 in cell microarray assays and flow cytometry analysis. We, therefore, designed a CAR construct using the h1218 scFv, 41-BB costimulatory domain in a lentiviral backbone. CAR-T cells were successfully generated using the Myltenyi Prodigy platform. In vitro cytotoxicity and interferon release assays showed potent effector functions of h1218 CAR-T cells that compared positively with FMC63-based CART19. Our group recently described the occurrence of a B-cell leukemia relapse characterized by the expression of the CAR19 itself on the cell surface due to accidental transduction during manufacturing (Ruella M., Nat Med, 2018). Interestingly, h1218 but not FMC63 CART19 were able to recognize and kill FMC63 CAR19-expressing tumor cells. In vivo, h1218 CART19 cells led to complete tumor regressions in both Raji (CD19+ B-cell lymphoma) and Nalm-6 (B-cell leukemia) xenograft models. Currently, h1218 CART19 is under advanced preclinical development and will be tested in a phase I-II clinical trial for relapsed and refractory B-cell lymphoma patients. Citation Format: Ki-Hyun Kim, Ruchi P. Patel, Yong Gu Lee, Soohwan Kim, Ji-Hyeon Choi, Sung-Min Kim, Gae-Baik Kim, Jong-Ho Lee, Hyun-Jong Lee, Ji-Ho Park, Guewha Lee, LeiGuang Cui, Min Yoon, Ki Hyun Kim, Soohyun Kim, In-Sik Hwang, Youngha Lee, Bong-Kook Ko, Jong-Seo Lee, Junho Chung, Marco Ruella. A novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 [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 LB030.

Currently both bispecific T cell engagers (BiTE) and innate immune checkpoint blockades (eg. Anti-CD47/SIRPα) have been under development. In this study, we developed a novel compound pegylated anti-CD3 x anti-CD47 which could act with dual mechanisms of both a BiTE and an innate immune checkpoint blockade. The compound did not induce hemagglutination of RBC cells at the extremely high concentration of 2250 ug/ml, while the corresponding monoclonal antibody Hu5F9-G4 did at the concentration as low as 16.67 ug/ml. Meanwhile, in a T cell apoptosis assay, the apoptotic T cells were 16.13%, only 5.9% higher than that of the control T cell sample at the concentration of 100ug/ml of the compound and were about the same as the control sample (10.23%) at concentration of 10 ug/ml of the compound. For comparison, the reported T cell apoptosis induced by 10ug/ml Hu5F9-G4 was as high as 70%. Furthermore, the target affinity by flow cytometry demonstrated that at the concentration 10ug/ml of the compound, stained RBCs were 0.551%, while the compound bound BxPC3 cells were 22.1%, a 40-fold difference in binding. At the concentration 100ug/ml of the compound, only 7.24% RBCs were stained positively, but stained BxPC3 cells were as high as 89.3%. Although not completely understood yet, unique structure of the compound (PEGylated scFv antibody v.s. Fc-bearing antibodies) may have contributed to the differential effect of the compound to healthy cells and tumor cells, resulting in high safety performance of the compound. Furthermore, pharmacokinetics study demonstrated that in vivo elimination half-life of the compound in wild type C57BL/6 mice was 18.4 hours for the single dose of 1mg/kg. Further preclinical study about the compound is still ongoing. Citation Format: Shumin Liu, Weidong Lyu, Yang Lei, Shuangyu Tan, Zibin Wu, Shuqiang Yin, Liling Zheng, Qiudong Zhuo, Yu (Yvonne) Wen, David Wu. A long acting bi-specific T cell engager differentially targeting CD47 positive malignant cells but not CD47 expressing healthy cells [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 1868.

SCLC is an aggressive neuroendocrine (NE) cancer with a 7% 5-year survival rate. Molecular characterization of this disease has lagged behind other cancer types and standard of care treatment has remained largely unchanged for over three decades. SCLC is treated as a clinically homogenous disease, but evidence of its intra- and inter-tumor heterogeneity is increasing. Part of this heterogeneity is attributed to expression of neurogenic transcription factors (TFs) ASCL1 and NEUROD1, which correlate with SCLC pathogenesis and NE status, and are now included in a consensus for SCLC classification [1]. Our Circulating tumor cell patient Derived eXplant (CDX) models recapitulate disease heterogeneity in terms of neurogenic TF expression; furthermore, we recently identified a previously unrecognized subset within our CDX model panel expressing another neurogenic TF, ATOH1, mostly within NEUROD1 expressing models [2]. I am investigating the roles of the three NE TFs in SCLC CDX, to refine disease classification and explore rationale for new therapeutic approaches stratified to SCLC phenotypes. To do so, we have characterized ASCL1 and NEUROD1 expression in CDX models with multiplex immunofluorescence and flow cytometry. We have begun to interrogate the biology of ASCL1 and NEUROD1 in established SCLC cell lines via CRISPR- and shRNA-mediated genetic depletion of these TFs. Furthermore, we have recently developed antibodies to ATOH1 to support interrogation of its under-researched role in SCLC biology. We have found that CDX models express ASCL1 (30/38), NEUROD1 (3/38) and ATOH1 together with NEUROD1 (4/38) [2]. ASCL1 and NEUROD1 are co-expressed in several CDX but (with the exception of one CDX model) in different cell subpopulations. In contrast with published literature, genetic depletion of ASCL1 and NEUROD1 in established SCLC cell lines did not influence cell proliferation and response to chemotherapy. I have recently generated specific antibodies to ATOH1 and ongoing studies are focused on characterization of ATOH1 expression in CDX models and its impact on the pathogenesis and chemotherapy responses in SCLC. In conclusion, ASCL1 and NEUROD1 are expressed in a mutually exclusive way in CDX models, with the exception of one CDX; manipulation of their expression does not influence cell proliferation or response to chemotherapy in SCLC cell lines. Studies looking at the consequences of genetic depletion of these TFs, including ATOH1, in CDX models are underway. [1] Rudin et al., Nature Reviews Cancer, 2019 [2] Simpson et al., Nature Reviews Cancer, 2020 Citation Format: Alessia Catozzi, Mitchell Revill, Fiona Blackhall, Kristopher Frese, Kathryn Simpson, Caroline Dive. Heterogeneity of neurogenic transcription factor expression in small cell lung 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 2436.

The purpose of this study is to develop a thorium-227 (227Th) antibody radioimmunoconjugate targeting glypican-3 (GPC3) and to test its therapeutic efficacy in a hepatocellular carcinoma (HCC) orthotopic xenograft model. GPC3 targeting antibody (αGPC3) was conjugated to bifunctional chelator p-SCN-Bn-H4octapa (octapa), and αGPC3-octapa binding affinity for GPC3 was evaluated by flow cytometry. 227Th radiolabeling of this conjugate was optimized, and in vitro stability of 227Th-αGPC3-octapa was evaluated in PBS with and without free radical scavenging agent over 14 days. For in vivo evaluation, an orthotopic xenograft model was generated by hepatic subcapsular injection of human HCC HepG2 cells. In vivo biodistribution was assessed in blood, tumor and organs at 1, 7 and 21 days after tail vein injection of 227Th-αGPC3-octapa (500 KBq/kg). To test therapeutic efficacy, tumor-bearing animals were injected with 227Th-αGPC3-octapa (250 kBq/kg or 500 kBq/kg) and compared to an irrelevant control antibody, 227Th-αBHV1-octapa (500 kBq/kg), and to a no-treatment control. Tumor burden was assessed with serial serum alpha-fetoprotein (AFP) measurements, a marker of tumor burden validated in this model. Toxicity to 227Th-αGPC3-octapa was measured by serum comprehensive metabolic panel obtained 21 days after injection. GPC3 binding affinity was highest after conjugation of αGPC3 with 10 equivalents of octapa. The protein recovery from the conjugation process was >85%, and mass spectral analysis indicated an average of 3.3 octapa moieties per molecule of αGPC3-octapa. After two weeks, >98% of αGPC3-octapa and αBHV1-octapa had 227Th bound in the presence of scavenging agent. αGPC3-octapa maintained high affinity for GPC3 as measured by flow cytometry, indicating the octapa conjugation reaction did not alter immunoreactivity of αGPC3. In vivo, 227Th-αGPC3-octapa accumulated in the tumor over time and cleared from normal tissues with a %ID/g of In conclusion, we report the development of a GPC3 targeted thorium conjugate and demonstrate its in vivo therapeutic efficacy in a murine orthotopic xenograft model of hepatocellular carcinoma. Citation Format: Kevin P. Labadie, Donald K. Hamlin, Aimee Kenoyer, Sara K. Daniel, Alan F. Utria, Andrew D. Ludwig, Heidi L. Kenerson, Delphine L. Chen, Johnnie Orozco, Raymond S. Yeung, Lily Li, Chris Orvig, Yawen Li, D Scott Wilbur, James O. Park. Glypican-3 targeted thorium-227 alpha therapy reduces tumor burden in an orthotopic xenograft model of hepatocellular carcinoma [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 928.

Cell-type abundance GE signatures are useful for informing drug mechanism of action and may be useful in-patient selection for cancer immunotherapies. Recently, we validated natural killer cell- and dendritic cell-type specific expression signatures using a tiered strategy that included both computational and ex vivo validation. Here, we built upon this strategy to validate expression signatures for immunosuppressive myeloid cells (IMC). IMC play a critical role in impairing anti-tumor immunity and increased levels of peripheral IMC have been associated with advanced tumor progression and poor prognosis in various cancers. However, their low abundance in normal tissues and heterogeneous surface expression render their profiling difficult. To address this, we focused on validating GE signatures for IMC abundance using mRNA expression methods which are more clinically tractable than established flow cytometry methods that require intact cells. We implemented a three-stage GE signature validation strategy. First, we generated GE data from human ex vivo differentiated IMC (LIN- CD11B+ CD33+ HLADR-). Then, we evaluated the concordance of 16 previously published myeloid signatures with cell type abundance in a series of spike-ins with varying but known quantities of ex vivo differentiated IMC in a background of undifferentiated peripheral blood mononuclear cells (PBMCs). Gene-set variation analysis (GSVA) was used to score the signatures and Spearman's rank (rS) correlation coefficient was calculated to assess the significance of correlations between GSVA scores and IMC abundance. With this method, we validated 3 of the 16 published myeloid signatures (1. MacB3/PMID:26873985 (rS = 0.87; p = 8.8e-07); 2. MacB2_3w/PMID:26873985 (rS = 0.83; p = 6.7e-06); 3. TAM/PMID:27424807 (rS = 0.80; p= 2.6e-05)). Next, we generated a ‘de novo' signature by performing differential GE analysis and identified up-regulated genes in IMC in comparison to PBMCs with log2 fold change &gt 2, p-adj &lt 0.05). We further refined this list by selecting genes with significant concordance to IMC abundance (rS &gt 0.80, p-adj &lt 0.05) and confirmed their specificity to IMC in published scRNAseq studies [PMID: 30979687; 31033233; 32302573]. This led to a 6-gene ‘de novo' signature (MRC1, APOE, C1QA, C1QB, MMP9, SPP1) associated with IMC function with highly significant concordance (rS = 0.96; p = 1.2e-11) to IMC abundance. Lastly, we showed that in whole blood baseline samples from HNSCC patients (n=53) treated with danvatirsen + durvalumab [NCT02499328], our ‘de novo' signature was significantly elevated (p=0.033) in patients with progressive disease compared to complete responders. Application to additional studies will be required to more fully determine its clinical utility. Citation Format: Srimathi S. Srinivasan, Gozde Kar, Deanna L. Russell, Peter Gathungu, Minoo Rafati, David Whitston, Grant Duclos, Ben Sidders, J Carl Barrett, Tim Slidel, Patricia McCoon. Identification of a novel immunosuppressive myeloid gene expression signature for clinical biomarker development [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 390.

CA125 represents the circulating subunit of the large transmembrane glycoprotein MUC 16. Elevated levels of CA125 are suggestive of ovarian cancer and the magnitude of elevation correlates with burden of disease. A rising CA125 level after treatment suggests cancer recurrence. Ovarian tumor cells produce and shed MUC 16 which can bind to certain peripheral immune cells. To study this, current techniques use labelled-fluorescent markers that bind to CA125 on cells and flow cytometry to detect mean fluorescence. A limitation is that this technique may not be able to distinguish low-binding events from autofluorescence. Our new technology improves the resolution for studying CA125 bound to immune cells by employing antibody-labelled gold nanoparticles with far-greater sensitivity than fluorescent markers. The cells are then examined using darkfied microscopy rather than flow, which also permits automated slide scanning and focusing for 3-dimensional counting. This enables both simultaneous use of fluorescent markers to identify the immune cell populations as well as automated image analysis to quantify the amount of bound CA125 on individual cells. Preliminary data show that MUC 16 is preferentially bound to NK and T cells in women with ovarian cancer compared to binding in healthy controls. These insights may help elucidate the function of CA125 in ovarian cancer and improve our understanding of immune-tumor interactions. Citation Format: Kornel Lakatos, Petra Krauledat, German Gonzalez Serrano, Peter Hansen, Daniel William Cramer, Kevin Elias, Manish Patankar, Jawad Hoballah. A research tool for elucidating the interaction between human mucin 16 (CA125) and immune cells in ovarian 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 3178.

Diffuse Intrinsic Pontine Glioma (DIPG) is the leading cause of pediatric death by brain tumors with a median survival of only 9 months, in spite of the current standard of therapy which includes radiation. The purpose of our study is to explore the use of oncolytic adenoviruses as a new therapeutic modality. Our group is leading a phase I clinical trial in naive DIPGs treated with the oncolytic adenovirus Delta-24-RGD, also named DNX-2401 (NCT03178032), which results are still pending. Of interest, clinical trials using Delta-24-RGD in adult gliomas resulted in encouraging results with long-term survivors and the presence of a strong anti-tumor immunity. In this study, we aim to test the effect of armed Delta-24-RGD with immune costimulatory ligands such as GITRL (Delta-24-GREAT) and OX-40L (Delta-24-RGDOX or DNX-2440) for the treatment of DIPGs. We used a panel of murine DIPG cell lines (NP53 and XFMA) to test the effect of these armed-oncolytic adenoviruses. Infectivity assay using an Ad-RGD-GFP vector and flow cytometry showed the expression of the ectopic GFP in more than 80% of the NP53 cells (50 MOIs) and XFMA cells (30 MOIs). Consistent results were obtained using flow cytometry and western blot to detect the expression of the ectopic ligands GITRL and OX-40L in these cultures. These cells were semipermissive for viral replication as assessed by the detection of the viral structural protein hexon in western blot analysis. Preliminary in vivo data using immunocompetent mice models where DIPG cells have been implanted into the pons showed a dose-dependent effect and the generation of immune memory. Our results suggest that oncolytic adenoviruses expressing positive immunocheckpoints might prove be a new effective modality for treating DIPG and hence, increase survival in children suffering this dismal disease. Citation Format: Sumit Gupta, Virginia Laspidea, Juan Fueyo, Oren Becher, Teresa Nguyen, Dong Ho Shin, Hong Jiang, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Joy Gumin, Frederick Lang, Candelaria Gomez-Manzano, Marta M. Alonso. Armed oncolytic virus for treatment of pediatric diffuse intrinsic pontine glioma [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 3053.

Background: Adoptive cell therapies using Chimeric Antigen Receptors (CARs) show durable clinical benefit for patients with hematologic malignancies, however, challenges remain for enabling this personalized treatment to be delivered in a timely, cost effective, and logistically friendly manner. Methods: Lentiviral vectors (LV) encoding CD19 and CD22 CARs with a synthetic driver element were packaged with a VSV-G envelope designed with the capability to target and activate CD3pos T cells in whole blood. LV were directly reconstituted in peripheral whole blood for four hours and total nucleated cells (TNC) or peripheral blood mononuclear cells (PBMC) were rapidly isolated utilizing two different closed systems. Immediately following the four-hour exposure event, isolated TNC or PBMC were injected subcutaneously into mice with disseminated Raji luciferase tumor cells. For further characterization, LV-exposed TNC and PBMC were cultured in vitro for six days and functionally examined. Results: Following four-hour exposure to CD3-directed LVs, more than 90% of T cells, including naïve/naïve derived (CCR7pos CD45ROneg) and central memory (CCR7pos CD45ROpos) T cells present within isolated TNC or PBMC exhibited a significant decrease in CD3 surface expression. Subcutaneous injection of gene modified TNC or PBMC resulted in the in vivo generation and expansion of large numbers of circulating CAR-T positive cells with complete eradication of disseminated Raji tumors. In parallel cell culture experiments, TNC or PBMC isolated following four-hour LV whole blood exposure exhibited robust expansion without additional T-cell receptor (TCR) or CD3 stimulation, while TNC or PBMC not exposed to virus did not show any expansion. Following six days in culture, immunophenotyping by flow cytometry demonstrated that more than 90% of the cells were CD8pos and CD4pos T cells with CAR-T expression present on central memory (CCR7pos CD45ROpos) and effector memory (CCR7neg CD45ROpos) T cells. CAR-T antigen specificity to CD19 and CD22 was measured by IFN-gamma release co-culture assays. Conclusion: We conclude that large numbers of functionally active CAR-T positive cells can be generated both in vitro and in vivo following a four-hour peripheral whole blood exposure to CD3-directed LVs encoding for CARs with a synthetic driver element. These results provide the basis for an autologous same-day peripheral blood draw to subcutaneous injection rapid point-of-care (rPOC) approach. Citation Format: Dongming Zhang, Frederic Vigant, Qun He, Anirban Kundu, Wei Zhang, Hongliang Zong, Ewa Jaruga-Killeen, Gregory Schreiber, Michelle Andraza, Alissa R. Kerner, Gregory I. Frost, Sid P. Kerkar. Subcutaneous injection of total nucleated cells rapidly isolated following four-hour peripheral whole blood exposure to CD3-directed CAR-T lentiviruses with a synthetic driver results in robust CAR-T proliferation and anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1511.

Natural killer (NK) lymphocytes exhibit potent responses against transformed and pathogen-infected cells. NK cells can act rapidly as they monitor the bloodstream and tissues for tumorigenic and infected cells that lack “self” markers of MHC class I. Upon recognition of a foreign invader or tumor cell, NK cells release cytotoxic proteins such as interferon-gamma and granzyme B. Because of their ability to recognize stressed cells lacking “self” markers, human NK cells have shown promise as therapeutics in clinical trials to reduce disease burden in patients with therapy-resistant or advanced-stage blood cancers. With more than one hundred NK cell- and induced NK cell-based clinical trials ongoing for blood cancers and refractory solid tumors, there is an ever-increasing need for NK cell therapies to treat cancer and immune diseases. We sought to develop novel, clinically relevant, and robust methods for human NK cell expansion. In addition, to simplify the processing of cells for characterization, we developed a flow cytometry based killing assay to monitor NK activity. Therefore, within the same experiment, we can analyze NK cell phenotype profiles with specific antibody panels while also assessing NK cell killing activity. Using optimized serum- and xeno-free conditions with minimal cell manipulations, we have expanded highly purified NK cells up to 500-fold in 14 days from human peripheral blood mononuclear cells (PBMCs) using optimized cytokines and Cloudz CD2/NKp46 microspheres. To characterize the resulting NK cell population, we used a unique antibody panel to phenotype the NK cells by flow cytometry. Within the same experiment, NK killing assays against K562 cells can be performed using a flow cytometry-based killing assay, which has the advantage of using one instrument for all analyses, shortened processing times, and reproducibility. Using this workflow, we can identify different culture conditions that adversely affect NK activity. Together, this data presents a comprehensive and clinically relevant workflow for the expansion of highly purified NK cells and assessing their killing potential. Furthermore, this workflow can be used for screening the activity of chimeric antigen receptor (CAR) expressing immune cells against specific cancer antigens. Citation Format: Jody Bonnevier, Christine Goetz, Li Peng, Jamie Van Etten, Bora Faulkner, Christopher Hammerbeck, Ariel Miller, Gabriella Perell, Christopher Johnson, Joseph Lomakin, David Hermanson, Kevin Flynn. Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay [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 LB141.