Combination In Preclinical Models Research Articles

Abstract 1658: CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis

Abstract Activating mutations in BRAF at residue V600 (typically V600E) lead to dysregulation of the MAPK pathway and occur in approximately 8% of all human cancers including 60% of melanoma, up to 12% of colorectal cancer (CRC), and 4% of NSCLC. Currently approved BRAF inhibitors (BRAFi) are selective for BRAF V600X mutant proteins and are typically used in combination with MEK inhibitors (MEKi) in melanoma and NSCLC, or anti-EGFR antibodies such as cetuximab in CRC. However, their activity is limited by primary or acquired resistance often mediated by RAF dimer-inducing mechanisms. Furthermore, progression of BRAF V600X melanoma after BRAFi/MEKi treatment frequently involves brain metastasis, and currently approved BRAFi have relatively poor brain penetration. CFT1946 is a potent, orally bioavailable, cereblon-based BiDACTM degrader that selectively degrades BRAF V600X mutant protein and is currently under investigation in a Phase I clinical trial. CFT1946 has the potential to overcome limitations of clinically approved BRAFi, as degradation of BRAF V600X should abrogate RAF dimer-driven resistance and paradoxical activation. Indeed, we have previously demonstrated that CFT1946 is efficacious in an A375 BRAF V600E/NRAS Q61K xenograft model of BRAFi resistant melanoma. Here we substantially expand our preclinical characterization of CFT1946 across multiple models of BRAF V600X-driven cancers including BRAF V600X-driven CRC and NSCLC, additional BRAFi-resistant melanoma models, and a brain metastatic melanoma model. Single agent CFT1946 outperformed the SOC encorafenib + cetuximab combination in a panel of BRAF-V600X CRC xenograft models. Pathway analysis in BRAF V600X CRC models revealed that CFT1946 suppresses EGFR-mediated MAPK pathway reactivation, a mechanism known to diminish the impact of approved BRAF inhibitors in this indication. CFT1946 also demonstrated regression in a BRAF-V600X NSCLC PDX model where the SOC dabrafenib + trametinib showed only modest tumor growth inhibition. Consistent with our previous results in the A375 melanoma model, single agent CFT1946 showed superior activity versus the SOC dabrafenib + trametinib combination in all additional melanoma models tested, with complete regression achieved using a CFT1946 + trametinib combination in a PDX model bearing a BRAF-V600E kinase duplication that showed minimal response to dabrafenib + trametinib. Finally, using an A375 intracranial model, treatment with CFT1946 gave robust, dose dependent efficacy and survival advantage over encorafenib. The promising activity of CFT1946 in a broad range of BRAF V600X preclinical models supports its ongoing clinical investigation in BRAF V600 mutant solid tumors (NCT05668585). Citation Format: Bridget Kreger, Jacob R. Stephenson, Mathew E. Sowa, Samantha A. Perino, Laura L. Poling, Eunju Hurh, Michael J. Thomenius, Yanke Liang, Stewart L. Fisher, Roy M. Pollock. CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1658.

Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.

Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients.

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Abstract 1615: NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models

Abstract PERK and GCN2 are eIF-2α serine-threonine kinases activated by endoplasmic reticulum (ER) stress and amino acid deprivation respectively. These kinases are key effectors of the unfolded protein response (UPR) and amino acid Response (AAR) branches of the integrated stress response (ISR). ISR downregulates protein synthesis while upregulating specific genes in response to various cellular stresses, thus allowing adaptation and survival. PERK and GCN2 cooperate to modulate survival under stress conditions in cancer cells, with GCN2 activation compensating when PERK is inhibited, therefore suggesting a certain degree of redundancy between the two. Multiple myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, due to their high protein load. Therefore, PERK inhibition represents a promising anticancer approach in these tumor types. Notably, in these contexts, dual PERK/GCN2 inhibition may be more effective than inhibiting the single kinase.NMS-03597812 is a novel, very potent, ATP-competitive PERK/GCN2 inhibitor, NMS-812 is preferentially active in vitro on multiple myeloma cell lines; activity has however been observed also on lymphoma and selected solid tumor cell lines, suggesting a broader but still specific role of PERK and GCN2 inhibition depending on cell context. In assays revealing the mechanism of action, as expected, a transient dose-dependent eIF2α pathway activation is observed, suggesting the attempt of GCN2 to compensate for PERK inhibition. However, at higher doses, NMS-812 strongly induces pathway downmodulation and apoptosis compared to prototype PERK selective inhibitors. NMS-812 treatment of a MM xenograft model significantly prolongs the median survival time as single agent and demonstrates synergistic activity with MM standard of care (SOC) agents. In lymphoma, NMS-812 shows activity both as single agent and in combination with SOC.NMS-812 shows a favorable in vitro ADME profile and good PK properties with oral bioavailability in preclinical species and an acceptable toxicity profile. NMS-812 is a novel dual, PERK/GCN2 inhibitor with potential for first-in-class, that validates dual PERK and GCN2 inhibition as a therapeutic approach for MM and other PERK/GCN2-dependent settings. Based on these promising results, a Ph1 study has been started in Multiple Myeloma with plans to expand to other malignancies in the near future. Citation Format: Claudia Perrera, Maurizio Pulici, Patrizia Banfi, Nilla Avanzi, Davide Carenzi, Daniele Casero, Antonella Ciavolella, Laura Gianellini, Fabio Gasparri, Grazia Saturno, Barbara Valsasina, Elena Ardini, Alessia Montagnoli, Arturo P. Galvani, Antonella Isacchi. NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1615.

Abstract 5510: TAS102 synergizes with regorafenib against colorectal and gastric cancer cells

Abstract TAS-102 is an oral formulation consisting of trifluridine (FTD) and thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI). Regorafenib is a multi-targeted tyrosine kinase inhibitor, it inhibits the activities of VEGFR2 and 3, Ret, Kit, PDGFR and Raf kinases and results in the inhibition of tumor angiogenesis and cell proliferation. TAS102 or regorafenib monotherapy can lead to an overall survival benefit in metastatic colorectal cancer (mCRC) patient previously treated with conventional chemotherapy and target therapy (Arnold et al., 2018). TAS102 in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGF-A), is associated with a significant and clinically relevant improvement in progression-free survival in colorectal cancer (C-TASK FORCE)(Kuboki et al., 2017; Pfeiffer et al., 2020). FTD can synergize with nintedanib to inhibit the growth of colorectal cancer xenografts(Suzuki, Nakagawa, Matsuoka, & Takechi, 2016). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in case reports of multidrug-resistant mCRC patients(Marks et al., 2015). Therefore, we hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers, and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. We found that TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, and inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. Collectively, TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The drug combination may be further tested in gastric and other GI cancers. Citation Format: Jun Zhang, Lanlan Zhou, Shuai Zhao, Wafik S. El-Deiry. TAS102 synergizes with regorafenib against colorectal and gastric cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5510.

Evaluation of Epcoritamab and Rituximab Combination in Preclinical Models of B-Cell Non-Hodgkin's Lymphoma (NHL)

Background: Epcoritamab (Epco) is a CD3 bispecific antibody that induces selective, potent T-cell-mediated killing of CD20-positive malignant B-cells. In the phase 1/2 monotherapy study, subcutaneous administered Epco showed a favorable safety profile and promising efficacy, including complete responses in heavily pretreated patients with relapsed / refractory non-Hodgkin's lymphoma (Hutchings et al, Lancet 2021; Thieblemont et al, EHA 2022, LB2364; EPCORE NHL-1). Ongoing clinical studies combining Epco with standard-of-care therapies including combinations with Rituximab show promising preliminary anti-tumor activity in diffuse large B-cell lymphoma and follicular lymphoma (Falchi et al, ASCO 2022 abstracts 7523 and 7524; EPCORE NHL-2 arm 1 and 2; NCT04663347). Here we evaluated the competition for CD20 binding and functional interactions between these agents and modeled its impact on Epco, T-cell, and tumor cell trimer formation that drives Epco clinical activity. Aims: Investigate the preclinical binding and functional interaction between Epco and Rituximab to establish the potential for the combination of these agents in the treatment of B-cell NHL. Methods: Competition between Epco and Rituximab for binding to CD20 cells was assessed by Flow Cytometry after incubating the single labeled agent in the presence of increasing concentrations of the competitor and vice-versa (across 0.1 - 300 ug/ml evaluated for each agent), on a range of CD20 expressing lymphoma cell lines. Epco-induced-T cell activation and redirected T cell cytotoxicity was determined by co-culturing T cells with lymphoma cells with varying expression of CD20. Rituximab antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by incubating fresh peripheral blood mononuclear cells with lymphoma cells in the presence of a CD3 control Epco antibody (DuoBody-ctrlxCD20). Data from binding and cytotoxicity was used to quantify the degree of binding curve shifts at different Rituximab:Epco concentrations as well as the impact on predicted trimer formation and cytotoxicity, based on modeling. Results: In binding assays, the addition of Rituximab resulted in a dose-dependent reduction of Epco binding to CD20 expressing lymphoma cell lines, with a significant reduction of Epco binding observed at clinically relevant Epco concentrations in cells with high CD20 expression levels (Figure 1). Similar binding interference interactions were observed for binding of Epco to cell lines with low and intermediate CD20 expression levels. Binding of Rituximab to cell lines expressing high CD20 levels was not significantly reduced by clinically relevant concentrations of Epco. In Epco functional studies, the addition of increasing concentrations of Rituximab did not affect Epco cytotoxic activity at clinically relevant range of Rituximab doses tested (Figure 2). Similarly, no effect of Rituximab on Epco-mediated T cell activation was observed at clinically relevant concentrations of Rituximab and Epco. ADCC activity at clinically relevant concentrations of Rituximab (10-250 ug/ml) was not impacted by incubation with the CD3 control Epco antibody (DuoBody-ctrlxCD20), even at concentrations exceeding the Epco clinical concentrations. Modeling and simulation of in vitro data showed that at clinically relevant concentration of Epco, the presence of clinically relevant concentration of Rituximab does not meaningfully impact Epco trimer formation, and the predicted trimer formation remains above EC90 relative to that needed for 50% cell kill. Conclusions: Our studies showed that while Rituximab exerts significant interference on Epco binding to CD20-expressing cells, tumor-cell cytotoxicity by Epco is minimally affected by clinically relevant concentrations of Rituximab. This conclusion is supported by preclinical modeling and simulations showing that Epco activity driven by trimer formation is not meaningfully impacted by clinical concentrations of Rituximab. Furthermore, we found a limited effect of Epco on Rituximab binding to CD20 and no impact on ADCC activity by Rituximab, thereby supporting potential synergy in the combination between these agents. Results from this study confirm the ability to combine Epco with CD20 antibodies, specifically with Rituximab, and the potential for synergistic activity of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

95 (PB085) - Preliminary results from FLAGSHP-1: A Phase I dose escalation study of ERAS-601, a potent SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors

Background: SHP2 is an oncogenic tyrosine phosphatase that transduces receptor tyrosine kinase signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors. ERAS-601 is a potent, selective, and orally bioavailable allosteric inhibitor of SHP2 that has demonstrated robust anti-proliferative activity in vitro and in vivo as monotherapy and in combination in preclinical models of cancer harboring EGFR, KRAS, BRAF class III and NF1LOF mutations.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

BackgroundTyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.MethodsKINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic–pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.ResultsIn mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.ConclusionsTaken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial registrationClinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Lysosomal Acid Lipase a (LIPA) Modulates Leukemia Stem Cell (LSC) Response to Venetoclax/TKI Combination Therapy in Blast Phase Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs.To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population.To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs.Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML.In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. DisclosuresPollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding.

EVT-701 is a novel selective and safe mitochondrial complex 1 inhibitor with potent anti-tumor activity in models of solid cancers.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia‐inducible factor 1α (HIF‐1α) levels putatively mediated by MC1 inhibition led to the development of HIF‐1α inhibitors, such as BAY87‐2243, with a more specific MC1 targeting. However, the development of BAY87‐2243 was stopped early in phase 1 due to dose‐independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF‐1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti‐cancer activity in solid tumors of lead compound EVT‐701. In addition, using murine models of lung cancer and of Non‐Hodgkin's B cell lymphoma we demonstrated that EVT‐701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT‐701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT‐701 alone or in combination in preclinical models and eventually in patients.

IMGN779, a Next Generation CD33-Targeting ADC, Combines Effectively with Cytarabine in Acute Myeloid Leukemia (AML) Preclinical Models, Resulting in Increased DNA Damage Response, Cell Cycle Arrest and Apoptosis In Vitro and Prolonged Survival In Vivo

Introduction: IMGN779, a next generation CD33-targeting ADC, is comprised of a novel DNA alkylating IGN payload, an s-SPDB cleavable linker and a humanized anti-CD33 antibody. IMGN779 is currently in Phase I dose escalation in patients with relapsed/refractory AML (NCT02674763). With improved patient outcomes in mind, we set out to evaluate the mechanism, antileukemia efficacy and tolerability of the combination of IMGN779 and cytarabine using in vitro and in vivo human AML preclinical models.Methods: DNA damage, survival and apoptotic responses to IMGN779 and cytarabine were assessed using flow cytometry and immunoblotting techniques. In vitro combinations of the free IGN payload and either an MDM2 antagonist or CHK1/2 inhibitors were assessed for impact on cell viability at 72 hours across a panel of 12 AML cell lines. IMGN779 and cytarabine were evaluated in vivo for antitumor activity, using disseminated (Molm-13 and MV4-11) and subcutaneous (SC; EOL-1) AML xenograft models. All mice were monitored for body weight loss, clinical signs, and SC tumor volume (EOL-1). Assessment was terminated for weight loss >20%, clinical signs, or, in the EOL-1 model, when SC tumor volume reached approximately 1000 mm3.Results: Exposure to cytarabine in vitro led to a dose- and time-dependent increase in CD33 transcription and cell surface expression (up to 2-fold increase at 150nM at 72 hours) in multiple human AML cell lines, revealing a novel mechanism by which cytarabine may potentiate the CD33-dependent uptake of IMGN779 by AML cells. Importantly, the in vitro combination of IMGN779 and cytarabine led to dose-dependent, additive antileukemia activity (Figure A), and enhanced levels of cell cycle arrest, DNA damage response (p21, CHK2) and apoptosis (Casp3/PARP1 cleavage). Moreover, the mechanistic importance of p53 and CHK2 in IMGN779-mediated antileukemia activity was demonstrated further using the IGN free payload in in vitro combination assays with a panel of 12 human AML cell lines. The results of these experiments suggest strong synergy between IGN free payload and an MDM2 antagonist and between IGN free payload and CHK1/2 inhibitors, pointing to additional combination regimens for IMGN779.In mice, the combination of single dose (0.253 mg/kg) or repeat dosing of IMGN779 (0.0534 or 0.0107 mg/kg, weekly x3) and high-dose cytarabine (75 mg/kg, at near MTD, daily x5) was highly active in all three AML xenograft models, and was well tolerated, with no body weight loss or other toxicities noted. The IMGN779 doses in these highly active combinations were far below the MTD (48 mg/kg). In MV4-11 and Molm-13 (both FLT3-ITD) disseminated xenografts, the combination resulted in a highly active 24-26 day tumor growth delay and 42-110% Increased Life Span (ILS), compared to minimally active IMGN779 (15-33% ILS) and inactive cytarabine (0-0% ILS) single agents. In the EOL-1 (chemo-resistant) SC model, 0.253 mg/kg single dose IMGN779 plus cytarabine was also highly active, resulting in 4/6 long-term (>90 days) complete remissions (CRs), compared to 2/6 CRs for IMGN779 and no CRs for cytarabine single agents (Figure B). Tumor growth delay for the EOL-1 tumors that persisted was 17 days for the combination, compared to 8 and 0.5 days for IMGN779 and cytarabine, respectively, supporting a highly beneficial combination effect on survival.Conclusions: IMGN779 and cytarabine are a highly effective and well tolerated combination in preclinical models of AML. Mechanistically, the IMGN779 plus cytarabine combination increased DNA damage response, cell cycle arrest, and apoptosis in vitro, which lead to increased survival in vivo . Interestingly, cytarabine increased cell surface CD33 levels on AML cells, indicating a novel mechanism by which cytarabine may potentiate IMGN779 uptake and possibly efficacy. In addition to the favorable combination with cytarabine, our in vitro combination studies also reveal the potential of combining IMGN779 with DNA damage response inhibitors. These results support rapidly advancing the combination of IMGN779 and high dose cytarabine in clinical trials. [Display omitted] DisclosuresAdams:ImmunoGen: Employment. Kelly:ImmunoGen: Employment. McCarthy:ImmunoGen: Employment. Wilhelm:ImmunoGen: Employment. Watkins:ImmunoGen: Employment. Lanieri:ImmunoGen: Employment. Sloss:ImmunoGen: Employment. Romanelli:ImmunoGen: Employment.

Combination therapy using nitro compounds improves the efficacy of experimental Chagas disease treatment.

Drug combinations have been evaluated for Chagas disease in an attempt to improve efficacy and safety. In this line, the objective of this work is to assess the effects of treatment with nitro drugs combinations using benznidazole (BZ) or nifurtimox (NFX) plus the sulfone metabolite of fexinidazole (fex-SFN) in vitro and in vivo on Trypanosoma cruzi infection. The in vitro interaction of fex-SFN and BZ or NFX against infected H9c2 cells by the Y strain was classified as an additive (0.5⩾ΣFIC<4), suggesting the possibility of a dose reduction in the in vivo T. cruzi infection. Next, the effect of combining suboptimal doses was assessed in an acute model of murine T. cruzi infection. Drug combinations led to a faster suppression of parasitemia than monotherapies. Also, the associations led to higher cure levels than those in the reference treatment BZ 100 mg day−1 (57.1%) (i.e. 83.3% with BZ/fex-SFN and 75% with NFX/fex-SFN). Importantly, toxic effects resulting from the associations were not observed, according to weight gain and hepatic enzyme levels in the serum of experimental animals. Taken together, this study is a starting point to explore the potential effects of nitro drugs combinations in preclinical models of kinetoplastid-related infections.

Activating chimeric receptor on IL-15 armored NK cells to improve in vitro and in vivo tumor response within the liver following regional delivery.

e14517 Background: Colorectal cancer liver metastases (CRCLM) are a major source of morbidity and mortality. Historically, curative therapy has been limited to surgical resection, but only a small fraction of patients are eligible. Cellular immunotherapy has shown promise in hematologic cancers, but challenges to solid tumor therapy remain, including lymphocyte trafficking, elevated interstitial fluid pressures, and immunosuppression. Regional intravascular infusion is a non-surgical, minimally invasive procedure commonly used in liver cancer to deliver therapeutics, which can be augmented by Pressure Enabled Drug Delivery (PEDD). We hypothesized that utilizing established regional delivery strategies to administer natural killer (NK) cells engineered to express a natural killer group 2, member D (NKG2D) activating chimeric receptor and membrane bound IL-15 (CAR NKG2D cells) could increase anti-tumor activity against liver cancer. Methods: In vitro cytotoxicity of CAR NKG2D NK cells was determined in co-culture systems. CRCLM-bearing NSG mice were treated with either CAR NKG2D, non-transduced NK cells (NT-NK), or vehicle via portal vein (PV) for regional PEDD or tail vein (TV) for systemic delivery (SD). Tumor burden was measured via tumor bioluminescence. Mann-Whitney tests were performed for statistical comparisons. Correlation of NKG2D ligand expression in tissue and serum was measured by CODEX and Luminex. Results: Multiple NKG2D ligands are highly expressed in hepatocellular and colorectal carcinoma cell lines (HCC and CRC respectively). As such, these cells lines are highly susceptible to NKG2D-mediated cytotoxicity. CAR NKG2D NK cells were 3- to 4-fold more potent in vitro than NT-NK cells against multiple HCC and CRC cell lines, including those bearing Ras pathway mutations. Using a mouse model of locoregional delivery under high pressure (10 mL/minute), we show that significant tumor reduction (p &lt; 0.05) is only achieved when CAR NKG2D NK cells, but not vehicle or NT-NK cells, were delivered via PV and not via TV. Recovery of CAR NKG2D NK cells in hepatic tissues was on average 2-fold higher after administration via PV than that observed after TV delivery (p = 0.0001). PV delivery of NT-NK cells did not result in appreciable liver engraftment or tumor growth inhibition. Conclusions: CAR NKG2D NK cells demonstrate enhanced in vitro and in vivo cytotoxicity against CRC and HCC cell lines. Significant tumor control using regional delivery in initial studies support continued clinical development. NKX101 is an investigational agent comprised of CAR NKG2D NK cells being evaluated in a phase 1 clinical study for treatment of relapsed/refractory acute myeloid leukemia or higher risk myelodysplastic syndrome. Studies are ongoing to understand NKX101 kinetics, role of delivery pressure, and activity in combination in preclinical models of CRCLM.

Early results of intratumoral INT230-6 alone or in combination with ipilimumab in subjects with advanced sarcomas.

11557 Background: Patients have limited treatment options following initial chemotherapy failure. INT230-6, a novel formulation of cisplatin (CIS) and vinblastine (VIN) with an amphiphilic cell penetration enhancer, is designed for intratumoral (IT) administration. Study IT-01 (BMS # CA184-592, NCT 03058289) evaluates INT230-6 alone or in combination with ipilimumab (IPI), an antibody to CTLA-4. INT230-6 dosing is set by a % of the volume of the tumor to be injected. The product has been shown to disperse throughout an injected tumor and diffuse into cancer cells. Cell death leads to recruitment of dendritic and T cells, the effect of which may be augmented by CTLA-4 inhibition as evidenced by increased efficacy of the combination in preclinical models. Historically, checkpoint inhibitors have limited activity in sarcoma. Considering the large volume of drug injected and retained in the tumor, coupled with immune infiltration on biopsies, RECIST response methodology may not capture the benefits of INT230-6 treatment. Methods: IT-01 is an open-label phase 1/2 study that is enrolling adult subjects with locally advanced, unresectable or metastatic sarcoma. INT230-6 was administered IT Q2W for 5 doses alone or with IPI 3mg/kg IV Q3W for 4 doses. The study objectives are to assess the safety and efficacy of IT INT230-6 alone and in combination with IPI. Results: 16 heterogenous sarcoma subjects (13 monotherapy, 3 IPI combination) having a median of 3 prior therapies (0, 8) were enrolled to date. The INT230-6 dose was up to 145 mL (72.5 mg of CIS, 14.5 mg VIN) in a single session (an amount of each agent in excess of standard IV doses). The most common ( &gt; 20%) related TEAEs in sarcoma subjects (n = 16) were localized pain (63%), fatigue (38%), decreased appetite (31%), nausea (31%), and vomiting (25%) most of which were low grade; with only grade 3 TEAE above 5% being anemia (13%). There were no related grade 4 or 5 TEAEs. In 11 evaluable monotherapy subjects, the disease control rate (DCR = CR+PD+SD) was 82%. Basket studies of sarcomas, including chordoma, with Royal Marsden Hospital index (RMHI) scores of 2 or higher report median overall survival (mOS) of 4 months. In this study 75% of monotherapy subjects had a RMHI score of 2 and preliminary estimates of mOS was 21.3 (4.67, NA) months. Pilot immunohistochemistry analysis of 5 paired (pre- and 28 days post-dose) biopsy samples showed substantial tumor necrosis, reduction of viable cancer, a decreased cancer proliferation as measured by Ki67, and increased TILs. Conclusions: Preliminary data shows that INT230-6 administered intratumorally alone or in combination with ipilimumab is well-tolerated in this small, heterogenous sarcoma population. The preclinical cancer cell death and immune infiltration mechanism of action appears to translate to sarcoma subjects. There are early signs of efficacy, DCR and potentially OS, that need to be confirmed in randomized studies. Clinical trial information: 03058289.

Synergistic Effects of Lenvatinib (E7080) and MEK Inhibitors against Anaplastic Thyroid Cancer in Preclinical Models.

Simple SummaryLenvatinib has been found to be effective against radioiodine-refractory thyroid cancer. However, compliance with lenvatinib therapy is poor due to cancer progression and adverse effects. To improve the success rate of lenvatinib treatment, we propose a combination of lenvatinib and another drug class that has a different target than lenvatinib. We tested the potential of this combination in preclinical models and found that it is effective in our models. The combination also does not cause significant adverse effects in our mouse model. Thus, our results suggest that the combination we propose in this study is a potential therapeutic approach for thyroid cancer that is not responsive to radiotherapy.E7080, known as lenvatinib, is an oral multitargeted tyrosine kinase inhibitor that has been shown to improve the survival rate of patients with radioiodine-refractory thyroid cancer. However, a majority of patients do not continue lenvatinib intake due to disease progression or significant toxicity. To improve treatment success rates, we propose the combination of lenvatinib with mitogen-activated protein kinase (MEK) inhibitors. To test this hypothesis, we tested the effects of lenvatinib with the MEK inhibitor U0126 in vitro using two human anaplastic thyroid cancer (ATC) cell lines, 8505C and TCO1, and with another MEK inhibitor, selumetinib (AZD6244), in an ATC mouse model. We found that the combination of lenvatinib with MEK inhibitors enhanced the antitumor effects of monotherapy with either agent in vitro and in vivo, and these effects may be through the AKT (Protein Kinase B) and extracellular signal-regulated kinase (ERK) signaling pathways. Furthermore, the combination does not have significant adverse effects in the ATC mouse models in terms of body weight, blood biochemical parameters, and histopathology. In conclusion, the combination of lenvatinib with an MEK inhibitor is a potentially viable therapeutic approach for ATC treatment.

CDK4/6 inhibition enhances antitumor efficacy of chemotherapy and immune checkpoint inhibitor combinations in preclinical models and enhances T-cell activation in patients with SCLC receiving chemotherapy.

BackgroundCombination treatment with chemotherapy and immune checkpoint inhibitors (ICIs) has demonstrated meaningful clinical benefit to patients. However, chemotherapy-induced damage to the immune system can potentially diminish the efficacy of chemotherapy/ICI combinations. Trilaciclib, a highly potent, selective and reversible cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor in development to preserve hematopoietic stem and progenitor cells and immune system function during chemotherapy, has demonstrated proof of concept in recent clinical trials. Furthermore, CDK4/6 inhibition has been shown to augment T-cell activation and antitumor immunity in preclinical settings. Therefore, addition of trilaciclib has the potential to further enhance the efficacy of chemotherapy and ICI combinations.MethodsIn murine syngeneic tumor models, a schedule of 3 weekly doses of trilaciclib was combined with chemotherapy/ICI regimens to assess the effect of transient CDK4/6 inhibition on antitumor response and intratumor T-cell proliferation and function. Peripheral T-cell status was also analyzed in patients with small cell lung cancer (SCLC) treated with chemotherapy with or without trilaciclib to gain insights into the effect of transient exposure of trilaciclib on T-cell activation.ResultsPreclinically, the addition of trilaciclib to chemotherapy/ICI regimens enhanced antitumor response and overall survival compared with chemotherapy and ICI combinations alone. This effect is associated with the modulation of the proliferation and composition of T-cell subsets in the tumor microenvironment and increased effector function. Transient exposure of trilaciclib in patients with SCLC during chemotherapy treatment both preserved and increased peripheral lymphocyte counts and enhanced T-cell activation, suggesting that trilaciclib not only preserved but also enhanced immune system function.ConclusionsTransient CDK4/6 inhibition by trilaciclib was sufficient to enhance and prolong the duration of the antitumor response by chemotherapy/ICI combinations, suggesting a role for the transient cell cycle arrest of tumor immune infiltrates in remodeling the tumor microenvironment. These results provide a rationale for combining trilaciclib with chemotherapy/ICI regimens to improve antitumor efficacy in patients with cancer.

Abstract 2269: ATR inhibitor M6620 enhances anti-tumor efficacy of the combination of the anti-PD-L1 antibody avelumab with platinum-based chemotherapy

Abstract Recently, combination of checkpoint inhibitors with chemotherapy demonstrated significant improvement of overall survival (OS) and progression-free survival (PFS) compared with chemotherapy alone in patients with metastatic non-squamous and squamous non-small cell lung cancer (NSCLC). However still a larger number of patients do not benefit of the combination treatment. Ataxia telangiectasia mutated and Rad3 related kinase (ATR) is one of the key players in the DNA damage response (DDR) to replication stress. M6620 is a potent and selective ATR inhibitor currently in Phase I clinical studies in combination with multiple DNA-damaging drugs. M6620 has been shown to sensitize cancer cells to the lethal effects of DNA-damaging chemotherapeutic agents. Here we tested the hypothesis that ATR inhibitor M6620 could further enhance anti-tumor efficacy of chemotherapy combinations with avelumab, an FDA approved human IgG1 anti-PD-L1 monoclonal antibody, in vivo. We profiled a panel of murine tumor cell lines to identify models sensitive to combination treatment of M6620 with cisplatin, carboplatin or oxaliplatin. Addition of M6620 to the chemotherapeutic agents in vitro potentiated growth inhibition effect and led to the increase in markers of immunogenic cell death (ICD) in the sensitive tumor cell lines. Based on these data we investigated anti-tumor efficacy of carboplatin or cisplatin in combination with M6620 and avelumab in MC38 murine colorectal tumor model. The triple combination regimens demonstrated a statistically significant better control of tumor growth, more frequent tumor regressions and increased overall survival compared to the double combination treatment groups. The triplet combination regimens were well tolerated as assessed by changes in body weight and clinical signs. Animals with complete tumor responses were re-challenged with MC38 tumor cells and were found refractory to the second tumor inoculation, indicating that durable, protective anti-tumor immunity was established in complete responders. Taken together, robust anti-tumor efficacy and acceptable toxicity of selected triplet combinations in preclinical models provide strong rationale for combining avelumab with M6620 and carboplatin in patients with PARP inhibitor resistant recurrent ovarian cancer. Citation Format: Marat Alimzhanov, Patricia Soulard, Astrid Zimmermann, Andreas Schroeder, Keyvan Tadjalli Mehr, Christiane Amendt, Geok Choo Sim, Andree Blaukat, Joern-Peter Halle, Frank T. Zenke. ATR inhibitor M6620 enhances anti-tumor efficacy of the combination of the anti-PD-L1 antibody avelumab with platinum-based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2269.

PS1310 THE NOVEL BISPECIFIC CD47‐CD19 ANTIBODY TG‐1801 POTENTIATES THE ACTIVITY OF UBLITUXIMAB‐UMBRALISIB (U2) DRUG COMBINATION IN PRECLINICAL MODELS OF B‐NHL

Background:Umbralisib is an orally available PI3Kδ inhibitor that has demonstrated activity both in preclinical models and primary B cell non‐Hodgkin lymphoma (B‐NHL) cells, and in patients with B‐cell malignancies. The doublet therapy of umbralisib used in combination with the glycoengineered anti‐CD20 monoclonal antibody (mAb) ublituximab (“U2” regimen), provides a non‐chemotherapy backbone regimen on which several novel multidrug combinations have been explored clinically. TG‐1801 is a novel, bispecific antibody in Phase 1 testing that targets CD47 selectively on CD19+ B‐cells, sparing red blood cells or platelets, blocking the CD47‐SIRPα macrophage checkpoint on mature B cells.Aims:We explored in vitro and in vivo potential synergies between TG‐1801 and ublituximab, umbralisib, and the U2 combination in preclinical models of B‐NHL.Methods:Human B‐cell lymphoma Raji and Jeko‐1 cells were cultured in vitro in the presence of different stromal and immune cell types including bone marrow derived stromal cells (BMSCs), M2‐polarized primary macrophages, and primary circulating PBMCs. Cells were analyzed by proliferation assay, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and fluorimetric evaluation of antibody‐dependent cell death (ADCC) and antibody‐dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor‐bearing mice for 17 days with ublituximab (5 mg/kg, qw) +/‐ umbralisib (150 mg/kg, bid), the combination of both and/or TG‐1801 (5 mg/kg, qw).Results:In the context of the B‐NHL co‐culture system, U2 combination treatment led to the decrease of anti‐inflammatory cytokines genes (IL‐6, IL‐10, IL‐1RA) and upregulation of pro‐inflammatory IL‐2, accompanied by a 2 fold increase of M2 dependent ADCP. When U2 was combined with TG‐1801, this 2‐fold increase in ADCP was doubled, brought to a 4‐fold increase. In addition, TG‐1801 alone activated ADCC 4.1‐fold above control, and when combined with ublituximab was 7.2‐fold above control. As expected, umbralisib did not impact ADCC. Transcriptomic analysis of CD20+ cells further revealed that U2 treatment was mainly associated with modulation of redox processes in Raji co‐cultures (NES = −0.57, p = 0.016). Although TG‐1801 single agent did not significantly affect the transcriptome of these cells, combination with U2 provoked the down‐regulation of genes associated to cell architecture homeostasis, including cellular membranes (NES = −0.57, p = 0.006), cytoskeleton (NES = −0.51, p = 0.046), and cell proliferation (NES = −0.44, p = 0.018). In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG‐1801 exhibited a 76% TGI with 1/8 tumor free‐mouse. Most importantly, the combination of TG‐1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor‐free mice 35 days after the last dose in these 3 groups. Interestingly, this superior anti‐tumor effect of the different TG‐1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling.Summary/Conclusion:The data presented here set the preclinical rationale and support a combination strategy of the novel CD47‐CD19 bispecific antibody TG‐1801 in B‐NHL with other B‐cell targeted mechanisms, including umbralisib and ublituximab.