Solid Tumor Xenografts Research Articles

Engineering potent chimeric antigen receptor T cells by programming signaling during T-cell activation

Programming cell signaling during T-cell activation represents a simple strategy for improving the potency of therapeutic T-cell products. Stim-R technology (Lyell Immunopharma) is a customizable, degradable synthetic cell biomimetic that emulates physiologic, cell-like presentation of signal molecules to control T-cell activation. A breadth of Stim-R formulations with different anti-CD3/anti-CD28 (αCD3/αCD28) antibody densities and stoichiometries were screened for their effects on multiple metrics of T-cell function. We identified an optimized formulation that produced receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeted chimeric antigen receptor (CAR) T cells with enhanced persistence and polyfunctionality in vitro, as assessed in repeat-stimulation assays, compared with a benchmark product generated using a conventional T-cell–activating reagent. In transcriptomic analyses, CAR T cells activated with Stim-R technology showed downregulation of exhaustion-associated gene sets and retained a unique subset of stem-like cells with effector-associated gene signatures following repeated exposure to tumor cells. Compared with the benchmark product, CAR T cells activated using the optimized Stim-R technology formulation exhibited higher peak expansion, prolonged persistence, and improved tumor control in a solid tumor xenograft model. Enhancing T-cell products with Stim-R technology during T-cell activation may help improve therapeutic efficacy against solid tumors.

Discovery of a Myeloid Cell Leukemia 1 (Mcl-1) Inhibitor That Demonstrates Potent In Vivo Activities in Mouse Models of Hematological and Solid Tumors.

Myeloid cell leukemia 1 (Mcl-1) is a key regulator of the intrinsic apoptosis pathway. Overexpression of Mcl-1 is correlated with high tumor grade, poor survival, and both intrinsic and acquired resistance to cancer therapies. Herein, we disclose the structure-guided design of a small molecule Mcl-1 inhibitor, compound 26, that binds to Mcl-1 with subnanomolar affinity, inhibits growth in cell culture assays, and possesses low clearance in mouse and dog pharmacokinetic (PK) experiments. Evaluation of 26 as a single agent in Mcl-1 sensitive hematological and solid tumor xenograft models resulted in regressions. Co-treatment of Mcl-1-sensitive and Mcl-1 insensitive lung cancer derived xenografts with 26 and docetaxel or topotecan, respectively, resulted in an enhanced tumor response. These findings support the premise that pro-apoptotic priming of tumor cells by other therapies in combination with Mcl-1 inhibition may significantly expand the subset of cancers in which Mcl-1 inhibitors may prove beneficial.

Targeting ROS-sensing Nrf2 potentiates anti-tumor immunity of intratumoral CD8+ T and CAR-T cells

Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8+ T and chimeric antigen receptor (CAR) Tcells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2-/- mice was significantly controlled and was reversed by Tcell depletion and further confirmed that Nrf2 deficiency in Tcells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8+ Tcells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-Tcells enhanced the survival and function of intratumoral CAR-Tcells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor Tcell responses, indicating that Nrf2 may be a potential target for Tcell immunotherapy strategies against solid tumors.

LP-184, a Novel Acylfulvene Molecule, Exhibits Anticancer Activity against Diverse Solid Tumors with Homologous Recombination Deficiency.

New agents with activity against DDR-deficient solid tumors refractory to standard-of-care therapies are needed. We report multiple findings supporting the potential for LP-184, a novel alkylating agent with three FDA orphan drug designations, to fill this void clinically: strong nanomolar potency; sustained, durable regression of solid tumor xenografts; synthetic lethality with HR defects. LP-184 adult phase IA trial to assess safety in advanced solid tumors is ongoing.

Abstract 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts [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 2616.

The use of L-asparaginase for the treatment of solid tumors: data from experimental studies and clinical trials

Drug therapy is one of the main strategies of cancer treatment. L-asparaginase, the enzyme that hydrolyzes asparagine, has been included in the treatment regimens for acute lymphoblastic leukemia and other hematological malignancies since more than 50 years ago, but its use for the treatment of solid tumors is still extremely limited. This review analyzes experimental data on the sensitivity of cell lines and xenografts of solid tumors to L-asparaginase, examines the results of clinical trials. Among the mechanisms of the cytotoxic effect of L-asparaginase on tumor cells, such processes as depletion of aspartic and glutamic acids, influence on the internal and external pathways of apoptosis, inhibition of cellular processes through a decrease in the activity of the mTOR protein, and weakening of the expression of the telomerase gene are discussed. Separately, molecular markers are considered, which can be used to suggest the effectiveness of future therapy with L-asparaginase in solid tumors. These markers include expression levels of asparagine synthetase and glutamine synthetase genes, degree of methylation of the ASNS gene promoter region, PTEN protein activity and autophagy, bone marrow environment of tumor cells, as well as expression of genes associated with asparaginase resistance (such as the μ1 opioid receptor gene and the huntingtin-associated protein 1 gene).

A First-in-Human, Open-Label, Phase I Dose Escalation Trial of Daily Oral Nmt Inhibitor Zelenirstat in Patients with Relapsed/Refractory B-Cell Lymphomas and Advanced Solid Tumors

Background Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. Zelenirstat (PCLX-001) is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC 50 of 5nM and 8nM, respectively). In vitro hematologic cancer cell lines were exquisitely sensitive to zelenirstat. Zelenirstat regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient (pt) derived xenograft model. Zelenirstat also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we conducted a phase I dose escalation trial to determine safety, tolerability, maximum tolerated dose (MTD), and pharmacokinetics (PK) of zelenirstat. Methods Pts with relapsed/refractory (R/R) B-cell lymphomas and advanced solid tumors were enrolled in a multicenter, open label, standard phase I dose escalation trial of once daily (OD) oral zelenirstat, administered in 28-day cycles until progressive disease or unacceptable toxicity. Seven dose levels were studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 210 mg, and 280 mg. Dose limiting toxicity (DLT), attributable to zelenirstat during cycle 1, was defined as: grade (Gr) 4 thrombocytopenia, Gr 3 thrombocytopenia with bleeding, Gr 4 neutropenia ≥ 7 days, febrile neutropenia, clinically significant > Gr 3 non-hematologic toxicity, or any significant toxicity warranting a hold of zelenirstat. Plasma PK were obtained on C1D1, C1D15, and trough concentrations C1D2, C1D16 and prior to each subsequent cycle. Response assessments occurred every 2 cycles to determine best overall response. Results Between Sept 2021 and Jul 2023, 21 DLT-evaluable pts [4 R/R B-cell lymphoma (3 DLBCL, 1 FL grade 3b); 17 advanced solid tumor (7 colorectal, 2 pancreas, 2 ovarian, 1 each of sarcoma, NSCLC, pleural mesothelioma, melanoma, CUP, bladder)] received zelenirstat at doses ranging from 20 mg OD to 280 mg OD. Dose escalation occurred without DLT until the 280 mg cohort where two DLTs were reported: a bladder carcinoma pt who developed nausea, vomiting, and drug-induced fever necessitating a hold of zelenirstat and a colorectal cancer (CRC) pt who had Gr 3 dehydration from nausea, vomiting, diarrhea, and oral mucositis. The maximal administered dose was 280 mg OD and the presumptive MTD was 210 mg OD. Drug compliance was high, and of 3 pts within the 210 mg cohort, none required dose reduction. Common treatment-emergent all-grade adverse events (AEs) at doses up to the 210 mg cohort were gastrointestinal and reported in 29% of pts. These were primarily diarrhea, nausea, or vomiting. Gr 2 thrombocytopenia occurred in 17% of patients. No other AEs were seen at a frequency of > 5%. There were no AEs of neuropathy, alopecia, or QT interval prolongation. Noncompartmental PK analyses demonstrated rapid oral absorption, a terminal half-life of approximately 10 hrs, rapid achievement of steady state, and no induction of metabolism. Zelenirstat trough plasma concentrations at 100 mg, 140 mg, and 210 mg doses markedly exceeded the EC 90 required to inhibit cultured cancer cells. Stable disease as best response was seen in five patients (pancreas at 40 mg; CRC at 140 mg; and two ovarian and one CRC at 210 mg). At the 210 mg dose, one of the ovarian cancer pts had a corresponding reduction of CA-125 (20%) while the CRC pt had a reduction in CEA (50%). Conclusions Zelenirstat is safe and well tolerated up to the 210 mg OD dose, which we have established as the presumptive MTD. Additional pts, including a phase IIA study in pts with R/R B-cell NHL, are accruing at this dose for further safety and activity exploration. The absence of severe toxicities, attainment of plasma concentrations highly active in preclinical models, and early evidence of antitumor activity support the ongoing development of zelenirstat for pts with R/R B-cell lymphomas and advanced solid tumors.

A first-in-human, open-label, phase I trial of daily oral PCLX-001, an NMT inhibitor, in patients with relapsed/refractory B-cell lymphomas and advanced solid tumors.

e15094 Background: Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. PCLX-001 is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC50 of 5nM and 8nM, respectively). In vitro, hematologic cancer cell lines were exquisitely sensitive to PCLX-001. PCLX-001 regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient derived xenograft model. PCLX-001 also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we hypothesized that PCLX-001 would be safe, tolerable, and active as a novel oral cancer therapeutic. Methods: Patients with relapsed/refractory B-cell lymphomas and advanced solid tumors are being enrolled in a multicenter, open label, standard phase I dose escalation design to determine feasibility, maximum tolerated dose (MTD), and pharmacokinetics of PCLX-001 monotherapy. Seven dose levels are being studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 200 mg, and 280 mg. Dose limiting toxicity (DLT) is defined as: grade (Gr) 4 platelets, Gr 3 platelets with bleeding, Gr 4 ANC ≥ 7 days, febrile neutropenia, or clinically significant > Gr 3 non-hematologic toxicity. Results: Eighteen evaluable patients (3 relapsed B-cell lymphoma; 15 advanced solid tumor) have been accrued through dose level six (200 mg cohort) without DLT. No attributable Gr 3 or 4 toxicities have been identified. Noncompartmental pharmacokinetic (PK) analyses demonstrate rapid oral absorption, a terminal half-life of approximately 10 hours, rapid achievement of steady state, and no induction of metabolism. PCLX-001 trough plasma concentrations at higher dose cohorts exceed the EC90 required to inhibit cultured cancer cells. Conclusions: PCLX-001 is safe and well tolerated up to the 200 mg daily dose. Our results support the ongoing development of PCLX-001 as an oral daily therapy for the treatment of patients with cancer. Updated study results will be presented, summarizing the safety, MTD, and PK outcomes in PCLX-001 treated patients. Preliminary clinical activity will be determined in 20 patient dose expansion cohorts of relapsed/refractory B-cell lymphomas, and selected advanced solid tumors following the identification of PCLX-001 MTD. Clinical trial information: NCT04836195 .

A Multicenter, Open-Label, Phase I/II Study of FN-1501 in Patients with Advanced Solid Tumors.

FN-1501, a potent inhibitor of receptor FMS-like tyrosine kinase 3 (FLT3) and CDK4/6, KIT, PDGFR, VEGFR2, ALK, and RET tyrosine kinase proteins, has demonstrated significant in vivo activity in various solid tumor and leukemia human xenograft models. Anomalies in FLT3 have an established role as a therapeutic target where the gene has been shown to play a critical role in the growth, differentiation, and survival of various cell types in hematopoietic cancer and have shown promise in various solid tumors. An open-label, Phase I/II study (NCT03690154) was designed to evaluate the safety and PK profile of FN-1501 as monotherapy in patients (pts) with advanced solid tumors and relapsed, refractory (R/R) AML. Pts received FN-1501 IV three times a week for 2 weeks, followed by 1 week off treatment in continuous 21-day cycles. Dose escalation followed a standard 3 + 3 design. Primary objectives include the determination of the maximum tolerated dose (MTD), safety, and recommended Phase 2 dose (RP2D). Secondary objectives include pharmacokinetics (PK) and preliminary anti-tumor activity. Exploratory objectives include the relationship between pharmacogenetic mutations (e.g., FLT3, TP53, KRAS, NRAS, etc.), safety, and efficacy; as well as an evaluation of the pharmacodynamic effects of treatment with FN-1501. Dose expansion at RP2D further explored the safety and efficacy of FN-1501 in this treatment setting. A total of 48 adult pts with advanced solid tumors (N = 47) and AML (N = 1) were enrolled at doses ranging from 2.5 to 226 mg IV three times a week for two weeks in 21-day cycles (2 weeks on and 1 week off treatment). The median age was 65 years (range 30-92); 57% were female and 43% were male. The median number of prior lines of treatment was 5 (range 1-12). Forty patients evaluable for dose-limiting toxicity (DLT) assessment had a median exposure of 9.5 cycles (range 1-18 cycles). Treatment-related adverse events (TRAEs) were reported for 64% of the pts. The most common treatment-emergent adverse events (TEAEs), defined as those occurring in ≥20% of pts, primarily consisted of reversible Grade 1-2 fatigue (34%), nausea (32%), and diarrhea (26%). The most common Grade ≥3 events occurring in ≥5% of pts consisted of diarrhea and hyponatremia. Dose escalation was discontinued due to DLTs of Grade 3 thrombocytopenia (N = 1) and Grade 3 infusion-related reaction (N = 1) occurring in 2 pts. The maximum tolerated dose (MTD) was determined to be 170 mg. FN-1501 demonstrated reasonable safety, tolerability, and preliminary activity against solid tumors in doses up to 170 mg. Dose escalation was terminated based on 2 DLTs occurring at the 226 mg dose level.

Abstract 1639: DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents

Abstract Background: The Integrated Stress Response (ISR) is one of the major adaptive stress response pathways in cancer and plays an important role in cell fate determination. Oncogene addicted solid tumors are under high stress levels, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR pathway activity to cope with the high demand for accelerated growth. The ISR is well known to be a double edge sword of survival and cell death and depending on context, the activation of the ISR kinase, GCN2, and downstream pathway can have either cytoprotective or cytotoxic effects. Given the context-dependent nature of the ISR pathway, the inhibition or stimulation of GCN2 in solid tumors can be pharmacologically leveraged to induce anti-tumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR pathway (phospho-GCN2, ATF4, CHOP) or the apoptosis pathway (PARP and Caspase3/7) was assessed via Western blot or ELISA. In vivo upregulation of tumoral ATF4 was determined in a fibrosarcoma PK/PD xenograft model. In vivo inhibition of tumor growth was determined in solid tumor xenografts. Results: Selective and potent modulators of GCN2 kinase with favorable drug-like properties were designed. These compounds were found to upregulate components of the ISR pathway (phospho-GCN2, ATF4, CHOP). The mechanism by which GCN2 modulator DP-9149 treatment induced the ISR pathway was found to be through the direct binding and activation of GCN2. Upregulation of the ISR pathway downstream of GCN2 led to induction of a programmed cell death pathway in oncogene-driven solid tumor cell lines in vitro. DP-9149-mediated activation of the ISR pathway led to cell growth arrest both as a single agent and in combination with standard-of-care (SOC) agents. Furthermore, oral dosing of DP-9149 in RAS mutant and other oncogene-driven xenograft models in vivo induced ATF4, and significantly inhibited tumor growth as a single agent and in combination with SOC agents. Additionally, therapeutic agents targeting the tumor microenvironment, including anti-angiogenic agents, synergized with DP-9149 to induce tumor regressions in vivo. Conclusions: The ISR is a targetable vulnerability in oncogene addicted solid tumors. Upregulating the ISR by paradoxical activation of the ISR family member kinase, GCN2, by DP-9149 can be leveraged as a novel mechanism to cause anti-tumoral effects in solid tumors in vitro and in vivo, likely through the induction of an unresolved stress response. In particular, DP-9149 exhibited robust activity in RAS mutant cancers and in VHL-mutant renal cancers as a single agent and in combination with SOC agents in vivo. Citation Format: Gada Al-Ani, Qi Groer, Kristin M. Elliott, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents [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 1639.

Abstract 3089: XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies

Abstract Enhancer of zeste homolog 2 (EZH2) is the histone methyltransferase component of polycomb repressive complex 2 (PRC2) that can regulate gene expression by H3K27 trimethylation (H3K27Me3). EZH2 also mediates PRC2-dependent non-histone protein methylation or directly methylates non-histone proteins to regulate gene expression independent of PRC2. EZH2 plays an important role in regulation of cell cycle, autophagy, apoptosis and cell senescence. Mutated or overexpressed EZH2 associates with cancer initiation, progression, drug resistance and immunity regulation in cancer. XNW5004 is a rationally designed selective EZH2 inhibitor which potently inhibits both wild type and mutant EZH2. XNW5004 significantly suppresses level of H3K27Me3, arrests cell cycle in G1/S phase and promotes apoptosis in tumor. XNW5004 exhibits strong antitumor activity in EZH2-mutant lymphoma xenograft models, such as Karpas422 (Y641N), Pfeiffer (A677G) and SU-DHL-10 (Y641F). Since inhibition of EZH2 synthetically lethal in SWI/SNF subunits mutated cancers, XNW5004 exhibits dose-dependent efficacy in solid tumor models such as G401 (SMARCB1 deficient) and COV434 (SMARCA2/SMARCA4 deficient) cancer cell lines and xenografts. In prostate cancer cells, EZH2 can bind to the promoter of androgen receptor (AR) gene and activate AR expression. In PARPi-treated cancer cells, inhibition of PARP (poly-ADP-ribose polymerase) diminishes PARylation of EZH2 to result in reactivation of EZH2 and drug resistance to PARPi. In tumor microenvironment, inhibition of EZH2 promotes differentiation of T cells and NK cells, enhances antigen presentation ability of dendritic cells, and increases cytokine/chemokine secretion to recruit T cells into tumor tissue. Based on the above mechanisms, XNW5004 could potentially improve efficacy of ARi, PARPi and anti-PD-1. We have verified the synergistic effects of those combinations in multiple solid tumor xenograft models. In pharmacokinetic studies, XNW5004 has excellent oral bioavailability and exposure. XNW5004 shows good toxicological profile as well, has a wide therapeutic window in rats and dogs, and the risk of clinical safety was controllable. In summary, XNW5004 is a potent EZH2 inhibitor with promising efficacy, pharmacokinetic and safety properties. XNW5004 is expected to be a potent therapeutic candidate in cancer treatment and is now in Phase II clinical trial for both liquid and solid tumors. Citation Format: Yonghan Hu, Wei Peng, Zhe Zhang, Zhenwei Wu, Xi Chen, Yan Huang, Xin Li, Cungang Liu, Xiaojun Liu, Qifeng Shi, Xufang Wang, Wengui Wang, Haiyang Wei, Yuchuan Wu, Jun Xian, Linfeng Xu, Lina Zhang, Jinfeng Zhao, Meijie Le, Jing Qiang. XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies [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 3089.

Abstract 1873: D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors

Abstract Background: HER2 is overexpressed in different solid tumors, including 10-20% breast and gastric cancers. Many HER2-targeted drugs, including trastuzumab, pertuzumab, lapatinib, trastuzumab emtansine (T-DM1), and trastuzumab deruxtecan (T-DXd), have been approved for treatment of HER2-positive early and metastatic breast cancer (BC). However, trials involving pertuzumab, lapatinib and T-DM1 have failed to provide significant clinical improvements in patients with HER2-positive gastric cancer (GC), suggesting differences in HER2 mediated disease biology between HER2-positive BC and GC. Recent studies have suggested efficacy of HER2 antibodies could be further enhanced by antibody-dependent cellular phagocytosis (ADCP) principally regulated by “don’t-eat-me” CD47 signals, which is overexpressed in many HER2-positive cancers and suppresses phagocytosis through binding to SIRPα. Methods: BC and GC cell lines were used. Monocytes were isolated from human PBMC and differentiated into macrophages, which were used for in vitro ADCP assays. In in vivo animal experiments, D3L-001 and comparator antibodies were examined for efficacy in solid tumor xenograft models. Results: D3L-001 was designed to have higher HER2 affinity (KD < 1 nM) than that of CD47 (KD >10 nM). With this unique design, D3L-001 showed preferential binding to HER2/CD47 double positive tumor cells as compared to CD47 single positive cells. D3L-001 showed better anti-tumor effect than trastuzumab in a panel of solid tumor models, including but not limited to BC and GC cell lines or patient derived xenografts. Immunohistochemistry (IHC) analysis was performed in tumor FFPE slides to evaluate expression of HER2 and CD47 in the efficacy tested models, and we observed that D3L-001 showed broad tumor growth inhibition in HER2 or CD47 high/low expression models. In both in vitro and in vivo studies using breast cancer HCC1954, JIMT-1 and gastric cancer N87 models, we observed a synergistic effect in the combination of Trastuzumab with Magrolimab. Moreover, D3L-001 showed a trend of better efficacy than the combination of Trastuzumab and Magrolimab. These results confirm the synergistic effect of dual-targeting HER2 and CD47 simultaneously and suggest advantage of using bispecific antibodies to confer such effect. D3L-001 also displayed potent and comparable in vivo efficacy as Enhertu in gastric N87 model, and it showed synergistic in vivo efficacy, when combining with Pertuzumab or Paclitaxel, in breast HCC1954, JIMT-1 and gastric cancer N87 models. Conclusion: D3L-001, a novel HER2×CD47 bsAb, demonstrated potent anti-tumor effect in a variety of solid tumor models via HER2 guided CD47 co-blocking. It also showed synergistic efficacy when combining with other anti-tumor agents. D3L-001 will be further evaluated in clinic for its potential as a novel treatment option in HER2-positive BC, GC and other solid tumors. Citation Format: Haopeng Rui, Xiaofeng Yang, Jiahao Chen, Jia Wang, Zhiqiang Zheng, Zhiqiang Chen, Jingtao Lu, Peggy Wu, Janet Chen, Allison Wang, George Chen. D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors [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 1873.

Abstract 2930: ADG153, a novel masked anti-CD47 IgG1 SAFEbody, demonstrates strong in vivo anti-tumor activities in preclinical solid tumor models and preferential CD47 target engagement in the tumor microenvironment

Abstract Background: Anti-CD47 therapies have demonstrated promising clinical activities; however, expression of CD47 on many different normal human cell types, including RBCs, serves as a large antigen sink for anti-CD47 antibodies. Blocking CD47 on RBCs, such as by magrolimab, has led to anemia, requiring a priming and maintenance dosing schedule in the clinic. ADG153 is a highly differentiated masked anti-CD47 SAFEbody in IgG1 isotype with enhanced ADCC and ADCP functions, in contrast to magrolimab of IgG4 isotype that lacks ADCC and has weaker ADCP effector functions. In normal tissues, the SAFEbody masking moiety is expected to shield ADG153 from binding to CD47; however, in the presence of highly upregulated CD47 in protease-rich tumor microenvironment (TME), the masked antibody can engage dynamically with highly expressed CD47 and can be cleaved, enabling efficient binding to CD47, blocking CD47/SIRPα signaling, and triggering strong anti-tumor activities in solid tumors. Methods: The in vivo anti-tumor efficacy of ADG153 or magrolimab analog (magrolimab) was assessed across several solid tumor xenograft models. Human CD47/SIRPα knock-in mice, harboring CD47-positive xenograft tumors, were used to determine CD47 receptor occupancy (RO) on normal tissues vs. tumors following dosing with ADG153 or magrolimab. Results: ADG153 monotherapy demonstrated strong anti-tumor activities in SCID mice in multiple solid tumor xenograft models, including gastric cancer, SCLC, TNBC, and ovarian cancer models, with mean tumor growth inhibition (TGI) ranging from 75%-99%. Notably, in 2 of these models, the ADG153 efficacy was significantly stronger than that of magrolimab at the same dose level, supporting the importance of IgG1 in introducing ADCC- and ADCP-mediated solid tumor killing effects. In the human CD47/SIRPα knock-in xenograft mouse model, ADG153 and magrolimab dosing resulted in <10% and ~75% CD47 RO on RBCs, respectively. Magrolimab dosing resulted in high RO on normal tissues, but very low RO on tumors. In contrast, ADG153 treatment resulted in low RO on the majority of the normal tissues and RO higher than that achieved by magrolimab on tumors, consistent with the significantly dampened antigen sink liabilities for the masked ADG153. Conclusion: ADG153 demonstrates significantly reduced antigen sink liabilities and favorable CD47 engagement in the TME, which are highly differentiated from other anti-CD47 agents. ADG153 is locally enriched and efficiently cleaved in tumors, engages its target, and elicits potent anti-tumor activities in multiple solid tumor xenograft models. These results indicate that the ADG153 SAFEbody is a novel anti-CD47 IgG1 antibody prodrug with strong ADCC and ADCP capacities and substantially reduced liabilities, supporting its advancement into clinical development. Citation Format: Aaron Nguyen, Peng Zou, Songmao Zheng, Jianfeng Shi, Guizhong Liu, Yan Li, Felix Du, Peter Luo, Jiangchun Xu. ADG153, a novel masked anti-CD47 IgG1 SAFEbody, demonstrates strong in vivo anti-tumor activities in preclinical solid tumor models and preferential CD47 target engagement in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2930.

Abstract 6264: NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts

Abstract The bromodomain and extraterminal domain (BET) family of proteins play a vital role in gene transcription, making it an attractive therapeutic target for cancer. BET inhibitors can also combine with many anticancer agents to enhance activity. The bromodomains of the BET protein, BD1 and BD2, have unique functions and inhibiting either domain can result in differential responses. To date, non-selective BET inhibitors have failed during early clinical development due to significant on-target toxicity and limited benefit; however, selectively targeting specific bromodomains may result in a more favorable benefit/risk profile. NUV-868 is a novel and highly selective BD2 inhibitor of the BET protein family with ~1500-fold selectivity for BRD4-BD2 relative to BRD4-BD1. Herein, we describe NUV-868 and its activity in multiple in vitro and in vivo solid tumor models. Target engagement, selectivity of bromodomain inhibition, and regulation of BET-mediated gene expression were examined. Additionally, the antitumor activity of NUV-868 in combination with enzalutamide or olaparib was studied in tumor xenograft models of prostate, breast, and pancreatic cancer. NUV-868 demonstrated high selectivity for BD2 and regulated expression of several BET target genes. NUV-868 in combination with enzalutamide inhibited growth of several prostate cancer cell line- and patient-derived xenografts. NUV-868 in combination with olaparib inhibited tumor growth in models of breast, ovarian and pancreatic cancer. Our preclinical data demonstrate that NUV-868, a BD2-selective BET inhibitor, inhibits growth of tumor xenografts in combination with enzalutamide or olaparib and provides rationale for examination of these combinations in the clinic. An ongoing, phase 1 clinical study (NCT05252390) is evaluating NUV-868 as a monotherapy and in combination with olaparib or enzalutamide in patients with advanced solid tumors. Citation Format: Hitisha Patel, Jennifer Hertzog, Laura Heller, Spandana Vootukuri, Yan Zhang, Chris Miller, Gary Hattersley. NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts [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 6264.

Methionine γ-Lyase-Daidzein in Combination with S-Propyl-L-cysteine Sulfoxide as a Targeted Prodrug Enzyme System for Malignant Solid Tumor Xenografts.

The purpose of this study was to determine the anticancer effect of dipropyl thiosulfinate produced in situ by the pharmacological pair: (1) conjugated with daidzein C115H methionine γ-lyase (EC 4.4.1.11, C115H MGL-Dz) and (2) the substrate, S-propyl-L-cysteine sulfoxide (propiin) against various solid tumor types in vitro and in vivo. The MTT test was used to calculate IC50 values for HT29, COLO205 and HCT116 (colon cancer); Panc1 and MIA-PaCa2 (pancreatic cancer); and 22Rv1, DU-145 and PC3 (prostate cancer). The most promising effect for colon cancer cells in vitro was observed in HT29 (IC50 = 6.9 µM). The IC50 values for MIA-PaCa2 and Panc1 were 3.4 and 3.8 µM, respectively. Among prostate cancer cells, 22Rv1 was the most sensitive (IC50 = 5.4 µM). In vivo antitumor activity of the pharmacological pair was studied in HT29, SW620, Panc1, MIA-PaCa2 and 22Rv1 subcutaneous xenografts in BALB/c nude mice. The application of C115H MGL-Dz /propiin demonstrated a significant reduction in the tumor volume of Panc1 (TGI 67%; p = 0.004), MIA-PaCa2 (TGI 50%; p = 0.011), HT29 (TGI 51%; p = 0.04) and 22Rv1 (TGI 70%; p = 0.043) xenografts. The results suggest that the combination of C115H MGL-Dz/propiin is able to suppress tumor growth in vitro and in vivo and the use of this pharmacological pair can be considered as a new strategy for the treatment of solid tumors.

A dose-escalation and expansion study of the safety and pharmacokinetics of XB002 in subjects with inoperable locally advanced or metastatic solid tumors (301)

<b>Objectives:</b> Cervical cancer and epithelial ovarian cancer (EOC) are two of the most prevalent gynecologic cancers associated with high mortality. Despite recent advancements in therapy, there is an unmet need for novel treatments to improve survival outcomes. XB002 is an antibody-drug conjugate (ADC) that targets tissue factor (TF). TF is a protein overexpressed in many solid tumors, including gynecologic cancers, and is associated with disease progression and poor prognosis. XB002 is a human mAb (25A3) directed against TF conjugated to a novel cytotoxic agent (payload) N-acyl sulfonamide auristatin, ZD02044. XB002 has been designed to improve the therapeutic potential of ADCs targeting TF as a new therapeutic strategy in cancers. XB002 has demonstrated activity in several solid tumor xenograft models. The objective of this ongoing first-in-human study is to evaluate the safety, tolerability, pharmacokinetics (PK), immunogenicity (anti-drug antibodies [ADA]), and preliminary antitumor activity of XB002 in patients with advanced solid tumors with limited treatment options. Presented here are the trial design and details of the cervical and EOC cohorts. <b>Methods:</b> In this phase 1, non-randomized, open-label, multicenter, dose-escalation, and dose-expansion study (NCT04925284), patients with advanced solid tumors will receive XB002 IV every three weeks at a starting dose of 0.16 mg/kg. In the dose-escalation stage, an i3+3 study design will be used to identify the maximum tolerated dose (MTD)/recommended dose (RD) and/or the maximum allowed dose of XB002. Dose level decisions will be made by the Cohort Review Committee after review of all available safety and PK data. In the cohort-expansion phase, the MTD/RD will be further evaluated for safety and preliminary efficacy in multiple tumor-specific expansion cohorts that will be enrolled using a Simon's 2-stage design. Patients will be treated until radiographic progression per RECIST v1.1 or unacceptable toxicity. Patients in the cervical and EOC expansion cohorts must have received ≤2 (cervical) or ≤3 (EOC) lines of prior systemic anticancer therapy for locally advanced or metastatic disease. Patients must have received prior platinum-containing therapy in both cohorts, and patients must have platinum-resistant disease in the EOC cohort. The primary endpoint of the cohort-expansion stage will be objective response rate (ORR) per RECIST v1.1 assessed by the investigator. Other endpoints will include duration of response (DOR) and progression-free survival (PFS) per RECIST v1.1 assessed by the investigator, and overall survival; ORR, DOR, and PFS may also be assessed by a blinded independent radiology committee in select expansion cohorts. The effects of XB002 on tumor and blood biomarkers, including TF expression, will be assessed in exploratory analyses. For patients with EOC, blood samples for CA125 assessment will be collected at screening and throughout treatment in both stages of the study.

Abstract 4174: Augmenting NK cell based immunotherapy by targeting mitochondrial apoptosis

Abstract Theoretically a broad spectrum of cancers, including those with low tumor burdens, could respond to NK cell therapy and NK cells might combat resistance to T cell-based therapies. Allogenic NK cells have also demonstrated a good safety profile in clinical trials. These promising features have led to increasing efforts to advance NK-based immunotherapies in recent years. However, efficacy of NK therapy remains limited. Strategies to augment NK efficacy are therefore much needed. To augment the efficacy of adoptive NK cells, most current studies revolve around two focal points: optimizing the source of NK cells and improving their functionality and persistence in vivo. In the current study, we took a different approach by studying how to make cancer cells more vulnerable to NK-mediated killing. It remains unclear to what extent mitochondrial apoptosis is required for NK-mediated killing. We found that primary human NK cells robustly induce mitochondrial apoptosis. Moreover, mitochondrial apoptosis is essential for efficient NK killing, especially at physiologically relevant low E:T ratios (Effector:Target ratios). It is traditionally believed that cytotoxic cell-cancer cell contacts are binary live/death events. We found that NK engagement is often sub-lethal and push cancer cells towards apoptotic threshold (i.e. priming cancer cells for apoptosis), making them more susceptible for killing by subsequent NK contacts. Upregulation of anti-apoptotic proteins has been widely implicated in cancer resistance to chemo- and targeted therapies. We found that overexpression of these proteins such as BCL-2, BCL-XL, and MCL-1 reduced mitochondrial priming for apoptosis and made cancer cells less susceptible to NK killing. We reasoned that additional agents that increase cancer cell mitochondrial priming for apoptosis (e.g., BH3 mimetics) might augment NK-induced killing, as long as NK cells can tolerate these agents. While primary resting NK cells are sensitive to BCL-2, BCL-XL, and MCL-1 inhibitors, unexpectedly, pre-activation with IL-2 conferred resistance of NK cells to these inhibitors. NK cells and BH3 mimetics synergized in both priming and killing cancer cells in vitro. BH3 profiling could also predict the ideal BH3 mimetics to be combined with NK cells for different tumor models. Using liquid and solid tumor xenograft models, we demonstrated that BH3 mimetics synergized with NK cells in suppressing tumor growth and prolonging mouse survival. In summary, we propose a rational strategy to sensitize cancer cells to NK cellular therapy. Moreover, we elucidate the mechanism underlying the apoptotic signaling that is the scientific basis for this strategy. Our results could potentially enable basic, pre-clinical, and clinical studies investigating the combined effects of BH3 mimetics with NK cells in cancers. Citation Format: Rongqing Aaron Pan, Jeremy Ryan, Deng Pan, Kai Wucherpfennig, Anthony Letai. Augmenting NK cell based immunotherapy by targeting mitochondrial apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4174.

Abstract 582: Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy

Abstract Introduction: Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses have been rare in solid tumors. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models1, and the anti-HER2 CAR-M CT-0508 is currently being evaluated in a Phase I trial. The use of myeloid cells as a platform for cell therapy provides the tools to overcome critical solid tumor challenges such as infiltration, immunosuppression within the tumor microenvironment, lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes. Experimental: Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR (CAR-mono) targeted against HER2. We established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product). Results: CAR-mono showed high CAR expression and viability (&amp;gt;90%), and efficiently differentiated into CAR-expressing macrophages. Adenoviral transduction led to pre-conditioning of CAR-mono, resulting in a strong M1 phenotype upon differentiation into CAR-M. CAR-mono derived macrophages demonstrated potent anti-tumor activity regardless of exposure to GM-CSF or M-CSF, and were protected against M2 switching by immunosuppressive factors. Treating CAR-mono with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs with an activated phenotype, indicating that these cells retained their myeloid differentiation potential. In vivo, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following IV administration, CAR-mono demonstrated the ability to traffic to both GM-CSFhigh and GM-CSFlow expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (&amp;gt;180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support. Conclusions: The CAR-mono platform enables an automated, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. The use of Ad5f35 for human monocyte transduction primes the cells toward M1 macrophage differentiation and produces a cell population phenotypically and functionally similar to our established CAR-M platform. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.1Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. March 2020. Citation Format: Daniel Blumenthal, Linara Gabitova, Brett Menchel, Patricia Reyes-Uribe, Sabrina Ceeraz DeLong, Sascha Abramson, Michael Klichinsky. Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 582.

Abstract 2754: LYL797, a ROR1 CAR T-cell therapy with genetic and epigenetic reprogramming for solid tumors

Abstract Chimeric antigen receptor (CAR) T-cell therapy has been shown to produce profound results in the treatment of certain hematologic malignancies, however treatment of solid tumors with CAR T cells has not been as successful. Studies have suggested that T-cell exhaustion plays a role in limiting the ability of CAR T cells to eradicate solid tumors. Additionally, stem-like qualities of T cells have been associated with better outcomes in patients treated with cellular therapies, including CAR T cells. Therefore, maintaining stem-like qualities and overcoming T-cell exhaustion may be key to improving clinical efficacy of CAR T cells in patients with solid tumors. ROR1 is a cell surface antigen expressed in several solid tumor types and chronic lymphocytic leukemia (CLL). ROR1 expression has been reported in 57% of triple-negative breast cancer (TNBC), as well as 42% of adenocarcinoma and 12% of squamous cell carcinoma subtypes of non-small cell lung cancer (NSCLC). These expression data of ROR1 in TNBC and NSCLC provide support for anti-ROR1 agents as a therapeutic strategy for these cancers. LYL797 is a novel, ROR1-targeted chimeric antigen receptor (CAR) T-cell product that incorporates genetic and epigenetic reprogramming technologies, Gen-R and Epi-R, to overcome barriers of CAR T-cell therapies in solid tumors. The ROR1-specific CAR contains a single-chain variable fragment (scFv) derived from an R12 rabbit monoclonal antibody that recognizes and binds with high specificity to human ROR1. Gen-R is ex vivo genetic reprogramming technology that engineers CAR T cells to overexpress c-Jun. Dysregulation of activator protein 1 (AP-1) has been implicated in CAR T-cell exhaustion, and studies have demonstrated that overexpression of c-Jun renders CAR T cells less susceptible to exhaustion, enhancing both anti-tumor efficacy and persistence in preclinical models of hematologic and solid tumors. Epi-R is a proprietary optimized manufacturing process that results in maintenance of stem-like phenotype and function of T-cell products. In preclinical studies LYL797 cells reprogrammed with Gen-R and Epi-R led to improved functional activity in the presence of ROR1+ tumor cells compared to conventional ROR1 CAR T cells. Additional studies are underway to determine the mechanisms by which antitumor activity of LYL797 in ROR1-positive solid tumor xenograft models is enhanced. LYL797 is anticipated to enter into Phase 1 clinical trials for TNBC and NSCLC in 2022. Citation Format: Spencer Park, Courtney Simianer, Sydney Spadinger, Xiao Wang, Purnima Sundar, Shobha Potluri, Rachel Lynn, Bijan Boldajipour, Grace Wang, Neeraj Sharma, Hajime Hiraragi, Veena Krishnamoorthy, Suman Kumar Vodnala, E-Ching Ong, Chang-Chih Wu, Martin Wohlfahrt, Byoung Ryu, Lisa Song, Brian D. Weitzner, Howell Moffett, Marc Lajoie, Scott Boyken, Tamer Shabaneh, Shivani Srivastava, Tina Albertson, Blythe Sather. LYL797, a ROR1 CAR T-cell therapy with genetic and epigenetic reprogramming for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2754.

Abstract 3306: Targeting HER3 receptor positive cancers with a novel anti-HER3 antibody radioconjugate (ARC)

Abstract Background: HER3 overexpression is reported to be associated with poor survival in breast, ovarian, lung, gastric and prostate cancer. In addition, upregulation of HER3 in response to HER1 or HER2 targeted therapies, is implicated in the acquired resistance against these therapies. Therefore, effective targeting of HER3 can potentially overcome resistance and enhance therapeutic efficacy. Although a number of anti-HER3 antibodies have failed clinical testing with the development focus being shifted to other approaches such as antibody drug conjugates and bispecific antibodies, there are currently no approved HER3-targeted therapies. Here we describe a novel approach that can enhance therapeutic efficacy in HER3+ cancer patients by conjugating an anti-HER3 antibody with the alpha-emitting cytotoxic radioisotope Actinium-225 (225Ac) to create an anti-HER3 antibody radiation conjugate (225Ac-HER3-ARC). Alpha emitting radioisotopes like 225Ac can cause double-strand DNA breaks for which there is no known resistance mechanism. Due to the cytotoxic properties of the radioisotope, lower levels of antibody may be needed, resulting in reduced incidence or less severe toxicities. We hypothesize that targeting HER3 in solid tumors with an ARC will result in tumor specific cell killing especially in a setting where HER-targeting agents are not a viable option. We developed a novel 225Ac-HER3-ARC and evaluated its efficacy in HER3+ in vitro and in vivo tumor models. Methods: AT-02, an anti-HER3 antibody, was conjugated with p-SCN-Bn-DOTA and radiolabeled with 225Ac. 225Ac-HER3-ARC specific binding to HER3 was assessed by ELISA using human recombinant HER3 and by flow cytometry on HER3+ cells. The cytotoxic effect of HER3 ARC was evaluated in a panel of HER3 expressing cells. We further evaluated the maximum tolerated dose and therapeutic efficacy of the ARC in nude mice bearing human HER3+ xenograft tumors. Results: In this study we successfully radiolabeled anti-HER3 with 225Ac. 225Ac-HER3-ARC showed similar binding properties to those of the native antibody by ELISA (HER3-ARC: EC50 = 0.0017 µg/ml, HER3 EC50 = 0.0022 µg/ml) and flow cytometry. Treatment with ARC was cytotoxic to HER3+ cells in a dose-dependent manner (EC50 = 54 kBq/ml). 225Ac-HER3-ARC showed potent in vivo efficacy in preclinical solid tumor xenograft models that was correlated with the in vitro cytotoxicity findings. Treatment with 225Ac-HER3-ARC (7.4 - 22.2 kBq, 200 - 600 nCi) led to complete responses and significantly prolonged survival compared to control groups (p &amp;lt; 0.0001). Conclusions: Our findings demonstrate that targeting HER3 with a novel 225Ac-HER3-ARC results in potent tumor cell cytotoxicity and complete anti-tumor response in HER3 tumor xenograft model. This approach provides a promising therapeutic strategy for HER3 positive tumors and warrants further assessment. Citation Format: Denis Beckford-Vera, Jason Li, Megan McCloskey, Caroline Jennings, Amanda Chin, Qing Liang, Jesse Hwang, Monideepa Roy, Mary Chen, Helen Kotanides. Targeting HER3 receptor positive cancers with a novel anti-HER3 antibody radioconjugate (ARC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3306.