Antitumor Activity In Preclinical Models Research Articles

Redirecting B7-H3.CAR T cells to Chemokines Expressed in Osteosarcoma Enhances Homing and Antitumor Activity in Preclinical Models.

Clinical efficacy of CAR T cells against pediatric osteosarcoma (OS) has been limited. One strategy to improve efficacy may be to drive chemokine-mediated homing of CAR T cells to tumors. We investigated the primary chemokines secreted by OS and evaluated efficacy of B7-H3.CAR T cells expressing the cognate receptors. We developed a pipeline to identify chemokines secreted by OS by correlating RNA-seq data with chemokines detected in media from fresh surgical specimens. We identified CXCR2 and CXCR6 as promising receptors for enhancing CAR T cell homing against OS. We evaluated the homing kinetics and efficiency of CXCR2- and CXCR6.T cells and homing, cytokine production, and antitumor activity of CXCR2- and CXCR6.B7-H3.CAR T cells in vitro and in vivo. T cells transgenically expressing CXCR2 or CXCR6 exhibited ligand-specific enhanced migration over T cells modified with nonfunctional receptors. Differential homing kinetics were observed, with CXCR2.T cells homing quickly and plateauing early, while CXCR6.T cells homed more slowly but achieved a similar plateau. When expressed in B7-H3.CAR T cells, CXCR2- and CXCR6 modification conferred enhanced homing towards OS in vitro and in vivo. CXCR2- and CXCR6-B7-H3.CAR treated mice experienced prolonged survival in a metastatic model compared to B7-H3.CAR T cell treated mice. Our patient-based pipeline identified targets for chemokine receptor modification of CAR T cells targeting OS. CXCR2 and CXCR6 expression enhanced homing and anti-OS activity of B7-H3.CAR T cells. These findings support clinical evaluation of CXCR-modified CAR T cells to improve adoptive cell therapy for OS patients.

Low level of tumor necrosis factor α in tumor microenvironment maintains self-renewal of glioma stem cells by Vasorin-mediated glycolysis.

Self-renewal of glioma stem cells (GSCs) is responsible for glioblastoma (GBM) therapy-resistant and recurrence. Tumor necrosis factor α (TNFα) and TNF signaling pathway display an antitumor activity in preclinical models and in tumor patients. However, TNFα exhibits no significance for glioma clinical prognosis based on Glioma Genome Atlas database. This study aimed to explore whether TNFα of tumor microenvironment maintains self-renewal of GSCs and promotes worse prognosis in glioma patient. Spatial transcriptomics, immunoblotting, sphere formation assay, extreme limiting dilution, and gene expression analysis were used to determine the role of TNFα on GSC's self-renewal. Mass spectrometry, RNA-sequencing detection, bioinformatic analyses, qRT-RNA, immunofluorescence, immunohistochemistry, single cell RNA sequencing, in vitro and in vivo models were used to uncover the mechanism of TNFα-induced GSC self-renewal. Low level of TNFα displays a promoting effect on GSC self-renewal and worse glioma prognosis. Mechanistically, Vasorin (VASN) mediated TNFα-induced self-renewal by potentiating glycolysis. Lactate produced by glycolysis inhibits the TNFα secretion of tumor-associated macrophages (TAMs) and maintains TNFα in a low level. TNFα-induced GSC self-renewal mediated by VASN provides a possible explanation for the failures of endogenous TNFα effect on GBM. Combination of targeting VASN and TNFα anti-tumor effect may be an effective approach for treating GBM.

Manuka Honey Inhibits Human Breast Cancer Progression in Preclinical Models.

Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.

Velcrin molecular glues induce apoptosis in glioblastomas with high PDE3A and SLFN12 expression

Abstract Background Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase SLFN12 and the phosphodiesterase PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumors cell lines from the Cancer Cell Line Encyclopedia (CCLE), including glioblastoma cell lines. We therefore aim to characterize velcrins as novel therapeutic agents in glioblastoma. Materials and Methods PDE3A and SLFN12 expression levels were measured in glioblastoma cell lines, TCGA tumor samples and tumor neurospheres. Velcrin-treated cells were assayed for viability, induction of apoptosis, cell cycle phases, and global changes in translation. Transcriptional profiling of the cells was obtained. Xenograft-harboring mice treated with velcrins were also monitored for survival. Results We identified several velcrin-sensitive glioblastoma cell lines and four velcrin-sensitive glioblastoma patient-derived models. We determined that BAY 2666605 crosses the blood brain barrier and elicits full tumor regression in an orthotopic xenograft model of GB1 cells. We also determined that the velcrins BAY 2666605 and BRD3800 induce tumor regression in subcutaneous glioblastoma PDX models. Conclusion Velcrins have anti-tumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

Disitamab vedotin, a HER2-directed antibody-drug conjugate, in patients with HER2-overexpression and HER2-low advanced breast cancer: a phase I/Ib study.

Disitamab vedotin (DV; RC48-ADC) is an antibody-drug conjugate comprising a human epidermal growth factor receptor 2 (HER2)-directed antibody, linker and monomethyl auristatin E. Preclinical studies have shown that DV demonstrated potent antitumor activity in preclinical models of breast, gastric, and ovarian cancers with different levels of HER2 expression. In this pooled analysis, we report the safety and efficacy of DV in patients with HER2-overexpression and HER2-low advanced breast cancer (ABC). In the phase I dose-escalation study (C001 CANCER), HER2-overexpression ABC patients received DV at doses of 0.5-2.5 mg/kg once every two weeks (Q2W) until unacceptable toxicity or progressive disease. The dose range, safety, and pharmacokinetics (PK) were determined. The phase Ib dose-range and expansion study (C003 CANCER) enrolled two cohorts: HER2-overexpression ABC patients receiving DV at doses of 1.5-2.5 mg/kg Q2W, with the recommended phase 2 dose (RP2D) determined, and HER2-low ABC patients receiving DV at doses of 2.0 mg/kg Q2W to explore the efficacy and safety of DV in HER2-low ABC. Twenty-four patients with HER2-overexpression ABC in C001 CANCER, 46 patients with HER2-overexpression ABC and 66 patients with HER2-low ABC in C003 CANCER were enrolled. At 2.0 mg/kg RP2D Q2W, the confirmed objective response rates were 42.9% (9/21; 95% confidence interval [CI]: 21.8%-66.0%) and 33.3% (22/66; 95% CI: 22.2%-46.0%), with median progression-free survival (PFS) of 5.7 months (95% CI: 5.3-8.4 months) and 5.1 months (95% CI: 4.1-6.6 months) for HER2-overexpression and HER2-low ABC, respectively. Common (≥5%) grade 3 or higher treatment-emergent adverse events included neutrophil count decreased (17.6%), gamma-glutamyl transferase increased (13.2%), asthenia (11.0%), white blood cell count decreased (9.6%), peripheral neuropathy such as hypoesthesia (5.9%) and neurotoxicity (0.7%), and pain (5.9%). DV demonstrated promising efficacy in HER2-overexpression and HER2-low ABC, with a favorable safety profile at 2.0 mg/kg Q2W.

FZ-AD005, A Novel DLL3-Targeted Antibody-drug Conjugate with Topoisomerase I Inhibitor, Shows Potent Antitumor Activity in Preclinical Models

FZ-AD005, A Novel DLL3-Targeted Antibody-drug Conjugate with Topoisomerase I Inhibitor, Shows Potent Antitumor Activity in Preclinical Models

Safety and efficacy of complement factor H (CFH) inhibitor GT103 in advanced non-small cell lung cancer (NSCLC): Results of a phase Ib first in human dose escalation study.

e14588 Background: CFH is a complement regulatory protein that protects cells from alternative complement pathway activation. Neutralizing antibodies against CFH on tumor cells increase deposition of C3b, facilitate antibody-dependent-cellular phagocytosis and complement dependent cytotoxicity, inhibit tumor growth, and modulate anti-tumor immunity. GT103 is a first-in class, fully human-derived IgG3 anti-CFH monoclonal antibody that recognizes a tumor specific epitope on CFH and has shown anti-tumor activity in preclinical models. Methods: We conducted a phase Ib, first-in-human, dose escalation trial evaluating safety and preliminary efficacy of GT103 in patients (pts) with advanced NSCLC refractory to standard therapies. Pts received GT103 intravenously across six dose levels/schedules following 3+3 design. Primary objectives were to determine maximum tolerated dose (MTD), recommended phase II dose (RP2D), and pharmacokinetic profile of GT103. Secondary objectives were to assess objective response rate, progression free survival (PFS), and overall survival (OS). Here we present updated safety and efficacy results of the study. Results: 31 pts were enrolled from 6/2020 to 9/2023 (median age 63yrs (range, 23-79), 61% had adenocarcinoma, 32% had previously treated brain metastasis). MTD was not reached. Treatment related AEs (TRAEs) observed in ≥10% pts included fatigue (19%), anemia (16%), diarrhea (16%), and nausea (13%). 3 pts developed ≥ grade 3 TRAE, including acute kidney injury, decreased lymphocyte count, and anemia. The best treatment response was stable disease in 9 (29%) pts. Median(m) PFS and mOS were 6 weeks (95% CI 6.0-6.1) and 25.7 weeks (95% CI 19.1-30.6), respectively. 24-week PFS and OS were 9.7% (95% CI 2.5-22.9%) and 50.6% (95% CI 31.9-66.6%), respectively. PD-L1 expression was available in 25 pts. No statistically significant difference (p=0.33) was noted in mPFS between PD-L1 positive (TPS > 1%) vs negative (TPS ≤1%) pts. KRAS and EGFR mutation status was available for 22pts. mPFS and mOS were numerically longer in pts with KRAS positive vs negative disease. While there was no difference in mPFS, 24-weeks PFS was 0 vs 16.7% (95% CI 4.1-36.5%) EGFR MUT vs wild type disease. Biomarker analysis of complement regulatory proteins, FcGR expression and polymorphisms, and changes in sC5b-9 is underway. Conclusions: GT103 was well tolerated across all dose levels. The RP2D of 10mg/kg IV every 3 weeks was selected, although PK modeling will be performed to optimize the dosing schedule. Encouraging anti-tumor activity was seen in heavily pretreated pts with advanced NSCLC with a subset of patients experiencing stable disease lasting for longer than 6 months. Additional biomarker analysis is ongoing. A phase II trial combining GT103 with pembrolizumab in pts with advanced NSCLC is currently enrolling. Clinical trial information: NCT04314089 .

IL2 Targeted to CD8+ T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity.

IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rβγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.

Abstract LB063: Anti-tumor activity in preclinical models utilizing a conditional bispecific redirected activation molecule (COBRA) targeting Her2

Abstract Among strategies to recruit cytotoxic T-cells into the tumor micro-environment (TME) are the use of T-cell engager molecules. One such therapeutic modality are molecules that recognize specific tumor associated antigens (TAA) but are activated preferentially in the TME. Conditional Bispecific Redirected Activation molecules (COBRA) are designed to be administered as a prodrug, and cleaved via matrix metalloproteinase to engage with both a tumor TAA and CD3 (Panachi, et.al. MAbs 2020 12(1) 1792130). In this study, we sought to determine the preclinical efficacy of a COBRA molecule targeting the Her2 receptor. Sub-cutaneous xenograft models (SCID mice using adoptive T-cell transfer) were utilized representing various levels of the TAA (HT-55, SNU-16, JIMT-1, and HCC827) to measure both efficacy and mechanism of action (T-cell extravasation). The COBRA molecule was dosed at 0.03, 0.1, 0.3, and 1 mg/kg using a Q3dx7 schedule. A non-cleavable COBRA molecule with the Her2 TAA was included as a control at 1 mg/kg. Efficacy was determined by caliper measurements of sub-cutaneous tumor size twice per week after dosing, and T-cell extravasation into the TME was measured by immunohistochemistry (IHC) at days 10, 19, and termination. IHC data was quantified using HALO imaging software. In our study, we measured dose-dependent tumor regression and/or stasis in all models tested. No anti-tumor activity was measured after dosing with the non-cleavable COBRA molecule. T-cell extravasation as measured by IHC and Halo analysis followed a dose and time dependent course. Comparison of Day 19 T-cell extravasation (one day after the last dose) showed a linear relationship to TAA expression. Our data indicate that treatment with a COBRA molecule in preclinical models with a Her2 TAA results in anti-tumor activity as measured by tumor stasis/regression, and exhibit the expected mechanism of action as evaluated by T-cell extravasation into the TME. Citation Format: Bradley Stringer, Johara Chouitar, Jacqualine Cerbone, Sarah Fortin, Alina Ainbinder, Cassie Kysilovsky, Brittany Scott, Antara Banerjee. Anti-tumor activity in preclinical models utilizing a conditional bispecific redirected activation molecule (COBRA) targeting Her2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB063.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract CT162: Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor

Abstract Background ZL-1218 is an anti-chemokine receptor 8 (CCR8) humanized IgG1 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. CCR8 demonstrates a preferential expression in tumor-associated Tregs over peripheral Tregs. Thus, targeting CCR8 may allow for specific depletion of intratumoral Tregs without impacting peripheral Tregs/other immune cells. ZL-1218 is intended to decrease the intratumoral Tregs by binding to both high- and low-expressing CCR8+ cells and eliciting potent ADCC activity. ZL-1218 blocked CCR8 ligand CCL1 from binding to CCR8, and reduced Treg recruitment by blocking chemotaxis of CCR8+ cells (unpublished data). Additionally, ZL-1218 demonstrated enhanced antitumor activity in preclinical models in combination with anti-PD-1. Methods This is a Phase I study of ZL-1218 as a single agent and in combination with pembrolizumab (P) to evaluate the safety, tolerability, pharmacokinetics/pharmacodynamics, and preliminary antitumor activity in subjects with advanced solid tumor malignancies. The study consists of Part 1 Dose Escalation and Part 2 Cohort Expansion. In Part 1, subjects will be treated with escalating doses of ZL-1218 alone and in combination with a fixed dose of P to identify the maximum tolerated dose (MTD) and/or the recommended expansion dose (RD). The Bayesian optimal interval (BOIN) design is used and adopted with overdose control. Upon progression in the single agent group, a subject may enroll into combination therapy of ZL-1218 with P. Subjects will start at their respective single agent dose or the cleared combination dose level, whichever is lower. Part 2 will explore the combination with pembrolizumab in subjects with selected tumor types, with or without prior CPI therapy. Major inclusion criteria include: (1) Subjects must have histologically confirmed and documented diagnosis of locally advanced unresectable/metastatic advanced solid tumor that is refractory/intolerant to standard treatment, or for which no standard treatment exists, and no greater than 3 prior lines of therapy in the relapse/refractory setting (2). Subjects must have at least one target lesion as defined by RECIST v1.1 on CT, PET/CT, or MRI scan. (3) Subjects must have a site of disease which is not previously irradiated and is safe and amenable to biopsy. Major exclusion criteria include: (1) Prior exposure to CCR8 inhibitor, or severe hypersensitivity (≥ Grade 3) to P and/or any of its excipients. (2) Subjects with symptomatic or uncontrolled brain metastasis requiring concurrent treatment, impaired major organ functions, active autoimmune disease, or active infections. Part 1 Dose Escalation is ongoing in the US and EU. Citation Format: Elena Garralda, Valentina Boni, David Vincente Baz, Arvind Chaudhry, Ignacio Gil Bazo, Martin Gutierrez, Jyoti Malhotra, Casal Guzman Alonso, Valentina Gambardella, Michael Chisamore, Renke Zhou, Maria Tea, Deborah Doroshow. Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT162.

Characterization of a novel anti-PVRIG antibody with Fc-competent function that exerts strong antitumor effects via NK activation in preclinical models

Poliovirus receptor-related immunoglobulin domain-containing protein, or PVRIG, is a newly discovered immune checkpoint that has emerged as a promising target for cancer immunotherapy. It is primarily expressed on activated T and natural killer (NK) cells, and once engaged with its ligand, PVRL2, it induces inhibitory signaling in T cells, thereby promoting the functional exhaustion of tumor-infiltrating lymphocytes (TILs). Here, we characterized IBI352g4a, a novel humanized anti-PVRIG antibody with Fc-competent function, explored the mechanism of its antitumor activity in preclinical models, and systemically evaluated the contribution of FcrR engagement to PVRIG blockade-induced antitumor activity. IBI352g4a binds to the extracellular domain of human PVRIG with high affinity (Kd = 0.53 nM) and specificity, and fully blocks the interaction between PVRIG and its ligand PVRL2. Unlike other immune checkpoints, IBI352g4a significantly induced NK cell activation and degranulation, but had a minimal effect on T-cell activation in in vitro functional assays. IBI352g4a induced strong antitumor effect in several preclinic models, through in vivo mechanism analysis we found that both NK and T cells contribute to the antitumor effect, but NK cells play predominant roles. Specifically, a single dose of IBI352g4a induced significant NK cell activation in TILs, but T-cell activation was observed only after the second dose. Moreover, the Fc effector function is critical for both NK cell activation and treatment efficacy in vitro and in vivo. Our study, for the first time, demonstrates that both NK activation and FcrR engagement are required for antitumor efficacy induced by PVRIG blockade.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models [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 6579.

Abstract 1656: ISM6331, a novel and potent pan-TEAD inhibitor, exhibits strong anti-tumor activity in preclinical models of Hippo pathway-dysregulated cancers

Abstract The Hippo-YAP/TAZ-TEAD signaling pathway plays critical roles in tumorigenesis and tumor progression. Genetic alterations of Hippo pathway regulators, such as NF2 or LATS1/2, have been shown to induce YAP/TAZ-TEAD transcriptional activity, promoting initiation and progression of many solid tumors. In addition, many studies have shown that YAP/TAZ-TEAD activation mediates resistance to multiple targeted therapies, particularly EGFR-KRas-MAPK pathway inhibitors. The TEAD family of transcription factors (TEAD1/2/3/4) share structural similarity and have overlapping functions in tumor development and TEAD auto-palmitoylation is critical for its interaction with YAP/TAZ and its transcriptional function. Therefore, blocking TEAD palmitoylation and allosterically disrupting YAP/TAZ-TEAD-mediated transcriptional regulation, is emerging as a promising approach for the treatment of tumors with Hippo pathway dysregulation. In this work, ISM6331, a novel and potent TEAD inhibitor with excellent inhibition against TEAD1/2/3/4 was identified leveraging an AI generative model. ISM6331 reversibly binds to the TEAD palmitoylation site and significantly suppresses TEAD transcriptional activity. ISM6331 potently and selectively inhibited the growth of many Hippo pathway-dysregulated tumor cells and dose-dependently suppressed the expression of TEAD target genes in vitro. In NF2-deficient or LATS1/2-mutant in vivo mesothelioma models, ISM6331 exhibited promising anti-tumor efficacy as monotherapy in a dose-dependent manner from 3 to 30mg/kg. ISM6331 also showed synergism with EGFR or KRasG12C inhibitors in inhibiting tumor cell growth and overcame drug resistance in in vitro and in vivo models. In addition, ISM6331 showed excellent pharmacokinetic properties in multiple preclinical species and was well-tolerated in GLP-toxicity studies. In summary, ISM6331 has promising potential to treat Hippo pathway-dysregulated tumors as monotherapy as well as in combination with targeted therapies. Citation Format: Qi Li, Jainfei Wan, Jinxin Liu, Jing Shang, Jiaojiao Yu, Wei Zhu, Celia X.-J. Chen, Junwen Qiao, Ling Wang, Man Zhang, Xiao Ding, Supriya Bavadekar, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM6331, a novel and potent pan-TEAD inhibitor, exhibits strong anti-tumor activity in preclinical models of Hippo pathway-dysregulated cancers [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 1656.

Abstract 20: KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications

Abstract Tumor Infiltrating Lymphocyte (TIL) therapy is an autologous adoptive cell therapy involving the isolation, ex vivo expansion and infusion of tumor infiltrating lymphocytes (TIL) into late-stage cancer patients for therapeutic benefit. While clinical responses to TIL therapy have been observed in metastatic melanoma, efficacy in other tumor types including non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC) [SM1] is limited by the intrinsic functionality and persistence of transferred T cells and the immunosuppressive tumor microenvironment. To improve the clinical outcome of TIL therapy, we have developed KSQ-001EX, an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene, for the treatment of solid tumors. SOCS1 is a negative regulator of cytokine signaling in T cells that we previously identified as a top target restraining T cell anti-tumor function and persistence using genome-wide in vivo CRISPR screens. To streamline and shorten TIL manufacture, we established a next-generation manufacturing process with resected tumors or core biopsies as starting materials that eliminates the use of feeder cells and REP, shortening the length of manufacture to 22 days. We demonstrated robust manufacture of cryopreserved KSQ-001EX at clinical scale from NSCLC, HNSCC, checkpoint refractory melanoma, and heavily pre-treated colorectal carcinoma (CRC). KSQ-001EX manufactured from clinical-scale runs were highly viable (>90%) following cryopreservation and thaw, with > 90% SOCS1 on-target editing and complete knockdown of SOCS1 at the protein level. Importantly, KSQ-001EX across all indications assessed showed high frequency of CD8 (median ~80%), an attribute associated with TIL clinical responses. KSQ-001EX also retained high diversity of TCR repertoire. Consistent with the biology of SOCS1 editing, KSQ-001EX exhibited greater cytotoxicity and higher IFNγ production against tumor spheroids in comparison to TIL and enhanced induction of pSTAT4 upon IL-12 stimulation, demonstrating heightened sensitivity of KSQ-001EX to cytokines. In conclusion, KSQ-001EX, an engineered TIL therapy with CRISPR/Cas9 mediated inactivation of SOCS1, can be manufactured at clinical doses from surgical resections or core biopsies within 22 days using a simplified feeder-free manufacturing process from multiple indications. KSQ-001EX exhibits heightened anti-tumor activity in preclinical models, with therapeutic benefit to be evaluated in a planned clinical trial in patients with treatment-refractory melanoma, NSCLC, and HNSCC. [SM1]Spell out these at first instance? Citation Format: Sharon Lin, Gustavo Martinez, Marie-Andrée Forget, Katrina Adlerz, Mitali Ghose, Leila Williams, Paul Dunbar, Frank Thompson, Fiona Sharp, Hugh Gannon, Conor Calnan, Ashish Yeri, Dipen Sangurdekar, Donald Sakellariou-Thompson, Samantha Fix, Tamara Griffith, Ellie Jafari, Jane Zulovich, Priya Balasubramanian, Erin Willert, MIcah Benson, Chantale Bernatchez, Karrie Ka Wai Wong. KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications [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 20.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [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 5988.

Abstract 1661: Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor

Abstract Background: Although therapeutic agents targeting alterations in the MAPK pathway have achieved great clinical success, the overall benefit is still suboptimal. Several studies have shown that reactivation of MAPK signaling is the main basis to compromise the efficacy. Thus, co-inhibition of ERK, the terminal master kinase of MAPK pathway, and the upstream targets may effectively shut down the MAPK signaling cascade and induce deeper and more durable anti-tumor activities. Herein, we present the preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT04908046) by HUTCHMED. Methods: The inhibition on ERK 1/2 kinase was determined using Z-LYTE™ kinase assay. The selectivity of HMPL-295 was evaluated against 394 kinases (KinaseProfilerTM) and 86 safety-related proteins (SafetyScreen87TM) by Eurofins. Cellular target inhibition on phosphorylation of RSK (p-RSK) was detected by ELISA. In vitro anti-proliferation activity of HMPL-295 as a single agent was measured by CCK-8 or CellTiter-Glo luminescent assay. Colony formation assay was used to evaluate the combination effect of HMPL-295 with targeted therapy. Multiple tumor models with RAS/RAF/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-295 as a single agent or in combination with either chemotherapy or targeted therapy. Results: HMPL-295 strongly inhibited ERK1 and ERK2 kinase activities with equal IC50 at 4 nM. In human colon cancer cell line COLO 205 harboring BRAFV600E mutation, HMPL-295 inhibited p-RSK, a direct substrate of ERK1/2, with an average IC50 of 180 nM from 3 independent experiments. With the inhibition of ERK and its downstream substrates, HMPL-295 treatment induced tumor cells arresting at G0/G1 phase via down-regulation of cyclin D1/p-RB signaling and consequently attenuated cell growth in a panel of MAPK pathway dysregulated tumor cell lines, but barely affected the growth of normal cells. Furthermore, HMPL-295 demonstrated favorable selectivity in the kinase and safety pharmacology profiling, indicating a low off-target risk. For in vivo studies, oral administration of HMPL-295 induced time- and dose-dependent inhibition of p-RSK and resulted in dose-dependent anti-tumor activity in several tumor models harboring KRAS or BRAF mutations. Moreover, HMPL-295 significantly improved the anti-tumor activity of standard-of-care chemotherapy as well as targeted agents in KRAS- or BRAF-mutant tumor models both in vitro and in vivo. Conclusion: HMPL-295 is a potent and selective ERK1/2 inhibitor which demonstrates strong anti-tumor activity in preclinical models, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Yuanzhi Lv, Wenjun Li, Hui Zhang, Xin Li, Ying Yu, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor [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 1661.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer [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 5909.

Anti-CD47 and Anti-PDL1 Immunotherapy Synergize for Anti-tumor Activity in Preclinical Models of Head and Neck Cancer

Anti-CD47 and Anti-PDL1 Immunotherapy Synergize for Anti-tumor Activity in Preclinical Models of Head and Neck Cancer

Phase I study of the Syk inhibitor sovleplenib in relapsed or refractory mature B-cell tumors.

Sovleplenib (HMPL-523) is a selective spleen tyrosine kinase (Syk) inhibitor with anti-tumor activity in preclinical models of B-cell malignancy. We conducted a dose-escalation and dose-expansion phase I study of sovleplenib in patients with relapsed/ refractory mature B-cell tumors. Dose escalation followed a 3+3 design; patients received oral sovleplenib (200-800 mg once daily [q.d.] or 200 mg twice daily [b.i.d.], 28-day cycles). During dose expansion, patients were enrolled into four cohorts per lymphoma classification and treated at the recommended phase II dose (RP2D) (clinicaltrials gov. Identifier: NCT02857998). Overall, 134 Chinese patients were enrolled (dose escalation, N=27; dose expansion, N=107). Five patients experienced dose-limiting toxicities: one each of amylase increased (200 mg q.d.), febrile neutropenia (800 mg q.d.), renal failure (800 mg q.d.), hyperuricemia and blood creatine phosphokinase increased (200 mg b.i.d.) and blood bilirubin increased and pneumonia (200 mg b.i.d.). RP2D was determined as 600 mg (>65 kg) or 400 mg (≤65 kg) q.d.. The primary efficacy end point of independent review committee-assessed objective response rate in indolent B-cell lymphoma was 50.8% (95% confidence interval: 37.5- 64.1) in 59 evaluable patients at RP2D (follicular lymphoma: 60.5%, marginal zone lymphoma: 28.6%, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, 0%). The most common (≥10% patients) grade ≥3 treatment-related adverse events in the dose-expansion phase were decreased neutrophil count (29.9%), pneumonia (12.1%) and decreased white blood cell count (11.2%). Pharmacokinetic exposures increased dose-proportionally with ascending dose levels from 200-800 mg, without observed saturation. Sovleplenib showed anti-tumor activity in relapsed/refractory B-cell lymphoma with acceptable safety. Further studies are warranted.