Preclinical Characterization Research Articles

Abstract PR006: EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic

Abstract Background: EG-70 (detalimogene voraplasmid) is a novel, investigational, non-integrating, non-viral gene therapy specifically engineered to elicit local stimulation of anti-tumor immune response in the bladder and to drive durable efficacy in patients with BCG-unresponsive high-risk non-muscle-invasive bladder cancer (NMIBC), while mitigating the risk of systemic toxicities from immune stimulation. Previous preclinical data indicate that intravesical instillation (IVI) of EG-70 results in local expression of transgene products in murine and non-human primate models. Here we describe the latest preclinical data to support the mechanism of action of EG-70 and Phase 1 clinical study results for EG-70 in the treatment of BCG-unresponsive NMIBC. Methods: Preclinical: A murine surrogate formulation of EG-70 (mEG-70) was administered locally to the bladder by IVI in C57BL/6 mice. Efficacy was assessed in a luciferase-expressing MB49 model of orthotopic bladder cancer. Following confirmation of cancer cell implantation by in vivo imaging, mice received two weekly IVIs of mEG-70. Immune profiling was evaluated by assessing protein expression levels and cell compositions (via MSD, flow cytometry, and immunohistochemistry). Immune cell depletion studies were conducted using specific antibodies directed against CD4 or CD8 antigen to assess T-cell contribution to efficacy. Clinical: In a Phase 1/2 study in high-risk BCG-unresponsive NMIBC with carcinoma in situ (CIS) (LEGEND; NCT04752722), EG-70 was assessed in four separate dose groups. Results: Preclinical: In a mouse model of orthotopic bladder cancer, IVI of mEG-70 caused striking remodeling of the tumor microenvironment from an immunosuppressive phenotype to a pro-inflammatory milieu supportive of tumor clearance. This was demonstrated by a significant decrease in myeloid cells and IL-4 cytokine levels, an increase in NK-cells and T-cells, and an increase in pro-inflammatory cytokines in tumor tissue. Accordingly, administration of mEG-70 was associated with marked reduction in tumor burden and significant improvement of survival. The anti-tumor response was largely immune-mediated as demonstrated by the critical role of T-cells, durable clearance of tumors, and protection against subsequent tumorigenic re-challenge, both locally in the bladder as well as distally, after flank implantation. Clinical: Results from the Phase 1 portion of the LEGEND study show that IVI of EG-70 was well-tolerated in patients with high-risk BCG-unresponsive NMIBC with CIS across all dose levels, with an overall complete response rate of 73%. Conclusions: The latest preclinical findings demonstrate that EG-70 has the potential to deliver immunostimulatory payloads locally to the bladder. The mechanism of action described preclinically has been translated into the clinic, in the Phase 1 portion of the LEGEND study in patients with BCG-unresponsive NMIBC with CIS. Citation Format: Marie-Line Goulet, Shauna M Dauphinee, Daniel Veilleux, Kristine S Louis, David Lazure, Sarah Stevenson, Darius Bilimoria, Fazmina Zamzameer, Ximin Chen, Sébastien Sublemontier, Sahar Amirkhani, Carlos Fleet, Richard Bryce, Matthew Bui, Shreyas Joshi, Susan J. Kalota, Anthony Cheung, James Sullivan. EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR006.

RE104: Synthesis and Activity of a Novel Serotonergic Psychedelic Prodrug of 4-Hydroxy-N,N-diisopropyltryptamine.

Results from randomized clinical trials of psilocybin in depressive disorders highlight the therapeutic potential of serotonergic psychedelic compounds in mental health disorders. The synthetic 5-hydroxytryptamine 2A receptor agonist 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DiPT) is structurally similar to psilocin but is reported to have a shorter duration (2-3 h) of psychedelic effects, suggesting the potential for psilocybin-like therapeutic activity with reduced clinical resource burden. Here, we describe the preclinical and translational characterization of RE104, a 4-OH-DiPT prodrug comprising a glutarate moiety designed to cleave rapidly in situ and thus provide reasonable bioavailability of the active drug. Plasma concentration of 4-HO-DiPT over time in PK experiments in rats was correlated with head-twitch intensity. The half-life of 4-OH-DiPT was 40 min after subcutaneous administration of RE104 in rats. In a forced swim test, a single dose of RE104 (1 mg/kg) significantly reduced mean immobility time at 1 week compared with vehicle (P < 0.001), confirming translational antidepressant potential. Taken together, these data with RE104 show that the glutarate ester can act as an efficient prodrug strategy for 4-HO-DiPT, a unique short-duration psychedelic with potential in depressive disorders.

Radiolabelling and preclinical characterisation of [89Zr]Zr-Df-ATG-101 bispecific to PD-L1/4–1BB

PurposeATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo.MethodsATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [89Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [89Zr]Zr-labelled antibodies. The specificity of [89Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies.ResultsThe Df-conjugation and [89Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [89Zr]Zr-Df-ATG-101 and [89Zr]Zr-Df-anti-PD-L1. Tumour uptake of [89Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo.Conclusion[89Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [89Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [89Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

Preclinical characterization of a novel investigational monoclonal antibody CM313 with potent CD38-positive cell killing activity.

CM313 is currently under clinical investigation for treatments of multiple myeloma, systemic lupus erythematosus, and immune thrombocytopenia. We aimed to report the preclinical profile of the novel therapeutic anti-CD38 monoclonal antibody (mAb) CM313, with an emphasis on the difference with other CD38-targeting mAb. The binding of CM313 to CD38 recombinant protein across species was assessed using ELISA. The binding of CM313 to CD38-positive (CD38+) cells was detected using flow cytometry assays. CM313-induced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis on different CD38+ cells were assessed by LDH release assays or flow cytometry assays. The effect of CM313 on CD38 enzymatic activity was measured using fluorescence spectroscopy. CM313 immunotoxicity in human blood was assessed using flow cytometry assays, ELISA, and LDH release assays. Anti-tumor activity of CM313 was assessed in multiple mouse xenograft models. Safety profile of CM313 were evaluated in cynomolgus monkeys and human CD38 transgenic (B-hCD38) mice. There exist unique sequences at complementarity-determining regions (CDR) of CM313, which facilitates its affinity to CD38 is consistently higher across a spectrum of CD38+ cell lines than daratumumab. In vitro studies showed that CM313 induces comparable killing activity than daratumumab, including ADCC, CDC, ADCP, apoptosis induced by Fc-mediated cross-linking, and effectively inhibited the enzymatic activity of CD38. However, CM313 showed more potent CDC than isatuximab. In vivo, CM313 dose-dependently inhibited xenograft tumor growth, both as a monotherapy and in combination with dexamethasone or lenalidomide. Furthermore, CM313 was well tolerated with no drug-related clinical signs or off-target risks, as evidenced by 4-week repeat-dose toxicology studies in cynomolgus monkeys and B-hCD38 mice, with the later study showing no observed adverse effect level (NOAEL) of 300mg/kg once weekly. CM313 is a novel investigational humanized mAb with a distinct CDR sequence, showing comparable killing effects with daratumumab and stronger CDC activity than isatuximab, which supports its clinical development.

The bispecific B7H3xCD3 antibody CC-3 induces T cell immunity against bone and soft tissue sarcomas.

Sarcomas are rare and heterogeneous malignancies that are difficult to treat. Approximately 50% of patients diagnosed with sarcoma develop metastatic disease with so far very limited treatment options. The transmembrane protein B7-H3 reportedly is expressed in various malignancies, including different sarcoma subtypes. In several cancer entities B7-H3 expression is associated with poor prognosis. In turn, B7-H3 is considered a promising target for immunotherapeutic approaches. We here report on the preclinical characterization of a B7-H3xCD3 bispecific antibody in an IgG-based format, termed CC-3, for treatment of different sarcoma subtypes. We found B7-H3 to be expressed on all sarcoma cells tested and expression on sarcoma patients correlated with decreased progression-free and overall survival. CC-3 was found to elicit robust T cell responses against multiple sarcoma subtypes, resulting in significant activation, release of cytokines and effector molecules. In addition, CC-3 promoted T cell proliferation and differentiation, resulting in the generation of memory T cell subsets. Finally, CC-3 induced potent target cell lysis in a target cell restricted manner. Based on these results, a clinical trial evaluating CC-3 in soft tissue sarcoma is currently in preparation.

Further preclinical characterization of molnupiravir against SARS-CoV-2: Antiviral activity determinants and viral genome alteration patterns

The SARS-CoV-2 pandemic has highlighted the need for broad-spectrum antiviral drugs to respond promptly to viral emergence. We conducted a preclinical study of molnupiravir (MOV) against SARS-CoV-2 to fully characterise its antiviral properties and mode of action. The antiviral activity of different concentrations of MOV was evaluated ex vivo on human airway epithelium (HAE) and in vivo in a hamster model at three escalating doses (150, 300 and 400 mg/kg/day) according to three different regimens (preventive, pre-emptive and curative). We assessed viral loads and infectious titres at the apical pole of HAE and in hamster lungs, and MOV trough concentration in plasma and lungs. To explore the mode of action of the MOV, the entire genomes of the collected viruses were deep-sequenced. MOV effectively reduced viral titres in HAE and in the lungs of treated animals. Early treatment after infection was a key factor in efficacy, probably associated with high lung concentrations of MOV, suggesting good accumulation in the lung. MOV induced genomic alteration in viral genomes with an increase in the number of minority variants, and predominant G to A transitions. The observed reduction in viral replication and its mechanism of action leading to lethal mutagenesis, supported by clinical trials showing antiviral action in humans, provide a convincing basis for further research as an additional means in the fight against COVID-19 and other RNA viruses.

Advances in preclinical TCR characterization: leveraging cell avidity to identify functional TCRs.

T-cell therapy has emerged as an effective approach for treating viral infections and cancers. However, a significant challenge is the selection of T-cell receptors (TCRs) that exhibit the desired functionality. Conventionally invitro techniques, such as peptide sensitivity measurements and cytotoxicity assays, provide valuable insights into TCR potency but are labor-intensive. In contrast, measuring ligand binding properties (z-Movi technology) could provide an accelerated processing while showing robust correlations with T-cell functions. In this study, we assessed whether cell avidity can predict functionality also in the context of TCR-engineered Tcells. To this end, we developed a flexible system for TCR re-expression by generating a Jurkat-derived Tcell clone lacking TCR and CD3 expression through CRISPR-Cas9-mediated TRBC knockout. The knockin of a transgenic TCR into the TRAC locus restored TCR/CD3 expression, allowing for CD3-based purification of TCR-engineered Tcells. Subsequently, we characterized these engineered cell lines by functional readouts, and assessment of binding properties through the z-Movi technology. Our findings revealed a strong correlation between the cell avidities and functional sensitivities of Jurkat TCR-T cells. Altogether, by integrating cell avidity measurements with our versatile Tcell engineering platform, we established an accelerated system for enhancing the invitro selection of clinically relevant TCRs.

Abstract LB438: Preclinical characterization of ONM-412, an ultra-pH sensitive nanoparticle encapsulated IL-12 fusion protein

Abstract Background: Interleukin-12 is a potent pleiotropic cytokine, but its clinical translation has been hindered by toxicities. To deliver IL-12 to tumors with high spatial and temporal precision while minimizing off-tumor effects, we have developed an ultra-pH sensitive nanoparticle platform - ON-BOARD - that shields payloads from systemic exposure and targets solid tumors by responding to tumor acidity. Herein, we report the preclinical efficacy and safety characterization of ONM-412, an ON-BOARD nanoparticle encapsulated IL-12Fc fusion protein, and muONM-412, an encapsulated murine IL-12Fc. Methods: Properties and stability profiles of ONM-412 were characterized. pH-specific bioactivity was determined in cell-based reporter and IFNγ induction assays while the activation of ONM-412 was compared to a protease-cleavable IL-12 prodrug. Efficacy and tolerability of muONM-412 were studied in vivo after intravenous injection in mice. Pharmacodynamic response was evaluated by measuring cytokine levels in plasma and tissue, and the changes in tumor immune microenvironment were characterized. Toxicity was measured by body weight loss, systemic cytokine levels and clinical chemistry. Anti-tumor efficacy of muONM-412 was evaluated in MC38 tumor-bearing mice. The safety and tolerability of ONM-412 is also undergoing evaluation in a toxicology study with cynomolgus monkeys. Body weight, hematology, clinical chemistry and urine analysis were performed. Results: ONM-412 showed high encapsulation efficiency (&amp;gt;85%) in uniformly distributed particles (Dh&amp;lt;50nm) with 6-month on-going shelf-life stability. pH-specific release was confirmed in vitro with a &amp;gt;100-fold activation window between the acid-activated and intact formulations by an HEK reporter assay and an IFNγ induction assay. ONM-412 showed rapid and complete recovery of IL-12 bioactivity in &amp;lt;10 minutes after acid-activation while MMP demasking of a prodrug required overnight incubation and compromised potency. In vivo, mice treated with muONM-412 demonstrated significantly improved tolerability compared to unencapsulated IL-12Fc: body weight loss was reduced, no liver toxicity was observed, and plasma IFNγ levels were &amp;gt;1,000-fold lower. muONM-412 induced tumor immune remodeling, with increased infiltration by IFNγ+ and GzmB+ CD8 T and NK cells, and demonstrated strong dose-responsive anti-tumor efficacy in MC38 tumor-bearing animals. Preliminary results from an on-going study in cynomolgus monkeys suggest ONM-412 is well-tolerated at equivalent doses to those that induced maximum antitumor response in mice. Conclusions: ONM-412 significantly improved tolerability over unencapsulated IL-12Fc and showed potent anti-tumor efficacy in mice. It was well-tolerated in mice and non-human primates, showing potential for clinical translation. Citation Format: Qingtai Su, Stephen Gutowski, Austin Burcham, Bhargavi Allu, Zirong Chen, Fiona Stavros, Ruolan Han, Jason B. Miller, Tian Zhao. Preclinical characterization of ONM-412, an ultra-pH sensitive nanoparticle encapsulated IL-12 fusion protein [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 LB438.

Abstract ND09: Actinium-225 -PSMA-Trillium (BAY 3563254): Preclinical evaluation and clinical imaging study of a novel 225Ac-labeled PSMA-targeting small molecule triad for the treatment of mCRPC

Abstract With the approval of a PSMA-targeting small molecule labeled with the beta-emitter lutetium-177, PSMA is considered a clinically validated target for the treatment of mCRPC. Although effective, many patients treated with this therapeutic experience only transient clinical benefit, and G1/2 xerostomia is common due to salivary gland toxicity. Since the linear energy transfer and degree of induced DNA damage of alpha particles far exceeds that of beta particles, several investigational PSMA-targeting small molecules labeled with the alpha-emitter 225Ac have generated encouraging preliminary efficacy signals, but with severe xerostomia as a common dose-limiting toxicity. 225Ac-PSMA-Trillium is a novel PSMA-targeting radioligand consisting of a triad of important features: (1) a high-affinity PSMA inhibitor for specific tumor-targeting; (2) a customized albumin-binding domain designed to prolong plasma residence time and increase the therapeutic index by improving tumor uptake while reducing salivary gland uptake; and (3) a “macropa” chelator for efficient and stable radiolabeling with actinium-225. A series of compounds containing the same high-affinity PSMA-targeting moiety, but different chain lengths and albumin-binding domains, were evaluated to identify the optimal combination of features required to achieve the best therapeutic index. In vitro preclinical characterization was performed to test affinities for PSMA and albumin, compound stability and cell-killing potential in LNCaP and 22Rv1 CDX models as well as KuCaP-1 PDX model. Preclinical in vivo investigation of selected compounds showed increased plasma residence time, reduced uptake in normal organs, strong uptake and retention in PSMA-expressing tumors and robust anti-tumor activity. These analyses led to the selection of compounds for further evaluation in a Phase 0 imaging and PK study in mCRPC patients (NCT05773703). In this Phase 0 study, PSMA-Trillium compounds were labeled with indium-111 via a DOTA chelator and used as surrogates to facilitate longitudinal imaging. In patients, 111In-PSMA-Trillium demonstrated targeting to PSMA-expressing tumors as defined by PET/PSMA scans and a plasma residence time consistent with albumin-binding affinities. Importantly, 111In-PSMA-Trillium showed robust uptake and prolonged retention in tumors, and reduced uptake and relatively fast washout from salivary glands. Based on these preclinical data and clinical imaging results, a therapeutic first-in-human trial to evaluate 225Ac-PSMA-Trillium in patients with prostate cancer is anticipated to begin enrolling patients in the first half of 2024. Citation Format: Sabine Zitzmann-Kolbe, Jacob Hesterman, Patricia E. Cole, Nicolas Salem, Alex Papple, Michael Jeffers, Goekben Koca, Thorsten Poethko, Miroslav Dostalek, Shawn Hillier, John W. Babich. Actinium-225 -PSMA-Trillium (BAY 3563254): Preclinical evaluation and clinical imaging study of a novel 225Ac-labeled PSMA-targeting small molecule triad for the treatment of mCRPC [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 ND09.

Abstract LB057: Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid tumors

Abstract Claudin18.2 is important for the formation and function of tight junctions between cells in epithelial tissues. Normally expressed only in gastric mucosa, Claudin18.2 is overexpressed in multiple types of solid tumors, including gastric, pancreatic, breast and colon cancers. Despite being a well-established tumor-associated antigen, the clinical value and feasibility of targeting Claudin18.2 by various modalities are yet-to-be fully demonstrated. IBI343 is a fully humanized anti-Claudin18.2 monoclonal antibody conjugated to Exatecan via site-specific glycol-conjugation with an average drug-to-antibody ratio (DAR) of 3.6. IBI343 showed robust antigen-specific in vitro cytotoxicity in cancer cell lines with varying levels of Claudin18.2 expression. In addition, due to the cleavable linker design and high hydrophilicity of the payload, IBI343 demonstrated potent bystander killing effects in vitro. The glycan-based conjugation technology leads to enhanced stability of the conjugated linker-payload and the entire ADC molecule, which results in superior pharmacokinetics. IBI343 showed potent in vivo anti-tumor efficacy with complete tumor regressions in multiple xenograft models. Furthermore, it demonstrated strong in vitro and in vivo synergistic effects with various anti-cancer agents, including immune checkpoint blockers and DNA damage repair pathway inhibitors. IBI343 displayed good safety profile in monkey GLP toxicology study (HNSTD = 30 mg/kg). These pre-clinical findings show that IBI343 is a promising anti-cancer agent with large therapeutic window, and support clinical exploration of IBI343 in patients with Claudin18.2-expressing tumors. A companion late-breaking abstract of clinical phase 1 study results (NCT05458219) will also be presented in AACR 2024. Citation Format: Shuaixiang Zhou, Xiong Yao, Jian Guan, Keke Fei, Jia Lu, Weiwei Wu, Yang Liu, Tianyu Zhu, Zhuangguang Liao, Shi Chen, Bingliang Chen, Kaijie He. Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid tumors [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 LB057.

Abstract LB165: GFH547: An orally bioavailable, cyclophilin A-hijacking panRAS(on) inhibitor with broad spectrum anti-tumor activities

Abstract KRAS has recently become druggable with the approval of two KRAS G12C inhibitors - sotorasib and adagrasib. However, current SIIP-based KRAS inhibitors preferentially target the inactive, GDP-bound KRAS and thus are susceptible to adaptive resistance due to MAPK pathway reactivation. Furthermore, RTK pathway alterations frequently emerge to cause acquired resistance. These drawbacks limit the rate and duration of anti-tumor response. Developing more robust small molecule RAS (on) inhibitors that target active, GTP-bound RAS proteins is a promising new approach to enhance clinical benefits. Here we report preclinical characterization of a novel RAS (on) inhibitor - GFH547, designed to treat cancers carrying multiple RAS mutations. In biochemical assays, GFH547 disrupted the binding of GMPPNP-bound G12V, G12D or wild type KRAS to RAF1 in a CypA-dependent manner, as the inhibitory effect was significantly abolished in the absence of CypA. GFH547 suppressed phosphor-ERK1/2 (p-ERK) levels in KRAS G12V- and G12D-mutated cells with IC50s &amp;lt; 1 nM, and demonstrated potent tumor-suppressive effect in 29 cancer cell lines carrying diverse KRAS (G12A, G12C, G12V, G12R, G12S or G13D), NRAS (G12D or Q61L) or HRAS (Q61H) mutations, with IC50s of 0.04-1.9 nM. In addition, GFH547 was also cytotoxic in 9 cancer cell lines carrying EGFR or FGFR2/3 amplification, mutation or fusion, with IC50s of 0.12-198 nM. Conversely, GFH547 was inactive against 4 BRAF V600E-mutated, RAS-independent cancer cell lines. In KRAS-mutated xenograft models, oral administration of GFH547 elicited rapid and potent inhibition of intratumoral p-ERK levels, and led to tumor regression at dose levels as low as 1 mg/kg QD. In contrast to sotorasib and adagrasib, GFH547 was not susceptible to RTK-reactivation following EGF stimulation, and remained effective against cell lines carrying KRAS mutations that confer acquired resistance to SIIP-based KRAS inhibitors. At 10 μM, GFH547 showed no off-target activity in 72-kinase selectivity and SafetyScreen44 panels. GFH547 has a bioavailability of 76% and was well tolerated in mice, with an estimated therapeutic index &amp;gt;20. Taken together, by hijacking CypA to block oncogenic RAS proteins broadly in their active forms, GFH547 represents a new generation of RAS inhibitors that are superior to the mainstream SIIP-based KRAS inhibitors in overcoming adaptive and acquired resistance. The compound demonstrated strong therapeutic activities in tumors with activating RAS and RTK mutations/alterations. Based on these promising results, we are advancing a CypA-binding panRAS(on) inhibitor into the clinic as targeted therapy for cancers addicted to RAS pathways. Citation Format: Feng Yan, Jichen Zhao, Tao Liang, Tao Jiang, Chonglan Lin, Ling Peng, Kai Ma, Huabin Yang, Wan He, Zhen Li, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaohui Zhang, Yonghong Ni, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Xiaoling Lan, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH547: An orally bioavailable, cyclophilin A-hijacking panRAS(on) inhibitor with broad spectrum anti-tumor activities [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 LB165.

Abstract LB163: Discovery and characterization of QTX3544, a potent, selective, and orally bioavailable allosteric G12V preferring multi KRAS inhibitor

Abstract Oncogenic KRAS mutations occur in approximately 25% of all human cancers with KRAS G12V being one of the most frequently occurring variants, particularly in pancreatic, colorectal, and lung cancers. These oncogenic mutations cause sustained activation of the mitogen activated protein kinase (MAPK) pathway, which ultimately drives tumorigenesis. While newly approved covalent inhibitors to KRASG12C have demonstrated clinical benefit to patients harboring this mutation, there are no approved drugs targeting mutations beyond KRASG12C, highlighting a significant clinical need. Here we show that QTX3544 is a potent and orally bioavailable KRASG12V preferring multi KRAS inhibitor. QTX3544 exhibited picomolar binding affinity (0.08 nM) and SOS mediated nucleotide inhibition (0.07 nM) to the inactive form of KRASG12V. Furthermore, QTX3544 inhibited “on state” KRAS activity in both biochemical and cellular assays. In vitro, QTX3544 significantly inhibited downstream ERK phosphorylation and cell proliferation in KRASG12V driven cancer cell lines. Similar inhibitory effects were observed across additional mutant KRAS cancer cell lines, including KRAS G12A, KRAS G13D , and KRAS Q61H, among others. Notably, QTX3544 displayed no impact against HRAS, NRAS or BRAF mutant cells. In vivo, oral administration of QTX3544 achieved sustained systemic exposure required for efficacy, indicating potential durable inhibition of KRAS signaling. QTX3544 demonstrated dose dependent anti tumor efficacy and significant tumor regression in various KRASG12V driven xenograft models when administered orally twice daily (BID). The physiochemical profile of QTX3544 is favorable with good measured LogD, solubility, and permeability. QTX3544 exhibits weak Cytochrome P450 inhibition (direct and time dependent) with a favorable in vitro off target selectivity/safety profile. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3544 was generally well tolerated in rat and dog repeat dose tolerability studies. In addition, QTX3544 showed good brain penetration in mice. Taken together, this preclinical characterization supports the advancement of QTX3544 into IND enabling studies for KRAS G12V mutant cancers. Citation Format: Yang W. Zhang, Elizabeth Donohue Vo, Pei Gan, Dave Rominger, Jillian M. Silva, Yang J. Zhang, Christopher Pan, Greg Lee, John M. Micozzi, Ben Reid, Brooke McDonough, Arghyotri Sinha, Audrey Hospital, Doug Krauth, Juan I. Luengo, Mike Kramer, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3544, a potent, selective, and orally bioavailable allosteric G12V preferring multi KRAS inhibitor [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 LB163.

Preclinical characterization of Pan-NKG2D ligand-binding NKG2D receptor decoys

NKG2D and its ligands are critical regulators of protective immune responses controlling infections and cancer, defining a crucial immune signaling axis. Current therapeutic efforts targeting this axis almost exclusively aim at enhancing NKG2D-mediated effector functions. However, this axis can drive disease processes when dysregulated, in particular, driving stem-like cancer cell reprogramming and tumorigenesis through receptor/ligand self-stimulation on tumor cells. Despite complexities with its structure and biology, we developed multiple novel engineered proteins that functionally serve as axis-blocking NKG2D “decoys” and report biochemical, structural, in vitro, and in vivo evaluation of their functionality.

Abstract 3631: Preclinical and clinical pharmacodynamic characterization of BBI-355, a novel, orally bioavailable, and selective CHK1 inhibitor being evaluated in the first-in-human Phase 1/2 POTENTIATE clinical trial of patients with cancer harboring oncogene amplifications

Abstract Pharmacodynamic (PD) biomarkers play an integral role in the rational development of novel therapeutic agents. Critically, PD biomarkers enable the measurement and confirmation of a drug’s intended effect in early clinical trials. Integration of PD biomarker assessments in early therapeutic development can inform the relationship between drug dose and regimen, target modulation, and biological tumor response, which are fundamental to determining the efficacy and safety of novel therapeutic agents. Boundless Bio is evaluating a novel, orally available, and selective Checkpoint Kinase 1 (CHK1) inhibitor, BBI-355, in the Phase 1/2 clinical trial, POTENTIATE (NCT05827614), in patients with solid tumors harboring oncogene amplification including those on extrachromosomal DNA (ecDNA). ecDNA-enabled oncogene amplification endows cancers with significant genomic plasticity, which can facilitate resistance to targeted therapy (Nathanson et al., 2014), and which is also associated with significantly shorter survival (Kim et al., 2020). While ecDNA provides a fitness advantage to cancer cells, its unique features also confer certain liabilities, including elevated DNA replication stress (RS). Elevated RS results in DNA damage and makes ecDNA+ cancers hyper-reliant on the master regulator of RS, CHK1, which manages DNA replication and repair to maintain cell viability (Chowdhry, 2022). Due to their elevated RS and hyper-reliance on CHK1, ecDNA+ cancer cells are rendered more susceptible to CHK1 inhibition than cancer cells that lack ecDNA. Prior clinical CHK1 inhibitor programs have lacked effective clinical PD biomarker assays. Here, we present preclinical data supporting the use of phosphorylated-CHK1 Ser345 (pCHK1-S345), a proximal marker of RS, as a PD biomarker for CHK1 inhibition by BBI-355. In preclinical studies, BBI-355 treatment induced pCHK1-S345 protein expression by immunoblotting in tumor cell lines in vitro and in xenograft tumors in vivo, which corresponded with tumor cell cytotoxicity. We went on to demonstrate in rats the utility of skin as a tumor surrogate tissue to detect CHK1 inhibitor PD effect through induction of pCHK1-S345 expression by immunohistochemistry. Finally, and for the first time, PD activity of a CHK1 inhibitor in humans was demonstrated by increased pCHK1-S345 expression by immunohistochemistry in skin biopsies from patients enrolled on the POTENTIATE study and treated with BBI-355. In conclusion, pCHK1-S345 is a useful PD biomarker for confirming clinical on-target activity of BBI-355 and may be useful to inform the pharmacologically active range of BBI-355 to support clinical development. Citation Format: Debbie Liao, Ryan Hansen, Sudhir Chowdhry, Evan Holmes, Julie Wiese, Auzon Steffy, AnneMarie Pferdekamper, Edison Tse, Salvador Garcia, Rebecca Reynolds, Sara Weymer, Klaus Wagner, Chris Hassig, Shailaja Kasibhatla. Preclinical and clinical pharmacodynamic characterization of BBI-355, a novel, orally bioavailable, and selective CHK1 inhibitor being evaluated in the first-in-human Phase 1/2 POTENTIATE clinical trial of patients with cancer harboring oncogene amplifications [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 3631.

Abstract 2727: Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for multiple myeloma

Abstract Multiple myeloma (MM), characterized by clonal accumulation of malignant plasma cells in the bone marrow, is the second most prevalent hematopoietic malignancy. Despite emerging therapies, MM remains an incurable disease with a 5-year overall survival of 56% for relapsed and/or refractory disease (RRMM) patients. Thus, innovative approaches that would be safe and potent are required. GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D) is an orphan GPCR protein involved in keratin synthesis with limited expression in healthy tissues. GPRC5D is expressed at very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care agents such as anti-CD38, or BCMA-targeting therapies. The ability of NK cells to kill tumor cells leaving other cells unharmed and to induce minimal pro-inflammatory cytokine release as compared to T cell-based therapies, makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. Like Sarclisa®, other therapies engaging NK cells and mediating tumor cell killing through antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism have demonstrated their efficacy in clinic. Here we report the design, preclinical characterization, and development of a novel anti-GPRC5D antibody inducing potent cytotoxic activity against MM cells. SAR446523 is an IgG1-based antibody with an engineered Fc domain for enhanced ADCC function and targeting human GPRC5D on tumoral plasma cells. SAR446523 demonstrated high affinity for GPRC5D target and increased affinity for both high and low affinity FCGR3A-158V and 158F variants of CD16a receptor. The ability of SAR446523 to induce ADCC in vitro was assessed in a panel of MM cell lines exhibiting low, medium, and high GPRC5D expression. SAR446523 induced strong potency against all tested cell lines (picomolar range), regardless of the levels of GPRC5D. In addition, SAR446523 induced higher cytotoxic activity compared to a clinical benchmark compound. This anti-tumor activity was associated with very low IL-6, TNFa and IFNg pro-inflammatory cytokine release, much lower compared to a CD3xGPRC5D T-cell engager in in vitro assay using human peripheral blood mononuclear cells (PBMC) in presence of GPRC5D positive cells. In addition, the in vivo anti-tumor activity of SAR446523 was investigated in a NK humanized NOG huIL15 transgenic mouse model bearing disseminated MM cells. SAR446523 led to a robust antitumoral efficacy with a significant improved mouse survival. These data demonstrating potent anti-tumor activity and favorable safety profile of this novel anti-GPRC5D antibody suggest that SAR446523 may be a promising therapy for patients with RRMM and provide consistent support for clinical development. Citation Format: Helene Bonnevaux, Elisabeth Remy, Theo Pizette, Sophie Bouvier, Klervi Desrumeaux, Laurent Bassinet, Celine Nicolazzi, Pauline Rettman, Zandra Klippel, Marco Meloni, Marielle Chiron, Angela Virone-Oddos. Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for multiple myeloma [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 2727.

Abstract 3123: Preclinical characterization of AMB302/GQ1011, a first-in-class FGFR3 antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor against diverse FGFR3 overexpression or alteration

Abstract Background: Molecular alterations of oncogene FGFR3 in various cancers include FGFR3 gene fusions, activating mutations, and overexpression as a driver mutation. AMB302/GQ1011 is a novel FGFR3-targeting ADC conjugated with an innovative topoisomerase I inhibitor (TopoIx) using intelligent Ligase-Dependent Conjugation (iLDC) technologies developed by GeneQuantum (GQ), providing high homogeneity, excellent druggability, and superior linker stability. Based on preclinical characterization, AMB302/GQ1011 exhibits impressive anti-tumor activities against bladder cancer (BC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma (GBM) with either FGFR3 overexpression or FGFR3 alterations including gene fusions, demonstrating potential as a first-in-class FGFR3 ADC against FGFR3 relevant solid tumor indications. Methods: For biomarker analysis, patient tissue microarrays and tissue blocks were subjected to IHC staining for FGFR3 to assess its real-world intensity. To characterize AMB302/GQ1011, in vitro assays and ex vivo plasma stability were evaluated to confirm the anti-tumor effect and linker stability. In vivo anti-tumor effects of AMB302/GQ1011 were assessed on various CDX and PDX models with FGFR3 overexpression or alterations. The potential toxicity profile of AMB302/GQ1011 was evaluated in NHP via repeated intravenous infusions. Results: AMB302/GQ1011 conjugates a novel FGFR3-targeting antibody (Aimedbio Inc.) with TopoIx (GQ) via a cleavable linker through iLDC platform. In IHC analysis, we detected high and widespread FGFR3 expression in various tumors. In in vitro cytotoxicity assay, AMB302/GQ1011 showed excellent antitumor activity against FGFR3-expressing cells. In various CDX and PDX models with FGFR3 expression or alterations, AMB302/GQ1011 demonstrated superior efficacy compared with benchmark ADCs and FGFR pan-inhibitors. AMB302/GQ1011 dramatically prolonged survival in GBM orthotopic PDX models with F3-T3 fusion by &amp;gt;150% in combination with TMZ. Particularly, in combination with an immune checkpoint inhibitor (ICI), AMB302/GQ1011 exhibited an excellent synergistic antitumor effect. In a monkey toxicity study, AMB302/GQ1011 demonstrated a high tolerability up to 60 mg/kg dosing, without severe adverse effects. Conclusion: AMB302/GQ1011 demonstrated a potent antitumor efficacy in vitro and in vivo models with FGFR3 overexpression and alterations across various solid tumors. Notably, AMB302/GQ1011 suggests the potential for combination with ICI treatment. Moreover, AMB302/GQ1011 exhibited excellent tolerability at high doses in NHP, indicating its safety profile. Our data suggest that AMB302/GQ1011 has potential to become a first-in-class FGFR3-targeting ADC for solid tumors with FGFR3 overexpression or alterations. Citation Format: Byeongkwi Min, Sunghyun Lee, Wonsik Jung, Lina Wang, Yajun Sun, Hyejin Kim, Nam-Gu Her, Paul H. Song, Gang Qin, Do-Hyun Nam. Preclinical characterization of AMB302/GQ1011, a first-in-class FGFR3 antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor against diverse FGFR3 overexpression or alteration [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 3123.

Abstract 1364: Characterization of CHS-1000, an Fc-modified anti-ILT4 monoclonal antibody for reprogramming suppressive myeloid cells in solid tumors

Abstract Tumor resident immunosuppressive myeloid cells play a significant role in inducing resistance to cancer immunotherapy and are associated with poor prognosis in several solid tumors. The leukocyte immunoglobulin-like receptor B2 (LILRB2), also known as ILT4, is primarily expressed on myeloid cells and is a negative regulator of myeloid cell activation. ILT4 and its primary ligands (HLA-G, HLA-A) are widely expressed in various solid tumors and are associated with disease progression, tumor metastasis and poor clinical outcome. Abrogating inhibitory signals mediated through ILT4 and its ligands is a promising strategy to reprogram suppressive myeloid cells and enhance efficacy of immune checkpoint therapies. Here we report IND-enabling preclinical characterization of CHS-1000, a novel recombinant humanized Fc-modified IgG1 antibody with attenuated Fc-gamma receptor (FcγR) binding, that specifically targets immunoglobulin-like transcript 4 (ILT4). CHS-1000 selectively binds to human ILT4 with high affinity and potently blocks ILT4 interactions with its primary ligands (HLA-A, HLA-G). Functionally, CHS-1000 promotes re-polarization of suppressive myeloid cells to a pro-inflammatory phenotype and enhances activation of dendritic cells and T cells. Combinations of CHS-1000 with PD-1 inhibitors or other immunotherapy agents hold particular promise for enhanced anti-tumor activity. These preclinical characterization data support clinical development of CHS-1000 in combination with toripalimab (a commercial stage anti-PD-1 antibody) as an immunotherapy treatment for patients with solid tumors. Citation Format: Narendiran Rajasekaran, Xiaoguang Wang, Scott Klakamp, Sruthi Ravindranathan, Suzanna Tatarewicz, Julia Du, Brian Nguyen, Kate Widmann, Nessa Hawkins, Daniel J. Chin, Theresa LaVallee, Varun Kapoor. Characterization of CHS-1000, an Fc-modified anti-ILT4 monoclonal antibody for reprogramming suppressive myeloid cells in solid tumors [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 1364.

Abstract 2050: Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC

Abstract SLITRK6 is a type I transmembrane member of the SLITRK family of proteins and is highly expressed in certain epithelial tumors including urothelial, lung, breast cancer and glioblastoma, with minimal-to-low expression in normal tissues. Recent studies with a vedotin-based SLITRK6-directed ADC, AGS15E, have offered clinical proof-of-concept of harnessing this target with the ADC modality for addressing high unmet need at least in bladder cancer. PRO1106 is comprised of a proprietary humanized SLITRK6-targeting IgG1 (dubbed as “mAb”) and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1106 and the parent mAb exhibited strong binding (with low nanomolar affinity) to recombinant human or cynomolgus monkey SLITRK6 on bio-layer interferometry. No binding was observed with human SLITRK1 or SLITRK4 or in additional negative-binding assays. Potent cellular binding (with sub-nanomolar EC50) was also observed for the parent mAb and ADC with bladder, head and neck, and brain cancer cell lines as well as SLITRK6-overexpressing 293F cells. Target-binding was superior to sirtratumab (the parent antibody in AGS15E) and the binding epitope appeared to be distinct from that of sirtratumab in cross-block studies. PRO1106 displayed rapid internalization and elicited robust cytotoxicity (IC50 in the sub-nanomolar to low nanomolar range) in SLITRK6-expressing tumor cells but not target-negative cells. PRO1106 produced strong tumor growth inhibition in multiple cell-derived xenograft (CDX) models in mice representing bladder cancer and esophageal squamous cell carcinoma. PRO1106 showed a stable PK with much higher exposure compared to an in-house synthesized analog of AGS15E in rats. In an exploratory PK study in cynomolgus monkeys, PRO1106 at 5 mg/kg exhibited extended PK with minimal release of exatecan in circulation and with no apparent toxicity observed. In summary, PRO1106 is an ADC built on a clinically validated linker-drug directed at a clinically validated target, with promising data in preclinical pharmacology studies. As SLITRK6 expression in skin is negligible, PRO1106 may offer an exciting new option for patients with bladder cancer where the current standard-of-care ADC therapy (enfortumab vedotin) confers significant skin-related toxicity burden as well as for patients with additional cancer types. Citation Format: Wanwan Shen, Haotian Lu, Yang Xiao, Xuan Qiu, Haidong Liu, Lei Wang, Zhu Chen. Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC [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 2050.

Abstract 4592: Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor

Abstract Background: PRMT5 inhibitors are considered as a promising approach for cancer therapy due to their crucial role in the initiation and progression of many types of malignancies. Nevertheless, the crucial involvement of PRMT5 in controlling hematopoiesis, even in normal cells, resulted in significant on-target-off-tumor hematological toxicity when using first-generation, non-selective PRMT5 inhibitors in clinical settings, which hampered the clinical development of these drugs. The MTAP gene is deleted in around 10-15% of all human malignancies. As a result, the loss of MTAP leads to the buildup of MTA, which can bind to PRMT5 and partially inhibit PRMT5 activity. Inhibitors targeting the PRMT5-MTA complex become a promising strategy for treating MTAPnull cancer in a synthetically lethal manner. Herein, we report the preclinical feature of ATG-042-198, a novel MTAPnull-selective PRMT5 inhibitor. Methods: The in vitro activity and MTAP selectivity of ATG-042-198 on cell proliferation and cellular symmetric dimethylarginine (SDMA) expression were profiled using HCT116 MTAP wild type (wt) cells and HCT116 MTAP knock out (ko) cells, as well as in five MTAPnull cell lines. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with HCT116 MTAP wt cells, HCT116 MTAP ko cells and LU99 cells (MTAPnull). The in vitro and in vivo pharmacokinetic properties were assessed with corresponding assay methods. Results: ATG-042-198 demonstrated potent and selectively inhibitory effect on HCT116 MTAP ko cells’ proliferation (IC50=44nM) and SDMA expression (IC50=0.146nM), but very weak effect on HCT116 MTAP wt cells’ proliferation (IC50=4550nM) and SDMA expression (IC50&amp;gt;500nM). Consistently, ATG-042-198 showed excellent anti-proliferation activities on multiple endogenous MTAPnull cell lines (A549, IC50=2nM; LU99, IC50=6nM; U87MG, IC50=11nM; NALM-6, IC50=37nM; MIA PaCa-2, IC50=43nM). ATG-042-198 demonstrated high permeability (Caco2 assay, ER=2), good metabolic stability (Human liver microsomes assay, T1/2&amp;gt;145min), and low risk of drug interaction (CYP inhibition assay, 3A4/2C19/2D6, all IC50&amp;gt;10μM). In vivo PK study showed that ATG-042-198 was well absorbed, with a dose-dependent increase in plasma distribution and high oral bioavailability (F=108%). Plasma free drug concentrations were continuously higher than the IC50 of cell proliferation inhibition within 24 hours after 30mpk administration. In three CDX models (HCT116 MTAP wt, HCT116 MTAP ko and LU99), ATG-042-198 at a dose of 30 mg/kg QD potently and selectively inhibited tumor growth (TGI=-1%, 89% and 106%) without inducing body weight loss. Conclusions: In summary, ATG-042-198 is an oral MTAP null-selective PRMT5 inhibitor with potent efficacy against MTAPnull tumor. It also demonstrated good tolerability and preclinical PK profiles. Citation Format: Ya Kong, Ming Zhang, Lulu Jiang, Ruiting Xu, Bin Jiang, Guoqiang Dai, Yulong He, Huiling Liu, Jay Mei, Bing Hou, Bo Shan. Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 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 4592.