Pharmacokinetic Properties Research Articles

Abstract 5733: Design and synthesis of the novel camptothecin analog MF-6 for application into site-specific antibody-drug conjugate

Abstract Introduction: Antibody-drug conjugates (ADCs) have emerged as a revolutionary breakthrough in cancer treatment by precisely targeting tumor cells with high-potency payloads attached to specific antibodies. However, the efficacy and safety of ADCs largely depend on the design and optimization of their payloads. This study leverages the advanced MtoxinTm platform to develop a novel ADC payload, MF6, aiming to enhance the antitumor activity of ADCs through precise drug design. Methods: Supported by the MtoxinTm platform, we first modified camptothecin-based compounds by preparing a series of camptothecin analogs with different substituents at the core position. Through in vitro efficacy testing, we screened out a series of compounds with significant antitumor activity and further optimized their attached dipeptide linkers. Subsequently, we conjugated the optimized drug-linker conjugates with anti-Trop2 and anti-B7H3 monoclonal antibodies to form novel ADCs. To comprehensively evaluate the potential of these ADCs, we conducted multiple experiments in vitro and in vivo, including pharmacodynamics, pharmacokinetics, safety, and antitumor activity studies. Results: The experimental results showed that MF6, as a free molecule, exhibited good drug-killing activity and maintained significant efficacy in multidrug-resistant models resistant to DXd. ADCs constructed using the clinically validated site-specific conjugation technology IDDC™ and our novel payload MF6 demonstrated good uniformity and stability, and exhibited significant antitumor efficacy in multiple CDX models. Additionally, these ADCs showed good bystander killing activity, meaning they could kill nearby tumor cells, thereby further enhancing their antitumor effect. We also found that ADCs synthesized with MF6 had excellent serum and plasma stability and pharmacokinetic properties, providing strong support for their future clinical application. Conclusions: This study successfully developed a novel ADC payload MF6 based on the MtoxinTm platform and initially validated its antitumor activity and safety in multiple in vitro and in vivo models. These results provide a solid experimental foundation for the further development of MF6-based ADCs for the treatment of various cancers. In the future, we will continue to explore the potential of MF6 and anticipate that MF6-based ADCs will undergo further clinical validation and application, bringing more precise, effective, and safe treatment options to cancer patients. Citation Format: Xiaowei Cen, Hui Xu, Huikai Zhu, Hua Deng, Zhenzhen Wang, Junwei Xu, Xueying Li, Zhiheng Wu, Yiyan Zhang, Yuhang Chen, Xiaolin Chen, Li Chen, Wei Zhou, Hai Wu. Design and synthesis of the novel camptothecin analog MF-6 for application into site-specific antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5733.

Abstract 2888: DB-1418, a bispecific antibody-drug conjugate (ADC) targeting EGFR and HER3, demonstrates superior and broad antitumor efficacy and favorable safety in preclinical models

Background: EGFR and HER3 are co-overexpressed in a spectrum of tumors, including lung, breast, gastrointestinal, renal cell, ovarian, bladder, and prostate cancers. Importantly, HER3 expression is further enhanced upon inhibition of EGFR, suggesting that simultaneous targeting of both EGFR and HER3 could enhance tumor selectivity and improve therapeutic efficacy in EGFR tyrosine kinase inhibitors resistant tumors. This hypothesis has been supported by the clinical data of BL-B01D1, a bispecific ADC currently in clinical development. Here, we developed a differentiated bispecific ADC with stronger antitumor activity in preclinical studies. Methods: We generated DB-1418, an asymmetric IgG-like bispecific ADC, featuring a Fab arm targeting EGFR with medium affinity and a scFv arm targeting HER3 with high affinity. The antibody is conjugated to a linker-payload L101P1021 at a drug-to-antibody ratio (DAR) of 6. Results: The bispecific antibody showed low affinity on EGFR-only or HER3-only cell lines but demonstrated high affinity on tumor cells co-expressing both EGFR and HER3 compared to the parent monospecific antibodies. It also showed higher internalization capacity than the parent monospecific antibodies. DB-1418 exhibited broad anti-tumor activity across different expression levels of EGFR and HER3. Superior tumor inhibition was observed compared to a BL-B01D1 analog, based on both equal molar and mass concentrations. In the EGFR-dominant CAL-27 model, DB-1418 demonstrated a significant tumor growth inhibition (TGI) of 83% at a dose of 1.9 mg/kg Q3W, whereas the BL-B01D1 analog achieved only a 73% TGI at a dose of 3 mg/kg Q3W. In the HER3-dominant SW-620 model, DB-1418 treatment at a dose of 10 mg/kg Q3W resulted in complete tumor regression in 4 out of 5 mice and persisted up to 63 days after administration, whereas the BL-B01D1 analog at the same dose level and frequency failed to inhibit tumor growth. Notably, in an osimertinib-resistant NSCLC xenograft model with the C797S mutation, DB-1418 induced tumor regression with a TGI of 98% at a dose of 6 mg/kg Q3W, significantly higher than the BL-B01D1 analog at 6 mg/kg Q3W (TGI of 68%, p<0.01). DB-1418 demonstrated in vitro plasma stability in human, monkey, and rat models. In cynomolgus monkeys, the dose-range finding study showed favorable safety, with the highest non-severely toxic dose (HNSTD) reaching up to 45 mg/kg. Conclusion: DB-1418 has shown promising pharmacological and pharmacokinetic properties, differentiated from the current clinical trial bispecific ADC targeting EGFR and HER3, with a broader therapeutic window. DB-1418 offers additional treatment options for patients who have developed resistance to EGFR tyrosine kinase inhibitors (TKIs) and addresses other unmet medical needs. Citation Format: Yunhua Zhou, Jun Yao, Rong Shi, Yang Qiu, Haiqing Hua. DB-1418, a bispecific antibody-drug conjugate (ADC) targeting EGFR and HER3, demonstrates superior and broad antitumor efficacy and favorable safety in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2888.

Abstract 3013: GSC000829, a novel selective FGFR2/3 inhibitor, displays superior antitumor activity in preclinical FGFR-driven neoplasms models

Fibroblast growth factors (FGF) and their receptors (FGFRs) are pivotal in a myriad of cellular functions. Tumorigenesis onset due to FGFR genomic alterations is commonly observed across diverse solid tumors. Although several FGFR inhibitors targeting FGFR1-3 or FGFR1-4 have gained approval for treating various solid tumors, their widespread application is hindered by the development of resistant gatekeeper mutations in FGFR proteins and toxicities linked to FGFR1 and/or FGFR4, necessitating precise dosing or even drug withdrawal. Here, we report the discovery of a novel pre-clinical candidate compound GSC000829 with a strong inhibition towards FGFR2/3 and a significantly improved selectivity over FGFR1 and FGFR4. Furthermore, GSC000829 shows excellent efficacy in various biochemical and cellular assays corresponding to different FGFR fusions or mutations. It also demonstrates enhanced antitumor activity at lower dosage in FGFR3-driven CDX models compared to the pan-FGFR inhibitor Erdafitinib, as well as other FGFR3-selective inhibitors such as TYRA300 and LOXO-435. GSC000829 exhibits favorable physicochemical and pharmacokinetic properties along with an excellent preclinical toxicity profile with no obvious phosphate increase at quite high exposure level in rats. Taken together, these findings bolster the rationale for advancing GSC000829 into clinical trials as a potential best-in-class FGFR isoform-selective inhibitors, offering an approach for treating advanced solid tumors harboring FGFR2/3 aberrations. Citation Format: Tian Han, Donglin Fu, Tao Cheng, Yuanfeng Xia, Zhilong Hu, Fanglong Yang. GSC000829, a novel selective FGFR2/3 inhibitor, displays superior antitumor activity in preclinical FGFR-driven neoplasms models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3013.

Abstract 7018: Antibody-degrader conjugates unlock the therapeutic potential of heterobifunctional degraders through antibody-like pharmacokinetics and excellent in vivo efficacy

Abstract Heterobifunctional small molecules recruiting the proteasomal machinery enable the targeted protein degradation (TPD) of selected proteins and considerably broaden the druggable space for therapeutic use. Many such molecules have seen fantastic success in preclinical and clinical developments, e.g., for targeted degradation of specific oncoproteins that were previously inaccessible for cancer therapy. Even though early hits are quickly generated by adapting the modular concept of bifunctional degraders, most of such molecules are against the central rules of a “drug-like” molecule: they are usually larger in size (800-1500 Da) and often lack sufficient solubility. These early bifunctional degraders hits require exhaustive structure optimization in each case to optimize pharmacokinetics (PK) profiles and bioavailability. Here, we report Tubulis’ antibody-degrader conjugates that exploit bifunctional degraders as high-potency bioactive payloads to be released specifically inside cells that are targeted by the antibody. Our linker technology ensures high degrader-to-antibody ratios (DARs) and excellent linker stability in blood plasma. Moreover, we liberate the degrader after receptor-mediated uptake of the ADC and show high efficiencies in target protein degradation. Most importantly, our conjugates exhibit antibody-like PK properties that ensure long-lasting plasma circulation of high DAR constructs over weeks, even when bifunctional degraders are employed that have not been optimized for PK properties. Taken together, these factors allow impressive in vivo efficacy of the conjugates after single dose application in preclinical animal models. Citation Format: Jan G. Felber, Anil P. Jagtap, Philipp Ochtrop, Saskia Schmitt, Isabelle Mai, Sarah Herterich, Paul Machui, Dietmar König, Dominik simon vogt, Schumacher, Jonas Helma-Smets, Annette Vogl, Marc-André Kasper. Antibody-degrader conjugates unlock the therapeutic potential of heterobifunctional degraders through antibody-like pharmacokinetics and excellent in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7018.

Abstract 4854: Alleviation of low pH induced immunosuppression through antagonism of the pH sensor GPR65 in highly glycolytic NSCLC tumors

Abstract Out of more than 5683 clinical trials testing anti-PD1/PDL1 mAbs, on average only 20% of patients achieved an objective response, leaving an unmet need for patients who do not respond to immune checkpoint inhibitors (ICIs)(Zhao et al, 2020, Upadhyaya et al, 2022). Independent reports in NSCLC and melanoma suggest that non responders to ICI exhibit elevated levels of tumor “aerobic” glycolysis (Renner et al, 2019, Silva et al, 2022), a phenotype associated with solid and metastatic tumor cells (Hanahan, 2022). Aerobic glycolysis results in accumulation of lactate and acidification of the tumor microenvironment (TME), resulting in immune evasion through inhibition of pro-inflammatory/ anti-tumorigenic responses in surrounding immune cells (Li et al, 2022). GPR65 is a proton (pH) sensing GPCR, expressed on immune cells, signals via cAMP to activate anti-inflammatory pathways in multiple immune cell types (Robert and Mackay, 2018; Wang et al, 2023). In the context of solid tumors, GPR65 activation promotes the increase in anti-inflammatory TAMs, in addition to suppressing anti-tumor responses by NK and cytotoxic-T cell (Raker et al, 2016). GSK analysis of Tempus RW data conducted with TEMPUS LENS software indicated high glycolytic gene signature in NSCLC cohorts is associated with worse prognosis to both chemo and ICI therapies, providing a window of opportunity to target pH sensor GPR65. Ex vivo experiments demonstrated that low pH dampens the proinflammatory phenotype of monocyte-derived macrophages (MDMs), while knockout of GPR65 under these acidic conditions restores secretion of the proinflammatory cytokines CXCL9/10/11 in M1-like polarized MDMs. Compound A, an exemplar compound from our lead series, is a potent GPR65 antagonist with low nanomolar in vitro functional activity (pIC50=8.2) and excellent lipophilic ligand efficiency (6.9). It also features strong correlation between its functional and binding (nanoBRET) activities along with selectivity at GPR65 over GPR4 and GPR68. In vitro translational and cell-based assays demonstrated dose-dependent attenuation of proinflammatory cytokine release by primary CD8+T cells, M1 macrophages at pH 6.8 and in a NSCLC patient-derived tumor fragmentation assay. In vivo, compound A displays attractive pharmacokinetic (PK) properties in rodent with robust oral bioavailability. A preliminary estimate of projected human dose (PHD) is ∼245 mg qd based on liver blood flow scaling of its rat PK properties and a targeted 24 h free drug coverage over functional in vitro IC50. Additional In vivo PD and/or efficacy data in translational models would further refine this calculation. In summary, we hypothesize that restricting immune cells from sensing acidification in the TME via antagonizing GPR65 will prevent the activation of anti-inflammatory programs in immune cells and promote proinflammatory anti-tumor effector mechanisms. Citation Format: Prajna Behera, Samantha Buongervino, Kayla Guidry, Kyle Krois, Yanah Hossain, Victor Du, Tamara Ouspenskaia, Hongyu (Annie) Liang, Shu-Yun Zhang, Laura Johnston, Abby Lin, Tolu Aweda, Ramona Plant, Paul Bojczuk, Marica Speranza, Chris Hopson, Charles McHugh, Stacey Kendrew, Bradley Spencer-Dene, Susanne Moore, Deon Hildebrand, Jonathan Lea, Kerena Norris, Antonia Lewis, Jacob Brett, Paru Rao, Carol Harris, Joseph Pero, Laura Ator, Jing Fang, David washburn, Lisa Sloan, Catherine Alder, Richard Lonsdale, Nick Barton, Sue Griffin, Paul Redford, James Smothers. Alleviation of low pH induced immunosuppression through antagonism of the pH sensor GPR65 in highly glycolytic NSCLC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4854.

Abstract 4380: HBW-016-K, a novel orally available pan-KRAS inhibitor targeting both “ON” and “OFF” states, is potent, selective, efficacious, safe, and high tissue-penetrant

Purpose: This study aimed to develop a novel, orally available pan-KRAS inhibitor capable of targeting both the “ON” and “OFF” states of KRAS, addressing drug resistance and expanding treatment options for KRAS-mutant cancers. Methods: Through medicinal chemistry efforts, we designed and optimized a pan-KRAS inhibitor that selectively targets various KRAS mutant subtypes while sparing wild-type KRAS, NRAS, and HRAS. The lead compound was further evaluated for its pharmacokinetic (PK) properties and safety profile. Its anti-tumor efficacy was assessed in multiple KRAS mutant mouse models following oral administration. Results: HBW-016-K demonstrated potent inhibition of various KRAS mutant cell lines with excellent selectivity for KRAS wild-type and other RAS isoforms. This compound effectively targets both the “ON” and “OFF” conformations of KRAS G12D. Additionally, HBW-016-K exhibits favorable PK properties and significant tissue penetration, particularly in lung, intestine, and pancreas. In a KRAS G12V mouse xenograft model, HBW-016-K exhibited dose-dependent anti-tumor activity. At the highest dose of 30 mg/kg (BID, PO), tumors shrank by 84.1% compared to baseline, with sustained tumor regression. HBW-016-K also demonstrated potent anti-tumor activity in KRAS G12D and G12C mouse models. Preliminary toxicity studies revealed a favorable safety profile for HBW-016-K, including minimal off-target effects, negative mutagenicity, and excellent tolerability in a 14-day rat toxicity study. Conclusion: HBW-016-K is a promising novel pan-KRAS inhibitor with superior potency, selectivity, and efficacy. Its favorable PK profile and safety profile support its advancement into clinical development. HBW-016-K has the potential to significantly impact the treatment of KRAS-driven cancers, particularly those with limited therapeutic options, such as lung, colorectal, and pancreatic cancer. Citation Format: Ning Lee, Yingfu Li, Guanfeng Liu, Jiang Li, Mao Yang, Zhiguan Song. HBW-016-K, a novel orally available pan-KRAS inhibitor targeting both “ON” and “OFF” states, is potent, selective, efficacious, safe, and high tissue-penetrant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4380.

Abstract 4378: BBO-11818, an orally bioavailable, highly potent and non-covalent pan-KRAS inhibitor demonstrates robust anti-tumor activity in KRAS-mutant preclinical models

Abstract KRAS is the most commonly mutated oncogene, altered in approximately 15% of human cancers. While inhibitors of KRASG12C have shown promising clinical efficacy, there are currently no approved targeted therapies against other KRAS variants, resulting in a significant underserved patient population across several major cancer types, including pancreatic carcinoma (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). To address this unmet clinical need, we have developed BBO-11818: a potent, selective, orally bioavailable non-covalent pan-KRAS inhibitor with activity against multiple KRAS mutants, including KRASG12D and KRASG12V, in both their inactive GDP-bound and active GTP-bound states. BBO-11818 binds mutant KRAS with high affinity and selectivity in its active and inactive conformations, locking it in the inactive State 1 to disrupt the association of GTP-bound KRAS with its key effector RAF1. Cell-based assays confirm that BBO-11818 potently inhibits several driver KRAS mutants, resulting in the suppression of MAPK signaling and inhibition of cell proliferation with low nanomolar EC50 values. The selectivity of BBO-11818 for KRAS is demonstrated by its >1000-fold lower potency against NRAS- and BRAF-mutant cell lines. BBO-11818 exhibits favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties in mouse models of cancer. Single dose, oral administration results in strong dose- and time-dependent inhibition of pERK in KRASG12D and KRASG12V cell-derived xenograft (CDX) models. Importantly, BBO-11818 monotherapy induces strong anti-tumor responses, including regression at well-tolerated doses in CDX models of KRAS-mutant pancreatic, non-small cell lung, and colorectal cancer. In combination with either BBO-10203, a selective RAS:PI3Kα breaker that blocks RAS-mediated activation of AKT; or cetuximab, an anti-EGFR monoclonal antibody, BBO-11818 shows significantly enhanced efficacy in models harboring KRASG12D or KRASG12V mutations. BBO-11818 also shows a combination benefit with anti-PD-1 treatment, resulting in complete tumor regressions in the CT-26 syngeneic tumor mouse model. BBO-11818 is a potent pan-KRAS inhibitor with activity against both GTP and GDP forms of KRAS, presenting the opportunity to address, as monotherapy or combination, a large fraction of KRAS mutant tumors currently lacking targeted therapeutic options. Citation Format: Carlos Ernesto Stahlhut Espinosa, Kanchan Singh, Zuhui Zhang, Kyle Sullivan, Nadege Gitego, Megan Rigby, Roger Ma, Patrick Alexander, Saman Setoodeh, Jin Shu, Ken Lin, Dana Rabara, Erik Larsen, Alok Sharma, Albert Chan, Brian P. Smith, Andrew Stephen, Yue Yang, Jun Pei, Felice C. Lightstone, Dhirendra Simanshu, Rui Xu, Chunmei Ji, Keshi Wang, Bin Wang, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Anna E. Maciag, Pedro J. Beltran. BBO-11818, an orally bioavailable, highly potent and non-covalent pan-KRAS inhibitor demonstrates robust anti-tumor activity in KRAS-mutant preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4378.

Abstract 7010: HDB-82: A promising PROTAC degrader for KRAS G12D mutant cancers

Abstract KRAS G12D mutations, occurring in approximately 30% of solid tumors, represent a significant therapeutic target. While selective KRAS G12C inhibitors have achieved clinical success, effectively targeting the more prevalent G12D mutation remains an unmet medical need. This study investigates the potential of proteolysis-targeting chimeras (PROTACs) as a promising avenue for KRAS G12D-driven cancers, potentially offering advantages over traditional inhibitors through catalytic protein degradation. Targeted protein degradation of mutant KRAS may yield greater efficacy than simply inhibiting the protein. This study focuses on the design, synthesis, and biological evaluation of novel KRAS G12D-targeting PROTAC degraders. Through rigorous optimization, we developed HDB-82, a highly potent and selective PROTAC compound targeting KRAS G12D. HDB-82 effectively induced degradation of the KRAS G12D protein, leading to significant antiproliferative activity against KRAS G12D-mutant cancer cells in vitro and in vivo. HDB-82 demonstrated potent anti-cancer activity against various KRAS G12D-mutant cell lines, achieving low nanomolar IC50 values. It effectively degraded the KRAS G12D protein in various KRAS G12D-expressing cell lines (DC50 value ranges from picomolar to sub-nanomolar range). Mechanistic studies revealed significant suppression of downstream MAPK and PI3K/AKT signaling pathways, accompanied by robust induction of apoptosis, and superior antiproliferative activity compared to existing inhibitors. Furthermore, HDB-82 exhibited favorable pharmacokinetic and pharmacodynamic properties in mice. Intravenous administration of HDB-82 (10 mg/kg, q.w.) resulted in substantial tumor growth inhibition across multiple KRAS G12D-positive tumor models. These findings establish HDB-82 as a promising therapeutic candidate for KRAS G12D-mutant cancers, warranting further preclinical investigation and potential clinical development. Citation Format: Mohammad Hassan Baig, Yun Seong Jo, Sagar Dattatraya Nale, Chang Joong Kim, Tae Hwan Park, Ju Han Bok, Dong Min Kim, Hye Mi Kim, Ji Min Park, Jae June Dong, Jae Hee Cho, Sung Ill Jang, Byoung Gon Moon. HDB-82: A promising PROTAC degrader for KRAS G12D mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7010.

Abstract 3788: BBO-8520, a first-in-class direct dual inhibitor of GTP-bound (ON) and GDP-bound (OFF) KRASG12C, exhibits robust efficacy in non-small cell lung cancer preclinical models

Abstract Sotorasib and adagrasib, allele-specific KRASG12C covalent inhibitors that only target KRASG12C in the inactive GDP-bound (OFF) state, have been approved for patients with KRASG12C locally advanced or metastatic non-small cell lung cancer (NSCLC). While these inhibitors have improved the treatment paradigm for patients with tumors harboring KRASG12C mutations, they are limited in their depth and duration of response. The suboptimal efficacy of KRASG12C (OFF)-only inhibitors is likely driven by adaptation through increasing the population of drug-insensitive KRASG12C bound to GTP (ON). Patient responses may be improved with a compound that exhibits dual activity, capturing both GTP-bound (ON) and GDP-bound (OFF) forms of KRASG12C in the cell. To address this unmet clinical need, we have developed BBO-8520, a potent, selective, orally bioavailable, direct covalent inhibitor of KRASG12C, with dual activity against both the GTP-bound (ON) and GDP-bound (OFF) states of KRASG12C. BBO-8520 binds KRASG12C with high affinity in its active and inactive conformations, locking it in the inactive state 1 confirmation, in which it is unable to bind its key effector RAF1. Cell-based assays show that BBO-8520 potently and selectively inhibits KRASG12C in the single digit nanomolar range or below across a panel of NSCLC cell lines on pERK and 3D viability. Unlike KRASG12C (OFF) inhibitors, BBO-8520 rapidly disrupts the binding of KRASG12C to RAF1 and maintains potency in growth factor activated states. In vivo, BBO-8520 demonstrates favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties in mouse models of NSCLC. Single dose, oral administration results in dose- and time-dependent inhibition of pERK in KRASG12C-driven cell-derived xenograft (CDX) models. As a monotherapy, BBO-8520 demonstrates robust tumor growth inhibition in KRASG12C-driven NSCLC CDX models both sensitive and resistant to KRASG12C (OFF) inhibitors as well as in patient-derived xenograft (PDX) and genetically engineered mouse (GEM) models. BBO-8520 also exhibits strong combination benefit in vivo in the KRASG12 CDX model NCI-H2122 when combined with either BBO-10203, a selective RAS:PI3Kα breaker that blocks RAS-mediated activation of AKT in clinical development (NCT06625775), or the anti-EGFR antibody cetuximab. In summary, BBO-8520 is a first in class, potent and selective KRASG12C dual GTP-bound (ON) and GDP-bound (OFF) inhibitor that exhibits robust efficacy and may improve upon approved, KRASG12C GDP-bound (OFF)-only inhibitors in NSCLC. BBO-8520 has entered phase 1 clinical trials in patients with KRASG12C NSCLC that are either naïve or have experienced first-generation KRASG12C (OFF) inhibitors (NCT06343402). Citation Format: James P. Stice, Anna E. Maciag, Alok K. Sharma, Albert H. Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Cathy Zhang, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, John-Paul Denson, Michela Ranieri, Mary Meynardie, Rui Xu, Felice Lightstone, Kwok-Kin Wong, Andrew G. Stephen, Keshi Wang, Dhirendra K. Simanshu, Kerstin W. Sinkevicius, Dwight V. Nissley, Bin Wang, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class direct dual inhibitor of GTP-bound (ON) and GDP-bound (OFF) KRASG12C, exhibits robust efficacy in non-small cell lung cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3788.

Development and Application of Radioactive Ligands Targeting Fibroblasts with Albumin-Binding Sites.

Fibroblast activation protein (FAP) is overexpressed on cancer-related fibroblasts (CAFs), making it an important target for cancer diagnosis and treatment, but limited tumor retention hinders late-stage diagnosis and radionuclide therapy. In this study, three albumin-bound FAPI radioligands, 68Ga/177Lu-DOTA-ALB-01, 68Ga/177Lu-DOTA-ALB-02, and 68Ga/177Lu-DOTA-ALB-03, were synthesized and evaluated for their in vitro stability, binding affinity, in vivo biodistribution, and tumor uptake using 68Ga and 177Lu labeling. All radioligands are stable in saline and plasma and exhibit high FAP binding affinity. 177Lu-DOTA-ALB-02 has longer retention in circulation than 177Lu-FAPI-46 and other radioligands. Continuous tumor accumulation was observed during imaging with both 177Lu-DOTA-ALB-01 and 177Lu-DOTA-ALB-02. Notably, 177Lu-DOTA-ALB-02 had a significant tumor/ nontarget (T/NT) ratio as indicated by biodistribution data. The outstanding tumor retention properties of 177Lu-DOTA-ALB-02 have been demonstrated in small animal single photon emission computed tomography (micro-SPECT) imaging and biodistribution studies, therefore it is considered the albumin-binding FAPI with the most favorable pharmacokinetic and imaging properties, worthy of further clinical investigation.

Abstract 4281: Pharmacological inhibition of thymine DNA glycosylase (TDG) triggers an anti-tumor immunity to suppress p53-deficient cancer

Abstract As the most frequently mutated or silenced tumor suppressor, TP53 mediates cell-cycle arrest, apoptosis, and senescence in cancer development. Although patients with TP53 alterations are unfortunately poor in prognosis and survival, targeting p53 directly is still largely difficult to activate or restore p53 normal function specifically in cancer. Recently, synthetic lethality approach has been successfully applied to treat patients with mutations in tumor suppressor genes (TSGs) like PARPi in BRCA1/2 mutated tumors, potentiating the future direction of next generation p53 therapies. However, it remains a huge challenge to identify the bona fide synthetic lethal partners to TP53 mutations in cancer. In this study, we demonstrated that pharmacological inhibiting thymine DNA glycosylase (TDG), a multifunctional DNA modifying enzyme with essential roles in DNA base excision repair (BER), transcriptional regulation, and DNA active demethylation, can induce synthetic lethality specifically in various p53-deficient solid tumors including lung, breast, and skin cancer, etc. This offers a novel strategy to target mutated TP53 for cancer therapies. We discovered and developed from CytosinLab’s EpigenPLUS platform a series of small-molecule inhibitors that binds specifically and directly to TDG and disrupts its DNA-binding and modifying activities. Among the series, C-271 is an orally bioavailable front-runner with excellent pharmacokinetics (PK) properties and druggabilities and exhibits profound efficacy in suppressing growth of p53-deficient lung cancer cells both in vitro and in vivo, with minimal repression on p53 wide-type cells. In p53-deficient cancer cells, treatment with C-271 reduces transcription of the RNA helicase DHX9, thereby leading to the accumulation of SINE (short interspersed nuclear element) -derived dsRNA and subsequent activation of the anti-tumor immunity pathways. Additionally, TDG inhibition enhances anti-tumor immunity and displays significant synergy with anti-CTLA-4 antibody, the immune checkpoint blockade (ICB). Furthermore, we explored and validated the sensitivity of other p53-deficient cancer types to TDG inhibition, broadening the potential of TDG inhibition to treat a larger patient population. In conclusion, we identified inhibition of TDG as a brand-new synthetic lethality approach and an innovative synthetic immune activation anti-cancer mechanism in TP53-mutant tumors. It underscores the promise of profiling and developing C-271 or other preclinical candidate molecules of TDG into clinic study and extending the breakthrough first-in-class (FIC) therapy to be viable and beneficial for treating p53-deficient cancers. CytosinLab Therapeutics holds full intellectual property (IP) rights to C-271 and its series of TDG-targeting small molecules. Citation Format: Jiannan Guo, Meng Wang, Jiaxin Zhou, Yao Cao, Yiqin Wang, Yilin Liu, Youzhen Wang, Yuan Mi, Yarui Du, Guoliang Xu, Haiping Wu. Pharmacological inhibition of thymine DNA glycosylase (TDG) triggers an anti-tumor immunity to suppress p53-deficient cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4281.

Abstract 1567: GLR1059, next-generation nectin-4-targeted ADC with a novel mechanism-of-action payload, demonstrated significantly potent anti-tumor efficacy and reduced toxicity in preclinical evaluation

Abstract Background: Nectin-4 is a tumor-associated antigen, which was observed in multiple cancer types, including bladder, breast, lung, pancreatic and ovarian cancer. Enfortumab Vedotin (EV) is the first approved nectin-4 antibody-drug conjugate (ADC) with MMAE as its payload for the treatment of urothelial carcinoma (UC). However, beyond UC, EV hasn’t been approved for other indications. In addition, severe skin adverse reactions were reported in EV-treated patients. There are several new nectin-4 ADCs in development but haven’t entered confirmative clinical studies so far. To overcome the limitations of efficacy and safety, we developed a novel mechanism nectin-4 ADC GLR1059, with a payload that was more effective for potential indications with nectin-4 expression and had lower toxicity, including skin adverse reactions. Methods: For in vitro studies, binding affinity and specificity, internalization, cancer cell killing, bystander effect and serum stability were performed. For in vivo studies, efficacy studies were conducted on cell line-derived xenografts (CDX) of multiple cancer types, e.g. breast cancer (including triple-negative breast cancer), and urothelial carcinoma. Pharmacokinetic (PK) properties were studied in BALB/c mice and tumor-bearing nude mice, and safety studies were conducted in humanized nectin-4 mice. Results: GLR1059 is a nectin-4 ADC composed of a novel humanized IgG1 monoclonal antibody, a cleavable linker and a microtubule targeting agent (not auristatin derivatives). Moreover, to generate DAR4 ADCs with higher homogeneity, the payload was site-specifically conjugated to the antibody via a disulfide bond bridge. In surface plasmon resonance (SPR) assays, GLR1059 had strong and specific binding to human nectin-4, and it did not bind to nectin-1, nectin-2, or nectin-3. Furthermore, GLR1059 exhibited efficient internalization and potent activity in nectin-4 expressing cell lines and effective bystander activity. In a panel of CDX models, GLR1059 demonstrated compelling anti-tumor activity across bladder cancer and breast cancer (including TNBC), with tumor growth inhibition rates (TGI) significantly higher than EV (approximately 2-4 times). In the tumor-bearing mice PK study, the Cmax of free payload in tumor tissue was approximately 100-fold higher than that in the blood (131±14ng/g vs 1.04±0.27ng/mL). GLR1059 demonstrated longer survival and less weight loss in the single- and repeated-dose toxicity studies in humanized nectin-4 mice. Notably, GLR1059 significantly reduced the incidence and severity of skin toxicity compared with EV (1/6 vs 6/6). Conclusion: Preclinical data show that GLR1059 has the potential to achieve better efficacy and safety, and the different drug mechanisms provide new options to overcome drug resistance of current nectin-4 ADCs. Citation Format: Jiao Jiao, Yingjie Sun, Jiaxing Wang, Liming Che, Menghan Cui, Wenjie Shao, Hairui Yuan, Yan Huang, Nannan Zhai, Yuling Tang, Xiaomei Zheng, Siyao Li. GLR1059, next-generation nectin-4-targeted ADC with a novel mechanism-of-action payload, demonstrated significantly potent anti-tumor efficacy and reduced toxicity in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1567.

Abstract 6389: Unveiling the potent anti-tumor activity and underlying mechanism of action of the novel pan-RAF inhibitor exarafenib in BRAF-mutated NSCLC

Abstract Somatic BRAF alterations occur in up to 5% of non-small cell lung cancer (NSCLC) cases and comprise three mechanistically distinct molecular subtypes: Class I alterations (e.g., V600E) function as constitutively active monomers, Class II alterations that signal as RAS-independent mutant BRAF homodimers, and Class III alterations that activate MAPK signaling via RAS-dependent mutant BRAF/wild-type RAF heterodimers. While BRAF V600E (Class I) mutations are effectively targeted by approved therapies that are active against these oncogenic BRAF monomers, there are no established targeted treatments for both Class II and III BRAF mutations that operate as BRAF dimers in NSCLC. Analysis of the GuardantINFORM™ database revealed that these untreatable Class II and III BRAF mutations comprise 65% of BRAF-mutant NSCLC cases, with Class II mutations associated with significantly worse survival compared to Class I (median survival 514 vs 818 days, HR: 1.4, P=0.0395). Here we evaluate exarafenib, a novel type 2 pan-RAF inhibitor designed to target both monomeric and dimeric forms of RAF proteins by binding the αC-helix-IN and DFG-out conformations. Through comprehensive mechanistic studies, we demonstrate that while exarafenib induces RAF dimerization, it maintains potent inhibition of both monomeric and dimeric RAF proteins with high selectivity for RAF kinases, leading to effective suppression of downstream MAPK pathway signaling. In preclinical studies, exarafenib showed robust anti-tumor activity in BRAF-mutant NSCLC cell lines and patient-derived xenograft models, with favorable pharmacokinetic and pharmacodynamic properties. Clinical case studies revealed promising responses, including a partial response and sustained disease control for over two years in a patient with BRAF G469S mutation, and a significant initial response lasting approximately 32 weeks in a patient with TMEM106B-BRAF fusion. Through analysis of acquired resistance models, we identified RAS activation as a key resistance mechanism and biomarker that was accompanied by sustained MAPK pathway signaling. Based on this mechanism, we evaluated combination strategies and found that co-targeting MEK with binimetinib provided synergistic anti-tumor effects both in vitro and in vivo, while maintaining tolerability. These mechanistic insights provide strong biological rationale for the ongoing Phase 1 trial (KN-8701; NCT04913285) evaluating exarafenib as monotherapy and in combination with binimetinib in BRAF-altered solid tumors including NSCLC and NRAS-mutant melanoma, offering a potential therapeutic strategy for patients that currently lack effective targeted treatment options. Citation Format: Tadashi Manabe, Tim Sen Wang, Paul Severson, Nichol Miller, Catherine Lee, Elifnur Yay Donderici, Nicole Zhang, Yu-ting Chou, Daniel L. Kerr, Wei Wu, Kathryn B. Grandinetti, Liliana Soroceanu, Robert J. Pelham, Eric S. Martin, Eric A. Murphy, Vishesh Khanna, Joel W. Neal, Christopher T. Chen, Shumei Kato, Richard Williams, Trever Bivona. Unveiling the potent anti-tumor activity and underlying mechanism of action of the novel pan-RAF inhibitor exarafenib in BRAF-mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6389.

Fluorine in the Pharmaceutical Industry: FDA-Approved Fluorine-Containing Drugs in 2024.

Fluorine has become an essential element in the development of modern pharmaceuticals, due to its unique chemical properties that can significantly enhance the biological activity, metabolic stability, and lipophilicity of drug molecules. This review explores recent advancements in the synthesis and application of fluorine-containing drugs approved by the US Food and Drug Administration (FDA) in 2024. These novel drugs demonstrate improved efficacy and safety profiles, addressing a range of therapeutic areas including oncology, infectious diseases, metabolic disorders and genetic disorders that affect the adrenal glands. The incorporation of fluorine atoms into drug candidates has facilitated the development of molecules with optimized pharmacokinetic and pharmacodynamic properties, leading to better patient outcomes. The review further discusses the synthetic methodologies employed, the structural characteristics of these drugs, and their clinical implications, providing insights into the ongoing innovation within the pharmaceutical industry driven by fluorine chemistry.

Abstract 4208: Azenosertib is a potent and selective WEE1 kinase inhibitor with broad antitumor activity across a range of solid tumors

Abstract Due to high proliferative rate, cancer cells experience significant stress during DNA replication and mitosis, leading to DNA damage and increased genomic instability. To mitigate this, cancer cells rely on checkpoint kinases like WEE1, which halt cell cycle progression and allow damaged DNA to be repaired before cells enter mitosis. Damage that is too extensive leads to apoptosis. Targeting WEE1 can exploit vulnerabilities created by replicative stress and genomic instability. Given these features are common in cancer, we hypothesized a WEE1-targeted therapeutic would have broad antitumor activity across various cancer types. Azenosertib (ZN-c3) is a novel, selective, orally bioavailable WEE1 inhibitor with optimized pharmacokinetic and pharmacodynamic properties. Preclinical studies demonstrate Azenosertib inhibits cancer cell proliferation through a WEE1-dependent mechanism, marked by a reduction of pY15-CDK1 levels and increases in DNA damage markers. Herein, we evaluated its antitumor activity in 54 mouse xenograft models representing 9 solid tumor subtypes and observed robust tumor growth inhibition (> 60% TGI) in 24 of these models, including breast, colorectal, lung, ovarian, pancreatic, prostate, soft tissue sarcomas, and endometrial cancers. We also explored various dosing schedules for Azenosertib, identifying regimens that maintain efficacy with minimal toxicity in preclinical models. To further understand the mechanisms underlying Azenosertib’s antitumor activity, we analyzed cell cycle changes in four cancer cell lines with variable sensitivity to Azenosertib. Treatment with Azenosertib caused accumulation of EdU-negative cells in the S-phase, reduced cells in the G1 phase, and promoted premature entry into mitosis. These effects suggest Azenosertib bypasses the G1/S and G2/M checkpoints, leading to immature S-phase and Mitosis entry, consistent with WEE1 inhibition of CDK1 and CDK2. Further analysis of DNA repair revealed an increase in γH2AX-positive cells, primarily in G1, EdU-negative S, and G2/M, and the co-localization of mitotic marker pHH3 and DNA damage marker γH2AX. Notably, WEE1 plays a critical role in safeguarding the integrity of the cell cycle, and this regulation is disrupted by Azenosertib. As a result, cells with unresolved DNA damage are driven into premature mitosis, exacerbating DNA damage and leading to mitotic catastrophe. Azenosertib is currently being investigated as a single agent in a dose escalation Phase I clinical trial (NCT04158336) evaluating safety, tolerability, and efficacy in cancer patients with previously treated, advanced solid tumors. Early clinical data from heavily pretreated patients with different types of cancer show promising signs of activity. Our findings highlight the potential of WEE1 inhibition as a broad therapeutic strategy, for gynecological cancers and various malignant indications. Citation Format: Wen Liu, Jianhui Ma, Jiali Li, Daehwan Kim, Sangyub Kim, Alexandra Levy, Kimberly Blackwell, Alejandro Recio-Boiles, Jennifer M. Segar, Shirai Sen, Deborah Doroshow, Danielle Jandial, Olivier Harismendy, Stephan K. Grant, Ahmed Samatar, Mark R. Lackner, Kimberlee M. Fischer. Azenosertib is a potent and selective WEE1 kinase inhibitor with broad antitumor activity across a range of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4208.

Abstract 6954: Discovery and characterization of selective mixed-lineage kinase 3 PROTAC degraders for cancer therapy

Abstract Mixed-lineage kinase 3 (MLK3) is a member of the MLK family of serine/threonine kinases. MLK3 is well-known to regulate MAPK signaling by acting upstream of ERK, JNK, and p38 pathways. In addition to its canonical role in activating MAPK cascades, MLK3 regulates the activity of various proteins and signaling pathways, including NF-κB, Wnt/β-catenin, and PAK1. Previous studies demonstrated that MLK3 can promote the proliferation, invasive growth, and metastatic potential of human cancers. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is characterized by poor prognosis owing to its aggressive clinical course and limited targeted treatment options. MLK3 is often upregulated in TNBC and is involved in driving the tumorigenic potential of TNBC cells. Consequently, several small-molecule inhibitors targeting MLK3 were pursued as new investigational drugs for TNBC in preclinical studies. However, the utility of these compounds is limited by their low specificity and multiple off-target effects. Furthermore, recent studies suggested that the pro-oncogenic activity of MLK3 might result from its kinase-independent and scaffolding functions, which are not effectively targeted by conventional small-molecule inhibitors. To overcome the challenges associated with MLK3 inhibition, we developed and characterized first-in-class MLK3 Proteolysis Targeting Chimera (PROTAC) degraders. We synthesized a series of PROTAC compounds by connecting pan-MLK inhibitor CEP-1347 to molecules binding different E3 ligases (VHL, IAP, Cereblon). We found that CEP-1347-VHL-02, a PROTAC based on CEP-1347 and ligand for VHL E3 ligase, was the most potent MLK3 degrader among all candidate compounds. Next, we demonstrated that CEP-1347-VHL-02 effectively degraded MLK3 via the ubiquitin-proteasome system in breast cancer cell lines. By applying biochemical assays and proteomic approaches, we found that CEP-1347-VHL-02 selectively targeted MLK3 but no other MLK kinases. Furthermore, we showed that MLK3 degradation by CEP-1347-VHL-02 led to diminished oncogenic features of TNBC cells, as evidenced by reduced proliferation, migratory potential, cell cycle arrest, and increased apoptosis. These effects were not observed in other breast cancer subtypes upon MLK3 degradation, which indicates the subtype-specific oncogenic functions of this kinase. Collectively, our research provides new opportunities for the selective targeting of MLK3 in cancer and highlights MLK3 degradation as a potential novel treatment strategy in TNBC. In the ongoing studies, we further investigate the pharmacokinetic properties and therapeutic potential of MLK3-targeting PROTACs across various cancer cell lines with high expression of this kinase. Citation Format: Dawid Mehlich, Kamila Karpinska, Venkata R. Sabbasani, Michal Lomiak, Pedro Torres-Ayuso, Katarzyna Wrobel, Vi Nguyen-Phuong Truong, Remigiusz Serwa, Rolf E. Swenson, John Brognard, Anna A. Marusiak. Discovery and characterization of selective mixed-lineage kinase 3 PROTAC degraders for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6954.

Abstract 3016: Therapeutic efficacy of a novel combination of ck2 inhibitor cx-4945 with cytotoxic therapy in neuroblastoma preclinical models

Abstract Introduction: Neuroblastoma is a pediatric cancer of the sympathetic nervous system, which is often diagnosed at a clinically advanced stage with primary or acquired resistance to conventional chemotherapy. Protein Kinase CSNK2A1 (CK2) is an intrinsically active, ubiquitously present serine-threonine kinase overactive in various solid cancers, including neuroblastoma (NBL). Recurrent genetic alteration in CK2 is not detected in NBL. CX-4945 (Silmitasertib) is an ATP-competitive CK2 selective small-molecule irreversible inhibitor of CK2 with inhibition constant (Ki) values in the low nanomolar range and high selectivity (49 of 250 kinases) versus other kinases. CX-4945 has shown in vivo anti-tumor activity in solid tumors and leukemia has favorable pharmacokinetic properties and is well-tolerated in human subject studies. Here, we report the therapeutic efficacy of CX-4945 in combination with temozolomide and irinotecan NBL preclinical models. Methods: The susceptibility of NBL cell lines with and without MYCN amplification was tested against CX-4945. We measured CK2 mRNA expression, protein level, and CK2 kinase activity in NBL cell lines and NBL PDX samples (n=18). CK2 knockdown was achieved in SMS-KCNR and SK-N-BE2 cell line using shRNA against CK2α and phenotype was assessed using cell viability assay, apoptosis, spheroid formation, migration, invasion assay. Cells were also injected into the flank of athymic mice to assess engraftment potential. We performed gene expression analysis using RNA sequencing of CX-4945 treated and gene knockdown NBL cells. MYCN-NBL cell line-derived subcutaneous model was developed in athymic mice. Mice were treated with a combination of Irinotecan (IRN) and temozolomide (TMZ) with and without CX-4945 for up to 28 days. Cohorts were followed for tumor growth and survival. Results: CK2α shRNA silencing in NBL cell lines resulted in significantly reduced proliferation, increased apoptosis, reduced spheroid formation, and reduced migratory potential. The IC50 of CX-4945 (3-6uM) in NBL cell lines also showed synergy with the other standard chemo drugs, Temozolomide and Irinotecan. Gene expression analysis showed a decrease in the expression of several known MYC target proteins. The cohort receiving IRN + TMZ with CX-4945 showed significant tumor inhibition (50-60%) and prolonged survival time compared to the TMZ+IRN group. Conclusion: Protein kinase CK2 is intrinsically active and negatively impacts survival in HR-NBL patients. In MYCN-NBL xenograft models, CX-4945 is well tolerated and shows superior therapeutic efficacy when combined with cytotoxic drugs Irinotecan and temozolomide. The ongoing Phase 1/II study is evaluating the safety, tolerability, and efficacy of combining CX-4945 with TMZ+IRN in relapsed refractory NBL patients ((NCT06541262). Citation Format: Marudhu Pandiyan Shanmugam, Muhammed Danial, Rajesh Rajaiah, Koby Duke, Katie Mercer, Yasin Uzun, Sinisa Dovat, Giselle Saulnier Sholler, Chandrika Gowda Behura. Therapeutic efficacy of a novel combination of ck2 inhibitor cx-4945 with cytotoxic therapy in neuroblastoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3016.

Abstract 6875: Preclinical development of a novel small molecule multitarget inhibitor LCI133 for endometrial adenocarcinoma

Abstract Objective: Endometrial cancer (EC) is the most common gynecologic malignancy with rising mortality and limited targeted therapies. LCI133, an in silico-designed triple inhibitor of CDK4/6-CDK9-AURK, has demonstrated nano-molar potency against PTEN-mutant EC cells lines and favorable in vivo tolerability. Genetically engineered mouse model (GEMM) is the optimal approach to investigate cancer therapeutics in an immunocompetent setting. The objective of this study is to: 1) describe the physiochemical and pharmacokinetic properties of LCI133, 2) establish and characterize the Pgr-Cre; Pten fl/fl GEMM of EC, 3) assess the preliminary effect of LCI133 on GEMM disease burden. Methods: 1) LCI133 was profiled for target inhibition by cell-free screen and KinomeSCAN. In vivo PK/ADME properties were assessed as described by Veena Bala.5 In vitro potency was assessed by IC50 assay, cell cycle assay, qRT-PCR and immunoblotting at the indicated timepoints for target biomarkers. 2) Pgr-Cre;Ptenfl/fl mice with conditional PTEN deletion were generated by crossbreeding PTEN floxed mice (Ptenflox)with mice expressing Cre under the regulation of the progesterone receptor promoter (PgRCre). Alterations of PTEN/PI3K/AKT pathway were validated by qRT-PCR and immunoblotting. Histopathologic features of female reproductive tract (FRT) were characterized by H&E and immunohistochemistry staining. FRT relative size was normalized to mouse body mass. 3) Beginning at 4-5 weeks of age, formulated LCI133-HCl was administered to Pgr-Cre;Ptenfl/fl mice intraperitoneally daily for 21 days. Animal weight was recorded during treatment. Relative FRT size, pathology and target inhibition were assessed at endpoint. Results: 1) LCI133 demonstrates favorable preclinical PK/ADME profile. LCI-HCl salt form is superior to free form. 2) We confirmed loss of PTEN in Pgr-Cre;Ptenfl/fl FRT with concomitant increases in pAKT. Endometrial adenocarcinoma with serosal and lympho-vascular invasion in a background of hyperplasia was identified at 7-8 weeks of age with high penetrance. Conditional loss of PTEN correlates with expression of Pgr, predominantly in the endometrium and myometrium. 3) LCI133 treatment was associated with decreased relative FRT size, reduced angiogenesis and target suppression. Conclusion: In silico-designed LCI133, as a highly selective inhibitor against CDK4/6-CDK9-AURK with nano-molar potency and favorable PK/ADME properties, demonstrated promising in vivo therapeutic index in immunocompetent mouse models. Citation Format: Qi Zhang, Vaidehi Mujumdar, Ritchie Delara, Krishnaiah Maddeboina, Dhananjaya Pal, Bharath Yada, Hailey Dryden, Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Cody Mchale, Donald Durden. Preclinical development of a novel small molecule multitarget inhibitor LCI133 for endometrial adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6875.

Abstract 7019: Identification of first-in-class paralog selective SMARCA4 degraders for treatment of hematological and prostate malignancies

Abstract The BAF (SWI/SNF) chromatin remodeling complex comprises of two mutually exclusive ATPases, SMARCA2 (BRM) and SMARCA4 (BRG1), that affect the mobilization and positioning of nucleosomes on DNA and thereby regulates important cellular functions including transcription, DNA recombination, DNA repair and chromosome decatenation during mitosis. SMARCA4 is frequently overexpressed in several types of cancers. Overexpression has been linked to increased proliferation and survival, as well as aggressive tumors and poor prognosis. SMARCA4 knockdown in these tumors lead to inhibition of proliferation and increased sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. In addition, PTEN and SMARCA4 appear to share synthetic lethality relationship based on reported in vitro and in vivo knockdown studies. Considering this well-established biological rationale, selective degradation of SMARCA4 should offer significant clinical benefit in multiple disease settings. Here, we report first-in-class SMARCA4 selective degraders that demonstrate distinct cellular phenotype. As part of the initial design plan, selective SMARCA2/4 Bromodomain inhibitors and specific ligands of several E3 ligases were chosen to arrive at different degrader designs. Several linkers and different exit vectors were considered to construct a variety of hetero-bifunctional degrader molecules. Our proprietary ternary complex modeling algorithm, ALMOND4 (ALgorithm for MOdeling Neosubstrate Degraders) helped in prioritizing the designs. Shortlisted compounds were synthesized and profiled in multiple cellular assays to understand their potential to degrade SMARCA4 selectively. These compounds exhibited very potent anti-proliferative activity in cell lines of hematological and prostate cancer origin (e.g. MV-4-11, VCaP, etc.) whereas no significant potency is observed in SMARCA4 mutant cell lines. Identified compounds have shown selective degradation of SMARCA4 over SMARCA2 in multiple cell lines. Few potent SMARCA4 selective degraders were optimized for pharmacokinetic properties and further evaluation of tolerability and efficacy in rodents is in progress. Citation Format: Chandrasekhar Abbineni, Kiran Aithal, Bilash Kuila, Sandeep Vitthal Dukare, Arya Desai, Megha Goyal, Khaji Abdul Rawoof, Gauri Rahul Petkar, Gopinath CH, T Jagadeesh Kumar, Charamanna KB, Rahul B. Chavan, Nandish C, Suraj Tgore, Leena Khare, DS Samiulla, Subhendu Mukherjee, Thomas Antony, Kavitha Nellore, Sanjeev Giri, Rajesh Eswarappa, Girish Daginakatte, Susanta Samajdar, Murali Ramachandra. Identification of first-in-class paralog selective SMARCA4 degraders for treatment of hematological and prostate malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7019.

Abstract 7463: A linker platform for antibody drug conjugates (ADCs): expanding the therapeutic window

Abstract Although ADCs have demonstrated great clinical promise, their therapeutic window remains limited due to the complexity of this drug format. Factors contributing to this narrow safety window include the instability of the linkers, accelerated plasma clearance due to the hydrophobicity of the ADCs, and off-target toxicity caused by nonspecific uptake by non-antigen expression healthy cells, free payload pre-release in the circulation system, low tumor retention due to heterogeneity of tumor antigen level and ADC DAR value By preserving the essential functions of the antibody and the payload, an optimized linker can significantly enhance ADC therapeutic window by increasing hydrophilicity and stability while reducing non-specific endocytosis by non-tumor cells. We have designed a panel of novel linkers focused on improving hydrophilicity and stability of ADC, with a particular emphasis on reducing its off-target endocytosis. A key feature of our linkers is the inclusion of a negative charged side chain and hydrophilic chaperone, which not only minimizes non-specific uptake by non-tumor cells but also greatly enhances its hydrophilicity. The side chain linker with this special architecture allows efficient enzymatic cleavage to ensure rapid payload release intracellularly, and lead to superior bystander cell killing effect than the ADC with GGFG-Dxd linker system. One example exploiting the proprietary linker is Trastuzumab-BL-001-exatecan with a drug-to-antibody ratio (DAR) of 8, demonstrated hydrophilicity comparable to Trastuzumab-GGFG-Dxd (biosimilar) in HIC analysis, with no aggregation detected by analytical SEC. This ADC exhibited excellent stability, maintaining high solubility, and cytotoxicity under stressed conditions. It also resisted deconjugation in the presence of high concentration of N -acetylcysteine and showed exceptional stability in human and mouse plasma. Pharmacokinetic studies in rats indicated favorable pharmacokinetic properties, while efficacy testing in the NCI-N87 tumor model revealed comparable performance to Trastuzumab-GGFG-Dxd. Additionally, BLB-101 ADC, featuring the same linker, was well tolerated in non-human primates following repeated administration. In conclusion, our novel linker designs offer significant advantages, with features that could expand the therapeutic window of current ADCs by improving their stability, reducing off-target effects, increasing bystander activity, and maintaining efficacy. We are also expanding the utility of this class of linkers to enable ubiquitous design of dual-payload-ADC Degrader-Antibody-Conjugates. Citation Format: David Yi, Wen Zhang, John Liu, Suk Lee, Alice Chen. A linker platform for antibody drug conjugates (ADCs): expanding the therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7463.