Favorable Safety Profile Research Articles

Abstract 4702: MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space

Abstract Antibody-drug conjugates (ADC) are a type of the fastest growing anticancer drug modalities. However, the traditional ADC technologies constrain on therapeutic development, requiring overexpressed targets, high internalizing antigens and efficient intracellular processing. To this end, MediLink’s TMALIN platform was developed, with an irreversible pyrimidine coupling anchor and a valyl dipropyl-lysyl glycinamide-methylene (-VK*G-NHCH2-O-) cleavable sequence. The TMALIN platform is featured as a high hydrophilicity linker-payload, remarkable stability in circulation, coupled with an efficient payload release mechanism which is achieved through the cleavage both extracellularly in the tumor microenvironment and intracellularly within lysosomes. A site-specific and homogeneous conjugation using TMALIN platform provides highly stable ADC in circulation. It is evidenced by less than 2% payload drop-off after a 21-day incubation in plasma under physiological conditions in vitro. This feature is observed in over 7 ADCs developed using the TMALIN platform. Additionally, the high stability was also illustrated by the pharmacokinetic profiles in NHP studies and clinical trials, with the overlapped curves of ADC and TAb in all TMALIN platform-based ADCs. Apart from the stability, a battery of in vitro and in vivo studies showed the linker-payload efficiently releases the payload in both tumor cells and the tumor microenvironment. Due to the high stability in circulation and efficient cleavage within tumors, the TMALIN platform ADCs have shown promising efficacy and good safety windows in preclinical studies. In xenograft mouse models, including those with low antigen expression, these ADCs have demonstrated tumor regressions and good tolerability. This highlights the potential of the TMALIN platform in improving the efficacy of ADCs and expanding their therapeutic applications. Furthermore, favorable safety profiles have been observed in clinical trials and/or pivotal non-human primate (NHP) studies for ADCs developed using the TMALIN platform. No drug-related adverse effects in organs were identified including the lungs, liver, or kidneys for those ADCs. In summary, the TMALIN platform addresses the unmet need of ADCs by releasing the payload extracellularly in tumors and tumor microenvironments on top of the known intracellular lysosomal cleavage mechanisms. This enables the advancement of ADC development, and has the potential to expand the field of ADC therapy and improve patient outcomes. Citation Format: Tongtong Xue, Liang Xiao, Qigang Liu, Shuai Song, Ailin Shan, Xiaozhou Zhang, Sasha Stann, Jiaqiang Cai. MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space [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 4702.

Abstract 3815: Clinical efficacy and safety of HER2 targeted therapy in patients with metastatic colorectal cancer: A systematic review and meta-analysis

Abstract Background: Therapies targeting HER2 have shown clear benefit in patients with breast or gastric cancer,; and their use in metastatic colorectal cancer (mCRC) has been increasingly explored in recent years. HER2 overexpressed or amplified (HER2-positive) mCRC is seen in 2-8% of all mCRC cases and is enriched in RAS wild-type (WT) tumors, representing a patient population that may benefit from anti-HER2 therapies. Although, several single-arm and some non-comparative randomized clinical trials have now tested the use of diverse dual anti-HER2 therapies in mCRC, no summative analysis have been performed. We conducted a systematic review and meta-analysis to evaluate the overall clinical benefit (efficacy and safety) of dual-HER2 inhibition in patients with RAS-WT HER2-positive mCRC. Methods: A systematic review of available literature was performed for clinical trials involving patients with mCRC treated with HER2 targeting therapy. Two independent reviewers selected studies and extracted data from PubMed and abstracts published at major international oncology meetings during the past 5 years. Case reports and other studies not meeting inclusion criteria were excluded, therefore a total of 13 studies were used for analysis. Patients with RAS-mutant tumors within each trial were excluded due to known limited benefit of dual HER2 inhibition in this subset. Inverse variance method with random-effects model was used for meta-analysis to determine the overall effect of the therapy on overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) and treatment-related adverse events (TRAE). Results: Systematic review identified 9 study cohorts which were utilized in the meta-analysis, encompassing a total of 279 patients with mCRC who received Trastuzumab (T) in combination with either Pertuzumab (P), Tucatinib (Tu), Pyrotinib (Py), or Lapatinib (L). Collectively, dual HER2 inhibition showed an ORR of 35.1% (95% CI: 29.4-41.0%), a DCR of 68.3% (95% CI: 62.5-73.9%), and a PFS of 5.2 months (95% CI: 4.4-6.0 months). Pooled TRAE of grade ≥ 3 across studies was 29.9% (95% CI: 18.8-42.4%) but displayed high inter-study heterogeneity (p<0.0001, I2 = 79%), mainly driven by one trial (Pyro-HER2; TRAE = 75.0%, 95% CI: 50.9-91.3%). Conclusions: Overall, this comprehensive meta-analysis confirms that dual HER2 targeting therapies yield meaningful clinical efficacy, with favorable safety profiles, and improve outcomes in patients with RAS-WT HER2-positive mCRC. Citation Format: Oscar E. Villarreal, Lianchun Xiao, Joelle Allam, Alexey Sorokin, Ying Yuan, Scott Kopetz, Kanwal Pratap Raghav. Clinical efficacy and safety of HER2 targeted therapy in patients with metastatic colorectal cancer: A systematic review and meta-analysis [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 3815.

Abstract 3132: A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models

Abstract Antibody-drug conjugates (ADCs) are emerging therapeutic agents for targeted cancer treatment with remarkable clinical success. However, tumor heterogeneity and microenvironment complexity are still the major factors contributing to drug resistance, recurrence, and metastasis, which cannot be resolved with a mono therapy strategy. We’ve developed a novel dual drug ADC (BiADC), BR113, by conjugating an anti-hTrop2 antibody with a drug linker of a Topoisomerase I inhibitor payload, and another drug linker of an immune stimulator. Our studies show that the homogeneous ADC containing two distinct payloads is a new promising drug class, with two different anti-tumor MOAs, synergistic antigen-specific cell killing potency, desirable pharmacokinetic profiles, and minimal toxicity at therapeutic doses. In the cancer cells and human PBMC co-culture assay, BR113 exerts significantly more potent killing on cancer cells than two individual single-drug ADCs and the combination of two single-drug ADCs, which suggested a synergy on anti-tumor effects of this dual-drug ADC. In the xenograft mouse model studies, BR113 exhibits greater treatment effect and survival benefit than the combo of two single-drug ADCs at various doses. This robust superior anti-tumor activity is further confirmed in a hTrop2-MC38 syngeneic model with a prolonged anti-tumor protection against the tumor re-challenge. Finally, the novel BiADC, BR113, is well tolerated in hTop2 humanized mice after repeated administration. Our findings highlight the therapeutic potential of the BiADC format for treating refractory advanced solid tumors. The outstanding anti-tumor activity across various CDX tumor models and its favorable safety profile in mice support further evaluation of the innovative BiADC, BR113. Citation Format: Jie Zhu, Xiaobei Zhao, Eileen Li, Hong Xu, Lei Nie, Gang Chen, Haibing Wang. A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3132.

Abstract 1812: Development of novel topoisomerase 1 inhibitor PBX-7 payload-based ADC including tandem cleavable linker system

Abstract Antibody-Drug Conjugates (ADC) employing camptothecin-based payloads are gaining high interests as a new modality to treat various cancers, especially solid tumors. Among them, novel ADCs containing synthetic camptothecin-based payloads specially optimized for ADC applications (e.g., DXd) are of special interest due to their successful clinical results. Despite the remarkable success of ADCs exploiting these payloads, such as Enhertu (DS-8201a), there are still clear unmet needs, including the improvement of safety profile (minimization of ILD and neutropenia) and the development of novel ADCs with multiple MoA payloads to combat cancer heterogeneity. To address these unmet needs, we have synthesized and evaluated a series of PBX-7 payloads, derived from novel camptothecin FL118, a potent, dual inhibitor of Top 1/anti-apoptotic pathway known for its favorable safety profile. These compounds showed strong in vitro cytotoxicity across various cancer cell lines and inhibition of Top 1 comparable to DXd or other related camptothecin compounds. They had excellent safety profile in the preliminary toxicity study in mice that can be attributed to their physicochemical properties. PBX-7 series payloads based ADC exhibited superior cellular cytotoxicity and in vivo tumor regression compared to Enhertu in Her2-positive cell line and CDX mouse model. Notably, PBX-7 based ADC demonstrated remarkable efficacy in reducing tumor volume more than Enhertu in the T-DM1-resistant JIMT-1 xenograft mouse model. Furthermore, PBX-7 based ADC have bystander effects similar to Enhertu, leading to reduced viability of antigen-negative cells when co-cultured with antigen-positive cell lines. In NHP toxicity study in Cynomolgus monkeys, no sign of toxicity was observed when dosed with Trastuzumab-PBX-7016 up to 30mg/kg. As another way of addressing unmet needs, we synthesized novel linker including tandem cleavage site to reduce payload mediated toxicity. Two distinct enzyme, beta-glucuronidase and cathepsin B cleave linker sequentially. This linker system has a higher stability during circulation in the body and higher specific payload release in the tumor tissue than single cleavable linker. In conclusion, our research shows the promising potential of novel camptothecin PBX-7 based ADCs and tandem cleavable linker system as potent and safe ADC candidates for cancer therapy. These advancements hold great promise in addressing the current challenges in ADC development with their enhanced safety profiles and targeted efficacy. Citation Format: Younghwa Chun, Areum Go, Hyun Yong Cho, Byeong Sung Lee, Seunggun Shin, JaeHyeon Lee, Sohui Kweon, Hyemin Han, Suhyun Park, Doo Young Jung. Development of novel topoisomerase 1 inhibitor PBX-7 payload-based ADC including tandem cleavable linker system [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 1812.

Abstract 5913: TEAD1/4 inhibitors exhibit deeper biological impact and broader activity compared to TEAD1-only inhibitors in both monotherapy and combination without additional kidney toxicity

Abstract The TEAD transcription factors in association with the YAP/TAZ co-activators drive the expression of pro-proliferative and pro-oncogenic genes that underlies the transformed phenotype of many carcinomas. In addition, YAP-TEAD transcriptional activity is emerging as a major resistance mechanism for diverse precision oncology drugs, with the most extensive data for resistance to drugs targeting the MAPK pathway. There is a strong interest in generating inhibitors of YAP/TEAD transcriptional activity with TEAD palmitic acid site inhibitors having demonstrated encouraging pre-clinical activity and now clinical activity with confirmed objective responses with the TEAD1/2/3 inhibitor VT3989. However, the TEAD1-preferential inhibitor IK930 did not yield any objective responses but also showed a more favorable safety profile especially with respect to proteinuria. These contrasting clinical read-outs provide a good lead-in to a critical design challenge/question of TEAD palmitic acid site inhibitors: the optimization of inhibitory profile against the respective four TEAD paralogs (TEAD1-4). In depth bioinformatic analyses led Sporos investigators to conclude that while TEAD1 inhibition was a minimum requirement for anti-neoplastic activity, inhibition of other paralogs would be necessary for maximizing biological impact and a TEAD1/4 inhibitor would provide the best balance of anti-neoplastic activity and toxicity. Activity against TEAD2 was identified as counter-productive associated with a context-specific paradoxical stimulation of cell proliferation and tumor growth while activity against TEAD3 was flagged as a major driver of podocyte effacement and kidney toxicity. Here, we provide novel corroborating data supporting this selection of inhibitory profile. We provide an update on the pre-clinical efficacy and toxicology of SPR1, Sporos’s TEAD1/4 preferential inhibitor which favorably contrasts with other TEAD inhibitors such as the TEAD1/3/4 inhibitor VT3989 and the TEAD1 inhibitor VT103 and IK930 in both the monotherapy and combination setting. We show that 1) SPR1 displays broader and deeper cell-based activity and extends the utility of TEAD inhibitors outside of mesothelioma and NF2 mutants 2) SPR1 shows stronger activity than TEAD1-only inhibitors in combination with MAPK and EGFR inhibitors in vitro and in vivo 3) SPR1 does not cause proteinuria in mice; dogs or rats even above therapeutic doses 4) SPR1 does not show the context-specific stimulation of tumor growth in Lung PDX previously observed with VT3989 and other inhibitors that include TEAD2 in their profile. Taken together - the data suggests SPR1 is positioned to become a best-in-class TEAD palmitic acid site inhibitor with broad utility in both monotherapy and combination setting. Citation Format: Florian Muller, Selvi Kunnimalaiyaan, Parth Mangrolia, Jill Olson. TEAD1/4 inhibitors exhibit deeper biological impact and broader activity compared to TEAD1-only inhibitors in both monotherapy and combination without additional kidney toxicity [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 5913.

Abstract 6023: 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity

Abstract Despite significant developments over the last few decades, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Prostate specific membrane antigen (PSMA), a prostate cancer-specific membrane marker which directly correlates with androgen independence, metastasis, and disease progression, has been well-established as a radioligand target for the diagnosis and treatment of mCRPC. Lutetium-177 (177Lu)-PSMA-617 (PLUVICTOTM) was approved in 2022 for the treatment of progressive PSMA-positive mCRPC. However, only 30% of patients showed a radiological response in the registrational trial. Meanwhile, recent clinical experience has shown very limited efficacy in low PSMA expressing patients. These data collectively request for further improvement in the clinical benefit of PSMA-targeted radiotherapy. Actinium-225 (225Ac), an alpha emitter, is significantly more potent on cancer cell killing and has a shorter range in tissue penetration when compared to the beta emitter 177Lu. Such a profile supports the development of 225Ac based radiotherapies. Using our proprietary Clear-XTM technology platform, we developed 225Ac-FL-020, a novel 225Ac-based PSMA radioligand therapy candidate which has demonstrated prominent pre-clinical properties. We have assessed the binding affinity against PSMA of the non-labeled vector FL-020 in vitro. The in vivo biodistribution profile of FL-020 was characterized by SPECT/CT imaging and biodistribution using Indium-111 (111In)-FL-020 in PSMA high LNCaP tumor-bearing nude mice. In addition, anti-tumor activities of 225Ac-FL-020 were evaluated in the LNCaP xenograft model and directly compared to 225Ac-PSMA-617. FL-020 was found bound to LNCaP cells with an IC50 value of 51.55 nM. Meanwhile, the off-target screening showed that less than 50% inhibition of binding or activity was observed by FL-020 at 10 µM against 85 targets including receptors, ion channels, enzymes, and transporters, indicating the high selectivity of FL-020. Moreover, 111In-FL-020 displayed a very promising in vivo distribution profile with high and sustained tumor uptake and fast systemic clearance. Furthermore, 225Ac-FL-020 exhibited superior anti-tumor activity compared to 225Ac-PSMA-617 at the same dose level (10 KBq/mouse) in the LNCaP xenograft model with a favorable safety profile as indicated by body weight and hematological parameters. A mechanistic study was also conducted in 225Ac-FL-020-treated LNCaP tumor samples where DNA double-strand breaks and tumor cell apoptosis were observed, confirming the MOA of alpha emitters. Taken together, these results collectively demonstrate that 225Ac-FL-020 is a potent and selective PSMA-targeting radioligand therapy candidate with superior anti-tumor activity and a favorable safety profile warranting further clinical development. Citation Format: Fa Liu, Jiangwei Zhang, Junyu Yang, Kirstine T. Thrane, Mathias W. Hallund, Rikke V. Grønlund, Nicholas C. Wong. 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity [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 6023.

Abstract 1322: A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo

Abstract Introduction Multiple myeloma (MM) is the second most prevalent hematopoietic malignancy with an overall 5-year survival rate of 58%. B cell maturation antigen (BCMA) is selectively expressed on normal and malignant plasma cells, making it an attractive MM target. Anti-BCMA chimeric antigen receptor (CAR) T cell therapy and T cell engagers have shown promise as treatment options for certain MM patients; however, potential toxicities associated with these anti-BCMA therapies, such as cytokine release syndrome (CRS), have limited broader utilization. Much like T cells, natural killer (NK) cells are cytolytic and have demonstrated an innate capacity to reduce tumor burden while exhibiting a more favorable safety profile in clinical testing. To that end, we developed a cryopreserved allogeneic cell therapy comprised of genetically modified human umbilical cord blood-derived (CB) NK cells transduced with a gammaretroviral vector, which incorporates genes for an anti-BCMA CAR and soluble human interleukin-15 (sIL-15). Referred to hereafter as anti-BCMA CAR-NK, it exhibits both innate NK- and CAR-mediated killing in vitro and robust in vivo activity against established MM tumors. Methods CBNK cells were isolated from donor cord blood units, propagated using feeder cells, transduced with a gammaretroviral vector to express an anti-BCMA CAR and soluble human IL-15, and propagated further before harvest and cryopreservation. Donor-equivalent untransduced (UTD) NK cells were generated via the same process but without the transduction step. Cryopreserved anti-BCMA CAR-NK and UTD NK cells were used for in vitro studies, including short-term killing, as well as for evaluation of activity against established MM tumors in vivo using the MM.1S-Luc4 model. Results The anti-BCMA CAR was successfully expressed across all batches of anti-BCMA CAR-NK generated. Results from an in vitro cytotoxicity assay indicated that anti-BCMA CAR-NK kills BCMA expressing MM.1S-Luc4 tumor cells and secretes IFNγ, TNFα, and granzyme B at greater amounts compared to donor-equivalent UTD NK. Anti-BCMA CAR-NK and UTD NK cells also demonstrated equivalent cytotoxicity towards BCMA non-expressing cell lines, JJN3-Luc BCMA KO and NCI-H520, in an effector:target cancer cell dependent manner, highlighting the potential for treatment with anti-BCMA CAR-NK post-prior BCMA therapy. In in vivo studies, anti-BCMA CAR-NK exhibited robust anti-tumor activity in an MM.1S-Luc4 NSG mouse xenograft model, with no signs of anti-BCMA CAR-NK related body weight loss. Conclusion A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated killing in vitro and robust in vivo activity in MM tumor models. Preclinical data supports future clinical evaluation in relapsed/refractory MM patients who have received prior BCMA therapy and IND enabling studies are ongoing. Citation Format: LeeAnn Talarico, Christina Wong, Chunyan Pang, Taylor Hickman, Chenqi Hu, Amy Shaw, Emily Wisniewski, Shao-Chiang (Michael) Lai, Pranjal Sharma, Shaun Moore, Luan Nguyen, Kayla Rhuda, Saurin Patel, Paul Lin, Rafet Basar, Shawn Cogan, Kat Sofjan, Arun Ramamurthy, Aaron Handler, Kathryn Fraser, Yana Wang, Katayoun Rezvani, Michael D. Curley. A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo [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 1322.

Abstract 4532: Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors

Abstract Cancers exhibiting homologous recombination (HR) repair deficiencies due to mutations in genes like BRCA1 or BRCA2 rely on alternative DNA damage response (DDR) pathways to uphold genomic integrity. This vulnerability allows for the targeted disruption of these pathways through synthetic lethality approaches. DNA polymerase theta (Polθ, encoded by POLQ) assumes a crucial role in mending DNA double-strand breaks (DSB) via microhomology-mediated end-joining (MMEJ), one of the principal pathways for repairing DNA DSB. Notably, Polθ expression is minimal in normal tissues but becomes upregulated in HR-deficient cancer cells, positioning Polθ as a prime target for synthetic lethality in HRD cancers. Reversion mutations in HR-related genes frequently employ micro-homology deletion mechanisms. Consequently, the inhibition of Polθ holds the potential to thwart the emergence of PARPi resistance, primarily driven by BRCA reversion mutations. In this context, we introduce DAT-1000A, a novel small-molecule Polθ inhibitor exhibiting potent activity with a single-digital nanomolar IC50 value. Its efficacy in MMEJ functional assays validates its on-target cellular activity. Assessments of cell viability in HR-deficient BRCA2-KO DLD-1 cells, compared to their parental counterparts, reveal a striking synthetic lethality window exceeding 500-fold. Additionally, DAT-1000A displays synergistic anti-proliferative effects when combined with PARP inhibitors in BRCA1/2-mutated cells. In BRCA1/2-mutated xenograft models, DAT-1000A demonstrates robust and sustained tumor regression, as evidenced by the level of γH2AX, a common marker for double-strand breaks, which closely correlates with anti-tumor efficacy. Importantly, high-dose DAT-1000A treatment in mice elicits no significant abnormalities, underscoring its excellent tolerability. Collectively, DAT-1000A emerges as a novel Polθ inhibitor, offering both potent activity and a favorable safety profile, and holds promise for advancing therapeutic options in the realm of HRD cancer treatment. Citation Format: Dan Yan, Yao Zhang, Yan Zhang, Jingxi Zhang, Bin Wang, Huixian Chen, Jingxue Shi, Xiaoling Lin, Jincong Zhuo, Kevin Zhou. Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient 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 4532.

Abstract 6013: VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response

Abstract Background: Targeting specific DNA Damage Response (DDR) proteins has been worldwide studied and developed, with the example of PARP inhibitors being the only approved treatments in this field. Here, we describe the antineoplastic and immunomodulatory effects of VIO-01, a first-in-class DNA decoy-cholesterol conjugate, that operates as a pan repair proteins decoy, resulting in constitutive exhaustion of the DNA damage response. Methods: VIO-01-induced DDR proteins trapping and cell cytotoxicity were examined in both homologous recombination proficient (HRP) and deficient (HRD) cancer cells. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. RNAseq analysis in HRP/HRD ovarian cancer cells was employed to uncover the molecular mechanisms underlying VIO-01 effects. Effects on the innate and adaptive immune responses were assessed by following T-cell mediated anti-tumor cytotoxicity. VIO-01 antitumor efficacy and biodistribution were also assessed. Additionally, the interest of cholesterol as a vector for VIO-01 was analyzed compared to other ligands like the folate receptor ligand. Results: VIO-01 bounds to several DDR proteins, such as PARP1, KU70/80, MRN complex and MSH2/MSH3 with high affinity, resulting in an abrogation of single- and double-strand break repair. In line with this, and through global transcriptome analysis, VIO-01 treatment resulted in notable downregulation of Base Excision Repair, NHEJ and Nucleotide Excision Repair pathways in both HRP/HRD cell lines. Additionally, VIO-01 elicited the activation of the immune system and inflammatory responses in ovarian cancer cells. Conjointly, VIO-01 treatment induced a significant differentiation of monocytes into non-classical monocytes, leading to a shaping of the tumor microenvironment via CD8+ T cell recruitment in early stage of activation. The activity of VIO-01 was specific to tumor cells, while sparing healthy and immune cells, at odds with PARP inhibitors. In line with in vitro results, VIO-01 mediated antitumor efficacy in vivo coupled to tumor-targeting T-cell responses. These interesting effects were driven by a favorable ADME/PK profile, showing a long-lasting VIO-01 residence time VIO-01 into tumors (at least 3 days post treatment), coupled to a clear hijacking from the liver and a rapid blood clearance, ensuring a minimal toxicity. Moreover, regulatory toxicology studies demonstrated a favorable safety profile of VIO-01 in non-human primate with the major findings being a transient increase in complement factors induced by the binding of VIO-01 to factor H, which was not observed in human serum, predicting favorable safety profile in humans. Conclusions: Our results provide a preclinical rationale for using VIO-01 to trigger DNA damage exhaustion and an antitumor immune response, paving the way for rapid clinical application in patients bearing HRD or HRP tumors. Citation Format: Vlada Zakharova, Chloe Doizelet, Nicolas Babault, Marie-Christine Lienafa, Fetta Mazed, Megane Debiais, Pierre Vilela, Emilie Perroud, Wael Jdey. VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response [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 6013.

Abstract 5255: A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential

Abstract Background: CAR-αβT cells have made limited advancement in treating solid tumors. Vδ1 T cells, a subtype of γδT cells, are rare in human peripheral blood, but they are the main tissue-resident immune cells found in epithelial and mucosal tissues. This suggests that Vδ1 T cells might be a potential modality for treating solid tumors. To explore this possibility, we hypothesized that Vδ1 T cells modified with B7-H3 CAR (B7-H3-CAR-Vδ1 T) could have great potential of anti-solid tumor activity, making them a promising candidate for further development as a universal cellular therapy product. Methods: A proprietary manufacturing procedure to produce Vδ1 T cells from the leukopak of healthy donors has been established under GMP compliance. The immune checkpoint molecule B7-H3 was selected as the target. The allogeneic B7-H3-CAR-Vδ1 T cells carrying IL-2 (UTAA06), which displayed the most prolonged anti-tumor activity, were chosen for comprehensive functional validation. Single-cell sequencing and mass spectrometry flow cytometry were utilized to characterize the biology of UTAA06. In vivo effectiveness of UTAA06 was confirmed in animal models. Results: A clinical-grade manufacturing process for Vδ1 T cells is developed. This process results in a purity of over 95% of Vδ1 T cells, with an expansion of more than 20,000-fold, and a CAR lentiviral transduction efficiency of over 50%.žSingle-cell sequencing and mass cytometry analysis both demonstrated that the Vδ1 T cells produced by our approach exhibited high expression levels of natural cytotoxic receptors (including NKG2D, DNAM1, and NKp series receptors), chemokine receptors (such as CXCR3 and CXCR6), anti-tumor related molecules (such as CD27 and CD137), and low expression levels of inhibitory molecules (such as PD1 and LAG3).ž UTAA06 cells did not produce IL17A, which is a signal protein with pro-tumor activity. Meanwhile, UTAA06 demonstrated significant and specific inhibitory effects on the growth of various solid tumor cell lines.žUTAA06 cells were found to significantly suppress tumor cell growth in solid tumor mouse models. The complete remission (CR) for each tumor model was 83.3% of neuroblastoma (SK-N-AS, N=6), 66.7% of pancreatic cancer (SW1990, N=6), 100% of lung cancer (A549, N=6), and 70% of colorectal cancer (HCT-15, N=10) respectively. In addition, a potent capacity of UTAA06 to penetrate tumor tissues with a favorable safety profile in mice was observed. Conclusion: UTAA06 exhibited extraordinary anticancer properties both in vitro and in vivo against solid tumors. Additionally, the outstanding manufacturing process of UTAA06 will facilitate the clinical application of this product. Citation Format: Licui Jiang, Changsong Qi, Fengtao You, Shufen Xiang, Ping Zhang, Min Wang, Hai Wu, Kunmin Yan, Jiequn Huang, Huimin Meng, Nan Yang, Shirley Zhang, Lin Shen, Lin Yang. A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential [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 5255.

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI > 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

Abstract 4503: Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2

Abstract SWI/SNF (BAF) complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a bromodomain. SMARCA4 protein expression is lost in some cancers due to loss-of-function (LOF) mutations and homozygous deletions, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deficient cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have recently identified a series of orally bioavailable SMARCA2 selective degraders that demonstrate robust efficacy in pre-clinical animal models with favorable pharmacokinetic properties and safety profiles. Our nomination candidate PRT7732 exhibits >1000x selectivity for SMARCA2 over SMARCA4 in cell-based assays, with DC50 values in cancer cell lines in the low nanomolar range. The PRT7732-induced SMARCA2 degradation was rescued by a proteasome inhibitor and neddylation inhibitor, indicating ubiquitin-proteasome dependent degradation. Furthermore, PRT7732 does not change levels of known CRBN ligand neo-substrates such as IKZF2/3, GSPT1 and SALL4. PRT7732 inhibits only SMARCA4-deficient cancer cell proliferation with IC50 values ranging from 5.0-50 nM, but not SMARCA4 WT cells in vitro. Oral administration of PRT7732 resulted in near-total degradation of SMARCA2 protein with complete selectivity over SMARCA4 protein in a SMARCA4 WT lung cancer model in mice, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. PRT7732 oral daily administration showed significant tumor growth inhibition of SMARCA4-deficient lung cancer xenograft models at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for 24h post dosing. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Artem Shvartsbart, Koichi Ito, Joseph Rager, Michael Hulse, Anjana Agarwal, Komali Vykuntam, Jessica Burtell, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Nick Stahl, Monisha Sivakumar, Anthony Reichelderfer, Jack Carter, Alexander Grego, Andrew Moore, Neha Bhagwat, Ross Kuskovsky, Shanthi Ganesan, Stefan Ruepp, Tom Emm, Philip Pitis, Corey Basch, Klare Bersch, Yongchun Pan, Song Mei, Raul Leal, John Rose, Dani Roth, Chaoyi Xu, Ganfeng Cao, Kris Vaddi, Sang Hyun Lee, Sandy Geeganage, Andrew Combs, Peggy Scherle. Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2 [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 4503.

Abstract 5947: Discovery of ACE-47228211, a highly potent and selective SOS1 inhibitor

Abstract Dysregulation of receptor tyrosine kinase (RTKs) and KRAS signaling is the most frequent causes of cancer development and progression. SOS1 is one of the major guanine nucleotide exchange factors (GEFs) that facilitates the conversion of the inactive GDP-bound KRAS “off” state to the activated GTP-bound KRAS “on” state. SOS1 is therefore required for dysregulated RTKs, and wildtype and mutant KRAS proteins to drive cancer cells. In addition, SOS1 plays a crucial role in modulating the reactivation of the RAS/MAPK signaling upon the treatment of RAS/MEK/ERK inhibitors. Thus, blocking SOS1 and KRAS protein-protein interactions could be an effective strategy for targeting KRAS-MAPK driven cancers. Here, we report the discovery of a highly potent and orally bioavailable small molecule SOS1 inhibitor, ACE-47228211. This molecule has a unique novel scaffold. It exhibits an IC50 of 8.5 nM in the SOS1-KrasG12D (GDP) exchange assay and 5.0 nM in cellular ERK phosphorylation assay in H1957 cells. ACE-47228211 at 3 mg/kg (BID) demonstrated significant tumor growth inhibition in MiaPaca-2 KRAS G12C tumor model than MRTX0920 at 50 mg/kg (BID). When combined with Sotorasib (5 mg/kg QD), a Kras G12C inhibitor, ACE-47228211 at 3 mg/kg exhibited synergistic effect similar to MRTX0920 at 50 mg/kg. Further, ACE-47228211 demonstrated synergistic effects with EGFR and FGFR2 inhibitors in RTK mutant tumor models. ACE-47228211 has excellent ADME and physical properties with low risks of hERG, CYP inhibition and drug-drug interactions. The evaluation of ACE-47228211 in a safety panel of 87 targets and a kinase panel of >500 targets shows that this molecule has favorable selectivity and safety profiles. The molecule is currently in IND enabling stage of development. Citation Format: Junmei Wang, Wenqian Li, Sanjeev Kumar, Weitao Pan, Zhiming Wen, Tinggui Yin, Genshi Zhao, Kuo-Long Yu. Discovery of ACE-47228211, a highly potent and selective SOS1 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 5947.

Abstract 1414: HBM9014, a first-in-class fully human anti-LIFR antibody with excellent preclinical efficacy and safety profile

Abstract Background: Leukemia inhibitory factor (LIF) and cardiotrophin like cytokine factor 1 (CLCF1) are multi-functional cytokines from IL-6 family. They are reported to be highly expressed in a variety of human cancers such as pancreatic carcinoma, cholangiocarcinoma, NSCLC, GBM, head and neck cancer etc. Their expression is associated with poor prognosis and survival. LIFR is the key component of LIF/CLCF1 receptors. Antibody targeting LIFR has the advantage to block both LIF and CLCF1 pathways thus inhibiting tumor growth more effectively than products to block single cytokines. Methods: Fully human Anti-LIFR antibodies were generated from H2L2 Harbour Mice® transgenic platform. In vitro function of the antibodies was tested for their abilities to block LIF/CLCF1 induced downstream signaling pathway and tumor spheroid proliferation. In vivo efficacy was tested in multiple tumor models as monotherapy and in combination with cisplatin. DRF toxicokinetics was evaluated on NHP with a 4-week repeat-dose toxicity study. Results: HBM9014, a fully human anti-LIFR antibody generated by H2L2 Harbour Mice®, specifically binds LIFR thus blocks the interaction between LIF/CLCF1 and their respective receptors, inhibits downstream STAT3 phosphorylation, and reduces exogenous and endogenous LIF induced tumor spheroid proliferation in vitro. Furthermore, HBM9014 shows potent anti-tumor activity in multiple tumor models in vivo. In a GLP NHP 4-week repeat-dose toxicity study, HBM9014 is well tolerated up to 150 mg/kg with no discernable drug-related toxicity. Conclusions: HBM9014 specifically binds to the key component of LIF and CLCF1 receptor, LIFR, thus blocks both LIF and CLCF1 signaling pathways. HBM9014 shows significant cell proliferation inhibition alone or in combo with small molecule inhibitors in 3D culture of multiple cancer cell lines. It also shows significant in vivo antitumor efficacy as monotherapy, and enhanced efficacy in combination with Cisplatin in multiple mouse models. Importantly, despite the benefits of double blocking, HMB9014 has an excellent safety profile. Taken together, HMB9014 is a promising drug candidate for advanced cancer with favorable efficacy and safety profiles that warrant clinical validation. Citation Format: Jue Zeng, Dongxu Zhang, Louis Liu, Lingbing Zhang. HBM9014, a first-in-class fully human anti-LIFR antibody with excellent preclinical efficacy and safety profile [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 1414.

Abstract 1958: EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs

Abstract RET fusions are an oncogenic driver mutation occurring in 1-2% non-small cell lung cancer (NSCLC). Currently, RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved for this patient population, demonstrating favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC. However, tumors eventually become refractory to selective RET inhibitor monotherapy. We sought to identify signaling pathways which mediate RET-independent acquired resistance to RET inhibitors and effective therapeutic combinations to overcome resistance. Using LC-2/ad (CCDC6-RET) NSCLC cells, we assessed the expression and activation of critical signaling pathways at a proteomic level following RET knockdown using reverse-phase protein array. RET knockdown resulted in rapidly increased adaptive upregulation of EGFR, HER2, MET, mTOR and MEK pathway signaling; observations were confirmed by Western Blotting. To determine whether EGFR or MET signaling facilitate RET inhibitor resistance, LC-2/ad and NCCE-TH1101 (KIF5B-RET) NSCLC cells were treated with increasing concentrations of selpercatinib or pralsetinib with or without ligands for these receptors, EGF or HGF respectively, and after five days cell viability was measured by CellTiter Glo. EGFR and MET signaling promoted RET TKI resistance in both cell lines. Next, to evaluate whether blockade of EGFR or MET signaling could enhance the activity of RET inhibitors, we treated LC-2/ad and NCCE-TH1101 cells with RET TKIs alone or in combination with the EGFR TKI erlotinib or poziotinib, or the MET inhibitor SU11274. We observed an additive effect with EGFR but not MET inhibitors when combined with RET TKIs. Western blot analysis revealed that EGFR signaling could reactivate mTOR and MAPK signaling after RET TKI treatment. Therefore, we next tested whether inhibition of these downstream pathways might enhance RET TKI sensitivity. We observed that treatment of LC-2/ad and NCCE-TH1101 cells with an mTOR inhibitor (everolimus) or a MEK inhibitor (trametinib) in combination with RET TKIs increased the anti-tumor activity or RET inhibitors. Finally, we established cells with acquired resistance to selpercatinib and pralsetinib. RET TKI resistant cells did not harbor secondary RET fusion mutations but underwent epithelial to mesenchymal transition and exhibited pan-RET inhibitor resistance. Moreover, RET TKI resistant cells showed increased cell surface expression of EGFR and MET by flow cytometry. RET TKI resistant cells were sensitive to mTOR or MEK inhibitors in combination with RET TKIs. Collectively, our findings indicate that in RET-fusion positive NSCLC tumor cells, activation of bypass pathways including the EGFR, MAPK, mTOR pathways may facilitate RET inhibitor resistance and that blockade of bypass pathways may enhance the efficacy of selective RET inhibitors and overcome acquired RET TKI resistance. Citation Format: Tomohiro Takehara, Monique B. Nilsson, Ximeng Liu, Hibiki Udagawa, Naoto Morikawa, Alissa Poteete, Junqin He, Xiaoxing Yu, Thiru Arumugam, Koichi Goto, John V. Heymach. EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs [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 1958.

Abstract 10: Non-viral engineered T cell therapy specific for the hotspot mutation p53 R175H that integrates signal 1 (TCR), signal 2 (co-stimulation) and signal 3 (cytokine) and co-opts FasL-dependent apoptosis to achieve a coordinated antitumor CD4/8 T cell response

Abstract Adoptive T cell therapy (ACT) has demonstrated antitumor efficacy in patients with solid cancers but requires further optimization to become a reproducibly effective treatment. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Targeting mutated oncogenic drivers addresses many of the major obstacles of this modality, in that the antigenic epitope is: 1) tumor-specific, 2) essential for tumor survival, and 3) derived from a stably expressed protein. However, the immune-suppressive tumor microenvironment makes further optimization of engineered T cells necessary to bring long-term clinical benefit to patients. For an optimal anti-tumor response, T cells require three signals: TCR, co-stimulation, and cytokine signaling. The tumor suppressor TP53 is the most frequently mutated gene across human cancers, with a highly recurrent arginine to histidine hotspot alteration in codon 175 leading to novel tumor-dependent functions. Here we report the use of a novel CRISPR-Cas nuclease system to knock-in a six-parameter multi-cistronic cassette into the TRAC locus with high efficiency. We employed several strategies to maximize the potency and durability of a TCR-T cell product targeting the p53 R175H oncogenic driver, including: 1) A high-affinity TCR (α and β chains) specific for the p53 R175H mutation presented by HLA-A*02:01 permits the recognition of tumor cells expressing even low levels of the epitope (Signal 1), 2) Inclusion of the CD8αβ co-receptor drives stimulation of CD4+ T cells with the MHC class I restricted TCR, allowing for a physiologic coordinated immune response required for maximal efficacy, 3) A FAS-41BB switch receptor acts as a dominant negative to the FASL-inducing apoptotic signal in the tumor microenvironment and drives stimulation and persistence of the T cell product via 41BB co-stimulatory signaling (Signal 2), 4) A chimeric cytokine receptor (constitutive Interleukin Receptor) promotes expansion and survival while avoiding immunologic exhaustion (Signal 3). Together, these strategies deliver the three signals required for maximal T cell function: antigen-driven activation, co-stimulation, and growth/survival-promoting cytokine signaling. The non-viral TRAC-knocked-in T cells demonstrate robust and specific cytotoxicity against endogenously expressing HLA-A*02:01 and p53 R175H cell lines in vitro and effective anti-tumor activity in vivo while maintaining a favorable preclinical safety profile. These data support the planned clinical development of a novel non-viral TRAC-knocked-in T cell therapy for the treatment of p53 R175H-mutant solid tumors. Citation Format: Santosh Narayan, Ken Gareau, Ankit Gupta, Josh Ferrell, Nicholas Rouillard, Tyler Warner, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Joshua Francis, Xingyue He, Patrick J. Browne, Rebecca Lamothe, Meghan D. Storlie, Gregory J. Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Damien Hallet, Michael Gormally, Chistopher A. Klebanoff, Gary Shapiro, Kim Nguyen, Loïc Vincent. Non-viral engineered T cell therapy specific for the hotspot mutation p53 R175H that integrates signal 1 (TCR), signal 2 (co-stimulation) and signal 3 (cytokine) and co-opts FasL-dependent apoptosis to achieve a coordinated antitumor CD4/8 T cell response [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 10.

Abstract 3124: MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most challenging cancers with high unmet medical need. Glypican-3 (GPC3) is an oncofetal glycoprotein anchored to the cell surface via GPI (glycosylphosphatidylinositol) and frequently over-expressed in HCC with limited expression in normal adult tissues, which makes it an appealing antibody-drug conjugate (ADC) target for HCC. MRG006A is a potentially first-in-class GPC3-targeting ADC composed of a novel humanized IgG1 antibody conjugated to a proprietary linker-payload platform utilizing a potent topoisomerase 1 inhibitor via a peptide-based cleavable linker. Materials and Methods: As a GPC3-targeting ADC intended for clinical therapy of GPC3-expressing cancers, the mechanism of action and therapeutic potential of MRG006A were investigated in a series of preclinical studies, including (1) MRG006A and its antibody moiety binding to human GPC3 expressed on cells assessed by flow cytometry; (2) binding affinity to human and cynomolgus monkey GPC3 measured by SPR (surface plasmon resonance); (3) internalization in Huh7 cells assessed by pHAb Reactive Dye and confocal microscopy; (4) in vitro cytotoxicity against a panel of HCC cell lines (Huh7, HepG2, Hep3B and GPC3-negative SK-HEP-1); (5) anti-tumor activity of MRG006A evaluated in Huh7 and HepG2 cell line-derived xenograft (CDX) as well as a panel of HCC patient-derived xenograft (PDX) mouse models with a different expression level of GPC3; (6) plasma stability of MRG006A evaluated in human, cynomolgus monkey, rat and mouse plasma; and (7) an exploratory pharmacokinetic (PK) and toxicology study of MRG006A in cynomolgus monkey. Results: The GPC3-targeting ADC, MRG006A, and its antibody moiety exhibited superior binding activity to GPC3-expressing cancer cell lines than a clinical antibody benchmark and similar nanomolar binding affinity to both human and cynomolgus monkey GPC3, which makes cynomolgus monkey suitable for toxicological studies. Rapid internalization of MRG006A antibody was observed in GPC3-expressing Huh7 cells, and MRG006A maintained comparable internalization capability to the naked antibody. More importantly, MRG006A exhibited potent GPC3-dependent cytotoxic activity in a panel of HCC cells expressing varying levels of GPC3. Administrations of MRG006A resulted in a robust and dose-dependent tumor growth inhibition of multiple CDX models and HCC PDX models. Remarkable stability of MRG006A was shown in the plasma of various species tested. MRG006A demonstrated a favorable PK and safety profile with good tolerability in the exploratory toxicology study. Conclusion: Overall, the preclinical study results suggest that MRG006A is a feasible ADC drug candidate for treating GPC3-expressing cancers in clinical studies. MRG006A was aimed to submit IND in China and the U.S in 2024. Citation Format: Yanchun Wang, Hongfeng Li, Hao Shen, Wenchao Liu, Shoujia Liu, Kequan Yin, Haili Xu, Xueyuan Cui, Wei Li, Wei Liu, Xiangyu Wu, Liu Yang, Tian Ma, Zhongrun Zhao, Jun Wang, Feifei Cui, Lei Fang, Minmin Qin, Chaohong Hu. MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies [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 3124.

Abstract 7141: The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents

Abstract Our previous work showed that the prenylated hydroxy-stilbene AUS_001 exerts a strong safety profile and potent anti-proliferative responses, as assessed in 30 types of cancer cell lines, stemming from its microtubule destabilization activity. The aim of the current study was to delineate the molecular mechanism of microtubule destabilization by AUS_001 and to provide insight into its high potency and low toxicity. Surface plasmon resonance (SPR) experiments revealed the direct interaction of AUS_001 with tubulin with an apparent equilibrium dissociation constant, Kd, of 2.13 × 10−5M. Size exclusion competition assays and an X-ray crystal structure of the tubulin-AUS_001 complex at 2.1 Å resolution establish ligand binding within the colchicine site on tubulin. Since colchicine-site ligands are well known to inhibit the curved-to-straight conformational transition of tubulin, which is an essential process for microtubule formation, these results readily explain the microtubule-destabilizing activity of the compound. Notably, while both combretastatin A4 (CA-4) and AUS_001 are both colchicine-site stilbene ligands, CA-4 binds in the cis configuration, while AUS_001 maintains a trans configuration leading to a substantially different pocket engagement. Comparison of the tubulin-AUS_001 complex structure with the ones of tubulin-colchicine and tubulin-plinabulin revealed that AUS_001 elicits different rearrangements in the T7 loop of β-tubulin versus colchicine and occupies a different zone of the pocket relative to plinabulin, further elaborating its unique binding properties. Interestingly, AUS_001 exhibits a higher relative affinity than colchicine itself, as assessed by i) an ultrafiltration-based tubulin-ligand release assay and ii) a radioligand colchicine competition assay. However, fitting steady state SPR data for AUS_001 binding to tubulin revealed fast association and dissociation rates and was indicative of reversible binding. The latter finding is corroborated by AUS_001-triggered reverse biological effects including morphological alterations, cell cycle G2/M arrest, and by a decrease of cell viability upon discontinuation of the drug treatment in glioma and pancreatic cancer cells. Conversely, colchicine, CA-4 or paclitaxel administered at the same doses as those applied for AUS_001 failed to reverse the drug-induced phenotypes upon removal resulting in sustained cytotoxicity. In conclusion, we fully characterized the unique binding mode of AUS_001 to the colchicine site of tubulin and elucidated the reversible nature of the target engagement. AUS_001 features a distinctive and reversible molecular interaction with tubulin which provides a plausible explanation for its favorable safety profile compared to other microtubule targeting agents. Citation Format: Herman Lelie, Yao-Chieh Chou, Alastair J. King, Zlata Boiarska, Andrea E. Prota, Michel O. Steinmetz, Marina Koutsioumpa. The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents [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 7141.

Abstract 7264: OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models

Abstract The Hippo pathway regulates critical cellular processes during development. In ~10% of human cancers, Hippo pathway dysregulation results in YAP/TAZ overactivation and increased TEAD-dependent transcription that promotes tumor growth and therapy resistance - often by supporting drug-tolerant persister survival. In 40% of malignant mesotheliomas (MM), neurofibromatosis 2 (NF2) gene mutations inactivate the Merlin protein, an upstream negative regulator of TEAD. Preclinical studies indicate high TEAD dependency in NF2-mutant MM. We have discovered an array of potent covalent and non-covalent small molecule inhibitors that occupy the palmitate-binding pocket of TEAD proteins, with varying paralog selectivity profiles. OPN-9840 is an oral non-covalent pan-TEAD inhibitor displaying dose-dependent and on-target in vitro and in vivo efficacy for NCI-H226, an NF2-mutant MM cell line. OPN-9840 showed equivalent binding in thermal shift assays (10°C delta Tm) for depalmitoylated TEAD1-4 proteins, and blocked TEAD reporter activity (IC50 100nM) and NCI-H226 cell growth (IC50 400nM). In an NCI-H226 xenograft model, OPN-9840 dosed orally for 14 days exhibited dose-dependent tumor growth inhibition (TGI) from 5 to 50 mg/kg PO (88 to >100% TGI) with tumor regression in the 15 (2/8 mice) and 50 mg/kg (4/8 mice) dose groups. qPCR of OPN-9840-treated tumors confirmed 50-70% downregulation of canonical TEAD targets, CTGF and CYR61. OPN-9840 shows a favorable safety profile, including a lack of cytotoxicity (IC50>50µM) in HEK293T and HepG2 cells. Additionally, OPN-9840 caused no body weight loss in the NCI-H226 xenograft model following 14 days of dosing. In vitro evaluation of OPN-9840 in the blood-brain barrier (BBB) specific parallel artificial membrane permeability assay exhibited high BBB penetration potential (-Log Pe = 4.7). High oral exposure was achieved at all dose levels (AUC0-24 > 100,000 h•ng/mL) after 14 days in the NCI-H226 study. We next investigated synergy between TEAD inhibition and targeted therapy in MM. Combined treatment of one of our pan-TEAD covalent compounds, OPN-9652 (from a separate chemical series to OPN-9840), at 50 mg/kg with 1 mg/kg trametinib resulted in NCI-H226 xenograft tumor regression (130% TGI) whereas trametinib alone only led to decreased tumor growth (74% TGI). RNA-Seq of treated tumors revealed that OPN-9652 and trametinib synergistically inhibited TEAD downstream target genes, including ANKRD1 and CTGF. OPN-9652 as a single agent caused significant downregulation of E2F targets, such as MYC and TP53, suggesting decreased proliferation. In summary, OPN-9840 is a potent pan-TEAD inhibitor that shows monotherapy efficacy in MM. As we observed synergy between another pan-TEAD inhibitor, OPN-9652, with trametinib, we are exploring synergy of OPN-9840 with additional targeted therapies in tumor types beyond MM. Citation Format: Pan-Yu Chen, Bernice Matusow, James Tsai, Peipei Li, Hoa Nguyen, Gaston Habets, Gideon Bollag, Somenath Chowdhury. OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7264.

Abstract 6354: The HER2/4-1BB bispecific antibody, YH32367 (ABL105) demonstrates optimal efficacy and safety for HER2-expressing tumors and exhibits synergistic efficacy when combined with anti-PD-L1

Abstract Overexpression/amplification of the human epidermal growth factor receptor 2 (HER2) is associated with clinically aggressive subtypes in various cancers. Currently, new HER2-targeted therapy like trastuzumab deruxtecan have improved outcomes not only for HER2-positive tumors but also for HER2-low tumors. However, curing cancer patients who develop resistance to current HER2-targeted therapies or experience unwanted side effects remains a significant challenge. Therefore, there is a pressing need for more advanced treatments, and one promising approach involves combination with immunotherapy. 4-1BB (CD137) is a key costimulatory receptor expressed on activated T-cells and natural killer cells, making it a promising therapeutic target in cancer. This study presents the anti-tumor efficacy of the HER2/4-1BB bispecific antibody, YH32367 (ABL105), against both HER2-positive and HER2-low tumors. Additionally, the study suggests that YH32367 could be a viable treatment option for combination with anti-PD-1 antibody (Ab).​ YH32367 has been designed to overcome the challenges with HER2 resistance via tumor-directed 4-1BB agonism. YH32367 induced IFN-γ secretion and thereby eliciting tumor cell death in co-culture condition with hPBMC and HER2-expressing tumor cells. YH32367 showed superior efficacy in eradicating tumors and triggered long-term anti-tumor immunity in MC38/hHER2-bearing h4-1BB KI mouse models, compared to benchmark 4-1BB/HER2-bispecific molecule and anti-HER2 Ab. Even in HER2-low tumor, YH32367 was more effective than benchmark 4-1BB/HER2-bispecific molecule. Additionally, when YH32367 was administered in combination with anti-PD-1 Ab to h4-1BB KI mice bearing HER2-low tumors, a synergistic effect was observed, surpassing the effects of anti-PD-1 Ab alone or anti-HER2 Ab-drug conjugate. The potent anti-tumor efficacy of YH32367 was also verified in HER2 expressing tumor-bearing hPBMC humanized mice. In terms of toxicity, YH32367 demonstrates a favorable safety profile, which was confirmed through a 4-week GLP toxicity study using cynomolgus monkeys at doses of 10, 30, and 100 mg/kg. In summary, YH32367 has demonstrated significant anti-tumor effects, impacting tumor immunity in h4-1BB KI mice with HER2-low and HER2-positive tumors. Furthermore, its combination with anti-PD-1 Ab has shown synergistic efficacy, indicating that YH32367 may offer treatment options not only as a monotherapy but also in combination therapy for HER2 expressing tumors. The phase 1 dose escalation study is currently underway (NCT05523947), and a dose optimization study to determine the recommended phase 2 dose (RP2D) is scheduled for the second half of 2024. Citation Format: Eunjung Lee, Young Bong Park, Minji Choi, Jangwoo Shin, Kyong Bae Kim, Ju Young Park, Junhwan Kim, Kyoung Kyu Ahn, Yangsoon Lee, Kyeongsu Park, Wonjun Son, Donghoon Yeom, Jaehyun Eon, Hanbyul Lee, Hanbyul Jung, Jonghwa Won, Se-Woong Oh, Yeul Hong Kim. The HER2/4-1BB bispecific antibody, YH32367 (ABL105) demonstrates optimal efficacy and safety for HER2-expressing tumors and exhibits synergistic efficacy when combined with anti-PD-L1 [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 6354.