Dose-dependent Antitumor Efficacy Research Articles

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer

BackgroundGastric cancer represents a highly lethal malignancy with an elevated mortality rate among cancer patients, coupled with a suboptimal postoperative survival prognosis. Nectin-4, an overexpressed oncological target for various cancers, has been exploited to create antibody-drug conjugates (ADCs) to treat solid tumors. However, there is limited research on Nectin-4 ADCs specifically for gastric cancer, and conventional immunoglobulin G (IgG)-based ADCs frequently encounter binding site barriers. Based on the excellent tumor penetration capabilities inherent in nanobodies (Nbs), we developed Nectin-4-targeting Nb drug conjugates (NDCs) for the treatment of gastric cancer.ResultsAn immunized phage display library was established and employed for the selection of Nectin-4-specific Nbs using phage display technology. Subsequently, these Nbs were engineered into homodimers to enhance Nb affinity. To prolong in vivo half-life and reduce immunogenicity, we fused an Nb targeting human serum albumin (HSA), resulting in the development of trivalent humanized Nbs. Further, we site-specifically conjugated a monomethyl auristatin E (MMAE) at the C-terminus of the trivalent Nbs, creating Nectin-4 NDC (huNb26/Nb26-Nbh-MMAE) with a drug-to-antibody ratio (DAR) of 1. Nectin-4 NDC demonstrated excellent in vitro cell-binding activities and cytotoxic efficacy against cells with high Nectin-4 expression. Subsequent administration of Nectin-4 NDC to mice bearing NCI-N87 human gastric cancer xenografts demonstrated rapid tissue penetration and high tumor uptake through in vivo imaging. Moreover, Nectin-4 NDC exhibited noteworthy dose-dependent anti-tumor efficacy in in vivo studies.ConclusionWe have engineered a Nectin-4 NDC with elevated affinity and effective tumor uptake, further establishing its potential as a therapeutic agent for gastric cancer.Graphical abstract

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

Abstract Oncogenic KRAS mutations occur in approximately 25% of all human cancers with KRAS G12V being one of the most frequently occurring variants, particularly in pancreatic, colorectal, and lung cancers. These oncogenic mutations cause sustained activation of the mitogen activated protein kinase (MAPK) pathway, which ultimately drives tumorigenesis. While newly approved covalent inhibitors to KRASG12C have demonstrated clinical benefit to patients harboring this mutation, there are no approved drugs targeting mutations beyond KRASG12C, highlighting a significant clinical need. Here we show that QTX3544 is a potent and orally bioavailable KRASG12V preferring multi KRAS inhibitor. QTX3544 exhibited picomolar binding affinity (0.08 nM) and SOS mediated nucleotide inhibition (0.07 nM) to the inactive form of KRASG12V. Furthermore, QTX3544 inhibited “on state” KRAS activity in both biochemical and cellular assays. In vitro, QTX3544 significantly inhibited downstream ERK phosphorylation and cell proliferation in KRASG12V driven cancer cell lines. Similar inhibitory effects were observed across additional mutant KRAS cancer cell lines, including KRAS G12A, KRAS G13D , and KRAS Q61H, among others. Notably, QTX3544 displayed no impact against HRAS, NRAS or BRAF mutant cells. In vivo, oral administration of QTX3544 achieved sustained systemic exposure required for efficacy, indicating potential durable inhibition of KRAS signaling. QTX3544 demonstrated dose dependent anti tumor efficacy and significant tumor regression in various KRASG12V driven xenograft models when administered orally twice daily (BID). The physiochemical profile of QTX3544 is favorable with good measured LogD, solubility, and permeability. QTX3544 exhibits weak Cytochrome P450 inhibition (direct and time dependent) with a favorable in vitro off target selectivity/safety profile. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3544 was generally well tolerated in rat and dog repeat dose tolerability studies. In addition, QTX3544 showed good brain penetration in mice. Taken together, this preclinical characterization supports the advancement of QTX3544 into IND enabling studies for KRAS G12V mutant cancers. Citation Format: Yang W. Zhang, Elizabeth Donohue Vo, Pei Gan, Dave Rominger, Jillian M. Silva, Yang J. Zhang, Christopher Pan, Greg Lee, John M. Micozzi, Ben Reid, Brooke McDonough, Arghyotri Sinha, Audrey Hospital, Doug Krauth, Juan I. Luengo, Mike Kramer, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3544, a potent, selective, and orally bioavailable allosteric G12V preferring multi KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB163.

Abstract 1890: HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies models

Abstract Introduction: Daratumumab (Dara), an anti-CD38 monoclonal antibody, has been widely used in the treatment of multiple myeloma (MM). However, some of MM patients exhibit primary or acquired resistance to Dara therapy. A CD38 targeting antibody-drug conjugate (ADC) HMA800067 is developed, in which Dara was conjugated with cytotoxic payload via a novel linker, aiming to have superior anti-tumor efficacy to Dara, even in the subjects with resistance to Dara treatment. Methods: HMA800067 was characterized by ELISA-based binding assay, cell internalization assay, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. The in vitro anti-tumor potency was assessed against a panel of 24 MM and B cell lymphoma cell lines with various CD38 expressions. Bystander effect of HMA800067 was evaluated in co-culture of CD38− SU-DHL-2 cells with CD38+ Ramos cells. In vivo anti-tumor efficacy was investigated in the multiple MM and Diffuse Large B-cell Lymphoma (DLBCL) subcutaneous xenograft models, including some non-responsive to Dara. Results: HMA800067 displayed comparable binding affinity and internalization capability to Dara in CD38+ Ramos cells. And HMA800067 maintained the ability of Dara to induce the death of CD38+ Daudi cells by ADCC and ADCP, which indicated that conjugation did not change the property of Dara in the ADC upon antigen binding. HMA800067 exhibited a CD38-dependent cytotoxic activity with median IC50 of 0.46 nM against a panel of tumor cell lines, with a trend that the higher CD38 expression, the stronger potency could be seen. Bystander killing effect of HMA800067 was observed in CD38− cells when co-cultured with CD38+ cells in vitro. Furthermore, with the low release of payload in plasma and sustainable high exposure of payload in the tumor, HMA800067 demonstrated dose-dependent and superior anti-tumor efficacy to Dara in all tested models, and 10 mg/kg induced complete tumor regression in MM RPMI-8226, DLBCL SU-DHL-6 and SU-DHL-10 models. For example, in SU-DHL-6 xenograft model, the treatment of intravenous injection of HMA800067 at 10 mg/kg single dose induced complete tumor regression in all the animals within 10 days of administration, which was maintained until the end of study (day 49), while 10 mg/kg Dara twice a week for 2 weeks inhibited tumor growth within 20%-40% without any tumor regression during a four-week observation post initial dosing. Conclusion: HMA800067 demonstrated potent anti-tumor activities in vitro and in vivo in multiple B-cell malignant tumor lines, including those with poor response to Dara. These results supported further development of HMA800067, as a superior therapeutic option for treatment of patients with CD38+ B cell malignancies. Citation Format: Yan Xu, Junqing Liang, Shuwen Jiang, Haibin Yang, Hui Zhang, Fangfang Mao, Jiahuan Zhu, Jianlin He, Na Yang, Jian Wang, WeiHan Zhang, Yongxin Ren, Weiguo Su. HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies 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 1890.

Abstract 6028: Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer models

Abstract Targeted radiotherapies represent an emerging treatment modality for aggressive cancers with limited treatment options, such as small cell lung cancer (SCLC). ABD147 is a Delta Ligand 3 (DLL3)-targeted antibody conjugate designed to carry and deliver cytotoxic radioactive isotopes to DLL3-expressing tumor cells. DLL3 is commonly expressed on the cell surface of neuroendocrine cancer cells, including SCLC, but has limited and predominantly intracellular expression in non-malignant tissues. The ABD147 antibody specifically binds human DLL3 with high affinity and is internalized by DLL3-expressing cancer cells. To minimize radiation exposure to normal tissue, ABD147 has been engineered to clear quickly from the blood compartment. Using indium-111 to determine ABD147 pharmacokinetics and biodistribution in mice, we demonstrate rapid blood and normal tissue clearance of 111In-ABD147, as designed, following a single intravenous dose administration. However, 111In-ABD147 retains high tumor accumulation (activity concentration of up to 30% ID/g) in xenograft mouse models of SCLC despite low DLL3 surface antigen expression on tumor cells (≤ 3000 copies). 111In-ABD147 shows a favorable tumor-to-normal tissue distribution with predominant liver clearance. Following ABD147 therapeutic radioisotope delivery of actinium-225 or lutetium-177 (225Ac-ABD147, 177Lu-ABD147), we demonstrate single-dose tumor regression and dose-dependent sustained anti-tumor efficacy, corresponding to an extension of survival out to 84 days in multiple SCLC xenograft models. In summary, ABD147 can preferentially deliver radionuclides to DLL3-expressing tumor tissue while substantially reducing the systemic radioactive exposure typical of conventional IgG radioconjugates. Following promising results in preclinical models, including potent anti-tumor activity, 225Ac-ABD147 is now progressing into clinical development. Citation Format: Iva Kulic, Etienne S. Melese, Emma Cummins, Alex Mandel, Raja Viswas, Michael Abrams, Adam Judge. Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer 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 6028.

Abstract 3295: Pharmacological characterization of CYRS1542: A potent and orally available GSPT1 molecular glue degrader for the treatment of neuroendocrine solid cancer with a favorable safety profile

Abstract Neuroendocrine cancers (NECs) are relatively rare compared to other malignancies, but their clinical behavior can be exceptionally aggressive. Two particularly challenging subtypes of NECs, Small Cell Lung Cancer (SCLC) and Neuroendocrine Prostate Cancer (NEPC), are worthy of attention due to their rapid progression, limited treatment options, and high mortality rates. At the molecular level, SCLC and NEPCs necessitate the presence of TP53 and RB1 mutations (Beltran et al., 2016; George et al., 2015). The frequency of TP53 mutations in SCLC is particularly noteworthy, with studies consistently reporting a high incidence (89~92%) (Chen et al., 2022; Sivakumar et al., 2023). A recent study has unveiled intriguing parallels between NECs and hematological malignancies, shedding light on their shared genetic features and analogous patterns of therapeutic susceptibility (Balanis et al., 2019). GSPT1, a translation termination factor, plays a key role in releasing the complete polypeptide from the ribosome. As cancer cells are dependent on translation processes to express elevated levels of oncoproteins, GSPT1 degradation would have lethal consequences. Specific CRBN modulators possess the capability to induce GSPT1 degradation via the ubiquitin-proteasome pathway. GSPT1 degradation leads to a halt in translation termination, activating the Integrated Stress Response (ISR) and subsequently triggering TP53-independent apoptosis. Despite GSPT1 degradation in cancer cells is appealing due to its potential to disrupt the production of critical proteins necessary for cancer cell survival and growth, the challenge lies in doing so without causing excessive harm to normal cells, as GSPT1 is also indispensable for their vital cellular functions. Remarkably, the ongoing development of GSPT1-targeted agents in clinical trials has raised intriguing questions about the mechanisms by which a therapeutic index can be established. Recently, we have identified CYRS1542 as an orally bioavailable GSPT1 molecular glue degrader (MGD) (Joo et al., 2023). Here, we present a pharmacological characterization of CYRS1542 as a potent and orally bioavailable GSPT1 MGD with the ability to effectively treat aggressive neuroendocrine cancers. In various NEC in vivo models, including an orthotopic model, CYRS1542 demonstrated dose-dependent and excellent anti-tumor efficacy comparable to the clinical stage GSPT1 MGD. Furthermore, it demonstrates improved safety profiles in a broad spectrum of normal cells, resulting in a higher therapeutic index compared to the clinical GSPT1 MGD. Citation Format: Min Sung Joo, JaeYung Lee, Joonhyung Lee, Eun-Jung Kim, Seogbeom Song, Jeonghyeon Lee, Joonwoo Nam, Heuijoon Park, Jaewoo Park, Keon Wook Kang, Wooseok Han. Pharmacological characterization of CYRS1542: A potent and orally available GSPT1 molecular glue degrader for the treatment of neuroendocrine solid cancer with a favorable 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 3295.

Abstract 3217: Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their anti-tumor activity

Abstract KAT6A and its paralog KAT6B are histone acetyltransferases (HATs) which direct chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement, resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and KAT6B entered phase 1 clinical trials (NCT04606446). With continuous daily dosing, durable partial responses were seen in estrogen-receptor positive (ER+) breast cancer patients who had progressed on a prior endocrine therapy and CDK4/6 inhibitor. In that study, evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies (Sommerhalder, ASCO2023). Isosterix has developed a series of structurally diverse small molecule KAT6A-selective inhibitors which engage at the active site in the acetyl CoA substrate binding pocket. Lead compounds are selective for KAT6A and have significant potency and cellular activity in a variety of assays; they inhibit KAT6A-mediated histone acetylation in a biochemical assay as well as pharmacodynamic dose-dependent inhibition of histone acetylation in the breast cancer tumor cell line ZR-75-1. These compounds reduce cell viability of ZR-75-1 in 10 days with continuous compound administration, confirming the dependence of this cell line on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Lead inhibitors are orally bioavailable with excellent pharmacokinetic properties in rodents. They demonstrate dose-dependent anti-tumor efficacy with minimal weight loss with continuous daily dosing in mouse xenograft tumor models. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in not only ER+ breast cancer but also other tumor indications which exhibit transcriptional induction dependent on KAT6A-directed chromatin remodeling. Selection of a lead candidate is planned for early 2024 with IND enabling studies to follow. Citation Format: Leslie J. Holsinger, Roopa Rai, Rahul R. Nagawade, Benoy K. Pal, Samir G. Pawar, Krishna Anand Putta, Somashekar S.C., Mahaboobi Jaleel, Jianhong Wang, Alison L. Hannah. Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their 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 3217.

Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024.

Abstract B055: Characterization of highly selective and CNS-penetrant PARP1 inhibitors

Abstract First-generation PARP1/2 inhibitors are FDA-approved for cancers such as breast, ovarian prostate and pancreas and provide significant therapeutic benefit to patients whose tumors harbor BRCA1 or BRCA2 mutations or homologous recombination deficiency (HRD). However, their use is associated with hematological toxicities that narrow their therapeutic index and restrict their application, particularly in combination with standard-of-care chemotherapy. All four FDA-approved PARP1 inhibitors are largely non-selective for the closely related enzyme PARP2, inhibition of which has been shown to drive hematotoxicity. Hence, the development of second-generation molecules highly selective for PARP1 over PARP2 offers a significant opportunity to 1) dramatically enhance therapeutic index, 2) enable additional precision medicine/combination approaches with chemotherapy, radiotherapy, immunotherapy and targeted agents and 3) expand the addressable patient population to those whose tumors harbour additional DDR defects. Utilising X-ray crystallography and structure-based drug design, we describe the characterization of exquisitely selective PARP1 inhibitors. These molecules demonstrate high potency in multiple biochemical and cell-based assays, including viability and colony forming unit read-outs in BRCA-deficient cancer cells, whilst sparing HR-proficient cancer cell lines and primary cells. The molecules are potent PARP1-DNA trappers and exhibit high selectivity across the PARP family and wider secondary pharmacology targets. The molecules potentiate the activity of ATR inhibitors when tested in ATM-deficient cancer cell lines. This chemical series generally exhibits highly desirable physico-chemical and in vitro ADME properties, which translate to high oral bioavailability and low clearance in rodent PK studies. Exemplar molecules also demonstrate high rat CNS penetrance using in vivo microdialysis. Lead molecules yield deep and durable dose-dependent anti-tumor efficacy in a variety of BRCAm xenograft models with responses continuing after cessation of treatment. The compounds demonstrate strong PK/PD relationships which correlate with anti-tumor efficacy using inhibition of tumor PARylation as a PD read-out. Strikingly, the molecules demonstrate compelling anti-tumor efficacy in BRCA-deficient xenograft models that are known to be largely resistant to FDA-approved non-selective PARP1/2 inhibitors. Taken together, these data predict low once daily therapeutic oral dosing based on a highly favorable pre-clinical PK profile and potent in vivo efficacy, with the potential to demonstrate improved efficacy and tolerability compared to marketed PARP inhibitors. In summary, we describe the characterization of next generation potent and selective CNS-penetrant PARP1 inhibitors. These molecules have the potential for an improved safety profile and greater combination potential with standard-of-care and targeted anti-cancer therapies. Citation Format: Phillip M Cowley, Barry E McGuinness, Gillian M Campbell, Alan Wise. Characterization of highly selective and CNS-penetrant PARP1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B055.

BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4+, CD8+, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

Efficacy of a HER2-Targeted Thorium-227 Conjugate in a HER2-Positive Breast Cancer Bone Metastasis Model.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-30% of breast cancers but has low expression in normal tissue, making it attractive for targeted alpha therapy (TAT). HER2-positive breast cancer typically metastasizes to bone, resulting in incurable disease and significant morbidity and mortality. Therefore, new strategies for HER2-targeting therapy are needed. Here, we present the preclinical in vitro and in vivo characterization of the HER2-targeted thorium-227 conjugate (HER2-TTC) TAT in various HER2-positive cancer models. In vitro, HER2-TTC showed potent cytotoxicity in various HER2-expressing cancer cell lines and increased DNA double strand break formation and the induction of cell cycle arrest in BT-474 cells. In vivo, HER2-TTC demonstrated dose-dependent antitumor efficacy in subcutaneous xenograft models. Notably, HER2-TTC also inhibited intratibial tumor growth and tumor-induced abnormal bone formation in an intratibial BT-474 mouse model that mimics breast cancer metastasized to bone. Furthermore, a match in HER2 expression levels between primary breast tumor and matched bone metastases samples from breast cancer patients was observed. These results demonstrate proof-of-concept for TAT in the treatment of patients with HER2-positive breast cancer, including cases where the tumor has metastasized to bone.

Abstract LB327: Discovery and characterization of ABSK112, a next-generation and potential best-in-class EGFR Exon20 mutant inhibitor with superior selectivity and brain penetration ability

Abstract Introduction: EGFR Exon20 mutations are clinically validated oncogenic alterations including a wide spectrum of mutations occurring in lung cancer and various other cancer types. Although several EGFR Exon20 inhibitors have reached clinical stage or received approval, there still leave large room for improvement in safety and efficacy, likely due to their limited selectivity against wild-type EGFR or other kinases, suboptimal mutation coverage, and lack brain penetrating ability. Herein, we have discovered a novel and next-generation EGFR Exon20 mutation inhibitor, ABSK112. It showed high selectivity over wild-type EGFR and other kinases, as well as a more comprehensive coverage over majority of EGFR Exon20 mutations in comparison with other EGFR Exon20 inhibitors. Method: Anti-proliferation experiments in cell lines harboring various EGFR Exon20 mutations were used to evaluate potency and spectrum of coverage for ABSK112 and other inhibitors. Efficacy studies and PK/PD study in multiple tumor models confirmed its in vivo activities. Cell proliferation and in vivo efficacies studies in models harboring mutated or wild type EGFR were used to demonstrate its selectivity. Kinome selectivity, safety profiles, PK and ADME profiles were also characterized. Results: ABSK112 showed potent inhibition of proliferation in multiple EGFR Exon20 mutation cell lines and superior wild-type EGFR selectivity compare to other inhibitors including mobocertinib (TAK788). In xenograft mouse models with various EGFR Exon20 mutations, oral dose of ABSK112 showed strong and dose-dependent anti-tumor efficacy. Moreover, ABSK112 exhibited much reduced in vivo inhibition against xenograft tumors with wild-type EGFR, compared to mobocertinib, confirming its superior selectivity. PK/PD analysis showed good correlation between ABSK112 exposure and inhibition of EGFR signaling. ABSK112 also demonstrated excellent preclinical brain penetration, bioavailability, and safety profile. Conclusion: ABSK112 is a novel and next-generation EGFR Exon20 mutation inhibitor with improved selectivity over wild-type EGFR and strong brain penetrating ability. It has demonstrated superior in vivo efficacy in several xenograft models with various EGFR Exon20 mutations, and excellent drug-like properties supporting its further development into clinical studies. Citation Format: Mei Ning, Shuqun Yang, Juan Peng, Shuangyun Hu, Weijia Lu, Cheng Dai, Baowei Zhao, Mingming Zhang, Hongping Yu, Zhui Chen, Yao-chang Xu. Discovery and characterization of ABSK112, a next-generation and potential best-in-class EGFR Exon20 mutant inhibitor with superior selectivity and brain penetration ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB327.

Abstract LB320: Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor

Abstract The RAS family of proto-oncogenes are the most frequently mutated genes in cancer, in which mutations in KRAS account for approximately 25% of all human cancers. RAS oncogenes impair the ability of RAS to convert from its active GTP-bound form into its inactive GDP-bound state leading to the sustained activation of the MAPK signaling pathway and ultimately driving tumorigenesis. Until recently, KRAS was regarded as undruggable, but newly approved covalent inhibitors of KRASG12C have demonstrated clinical benefit in patients harboring this mutation. However, patients with KRASG12C mutation only represent approximately 10% of the KRAS-mutated or amplified cancer population, and multiple RAS-mediated resistance mechanisms have emerged with these covalent compounds. Here we show that QTX3034 is a potent, highly selective, and orally bioavailable mutant KRAS inhibitor. Using the most common KRAS mutant, KRASG12D, QTX3034 demonstrated picomolar binding affinity (0.6 nM) to the inactive form of KRASG12D and inhibited SOS1/2 mediated nucleotide exchange with picomolar potency (0.2 nM). In a NanoBiT cellular target engagement assay, QTX3034 treatment disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner with an EC50 of 17 nM in HEK293T cells. Western blot analyses and CTG proliferation assays illustrated that QTX3034 treatment inhibited downstream ERK phosphorylation and cell proliferation in KRASG12D -driven cancer cell lines. Similar inhibitory effects were observed across KRASG12D and KRASG12V mutant cancer cell lines, albeit less potent against the latter. Notably, QTX3034 produced no effect against NRAS or BRAF mutant cells. In vivo, QTX3034 achieved sustained systemic exposure levels required for efficacy following single oral administration in mice, indicating potential durable inhibition of KRAS signaling in vivo. In a KRASG12D -driven xenograft model, oral administration of QTX3034 twice daily (BID) demonstrated dose-dependent anti-tumor efficacy and achieved tumor regression in 100% of tumors. In a KRASG12V -driven xenograft model, oral administration of QTX3034 showed significant tumor growth inhibition, consistent with its activity against KRASG12V in vitro. QTX3034 was well-tolerated in rodent, repeat dose toxicology studies and exhibited significant exposure in the brain. QTX3034 has good solubility, permeability, and moderate protein binding in human plasma. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3034 exhibited weak CYP inhibition (direct and time-dependent) with a favorable in vitro off-target selectivity/safety profile. Taken together, this preclinical characterization supports the advancement of QTX3034 into IND-enabling studies for KRAS mutant cancers. Citation Format: Yang W. Zhang, Dave Rominger, Elizabeth Donohue Vo, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB320.

Abstract 1780: TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements

Abstract The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1780.

Abstract 953: Development of a predictive biomarker signature for the highly potent SIK3 inhibitor OMX-0407

Abstract Using the iOTarg genetic screening platform, salt-inducible kinase 3 (SIK3), a serine/threonine kinase of the AMP-activated protein kinase (AMPK) family was recently identified as a novel cell signaling modulator in cancer biology. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to inhibit tumor growth in a distinct panel of tumor models in vitro as well as in vivo by decreasing downstream pro-survival signaling of SIK3, enhancing caspase-mediated apoptosis and repolarizing the tumor microenvironment by decreasing regulatory T cells. In a comprehensive viability screening, more than 200 murine and human cancer cell lines and patient derived tumor models of different indications were used to identify a distinct subset of cancers highly sensitive towards OMX-0407. Representative for these tumors, OMX-0407 demonstrated dose-dependent anti-tumor efficacy in the renal cell carcinoma model RENCA, the squamous non-small cell lung cancer model (sqNSCLC) KLN205 and the murine colorectal tumor model MC38. With significantly prolonged overall survival of more than 60% tumor growth inhibition in all tumor models, OMX-0407 demonstrated equivalent or superior efficacy compared to anti-PD-1 monotherapy. In addition, OMX-0407 enhances the therapeutic potential of the standard of care chemotherapy in a broad range of human cancer cells and murine tumor models of sqNSCLC. Using comprehensive transcriptomics and proteomics analyses the selective activity profile of OMX-0407 on individual cancer cell lines and PDX models was used to identify and validate a predictive biomarker signature. With this gene signature, 84% of the selected cancer cells lines are correctly classified and would benefit from OMX-0407 therapy. The predictive biomarker will be evaluated in the clinical Phase I trial of OMX-0407 scheduled to start enrollment shortly. In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in tumor indications with high unmet medical need equal or superior to anti-PD-1 checkpoint blockade. By screening sensitive and non-sensitive tumor cell lines and PDX models, we identified a response-prediction biomarker signature. In upcoming clinical studies, this predictive biomarker signature will be evaluated for it’s potential to enrich for patients highly responsive to OMX-0407 therapy. Citation Format: Ilona-Petra Maser, Sonja Lacher, Vincent Piras, Barbara Kracher, Kai Zanziger, Murray Yule, Hannes Loferer, Stefan Bissinger. Development of a predictive biomarker signature for the highly potent SIK3 inhibitor OMX-0407 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 953.

Abstract 4020: HMPL-760 is a highly potent and selective reversible BTK inhibitor, targeting BTK and BTKC481S in B-cell malignancies

Abstract Introduction: Bruton’s tyrosine kinase (BTK), a member of the Tec family, plays a crucial role in signaling through B-cell receptor (BCR). BTK inhibition blocks BCR signals and prevents B-cell activation and growth. First-generation BTK inhibitors such as ibrutinib covalently binds to a cysteine residue (C481) of BTK. Their most frequent acquired resistance is the development of a serine mutation in the binding site (C481S). Next generation BTK inhibitors such as LOXO-305 and ARQ 531 are being developed to overcome this resistance to first-generation inhibitors. Methods: HMPL-760 was tested in biochemical assays using recombinant human wild type (WT) and C481S mutant BTKs. Its selectivity was carried out using Eurofins Cerep KinaseProfilerTM panel. Cellular activity of HMPL-760 was evaluated in HEK293 cells stably transfected with BTKWT or BTKC481S, and other tumor cell lines, which are either human diffuse large B cell lymphoma (DLBCL) or mantle cell lymphoma (MCL) cell lines. The in vivo antitumor activity and PKPD correlation of HMPL-760 was studied in HBL-1 xenograft mouse models bearing BTKWT or BTKC481S respectively. Results: In biochemical assays, HMPL-760 strongly inhibits BTK kinase activities towards wild-type BTK (BTKWT) and C481S mutant (BTKC481S), and binds to BTK in a reversible way. HMPL-760 demonstrates high selectivity in a panel containing 413 kinases. In cellular assays, HMPL-760 displays strong anti-proliferative activities in B-cell lymphoma cells (TMD-8, OCI-LY10, REC-1, HBL-1 and HBL-1-BTKC481S) harboring either BTKWT or BTKC481S (GI50: 0.0015-0.046 μM). In human whole blood assay, HMPL-760 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 expression in CD19+cells. HMPL-760 shows ≥ 10-fold inhibitory potency than ARQ 531 in both BTKWT and BTKC481S cells, and ~3-fold higher inhibitory potency than that of LOXO-305 in BTKC481S cells. In cellular assay by detecting p-BTK after compound washout, HMPL-760 maintains a longer duration of target inhibition than LOXO-305 in both BTK wild type (HBL-1) and BTK mutant (HBL-1-BTKC481S) cell lines. HMPL-760 displays dose-dependent antitumor efficacy in multiple human B cell lymphoma xenograft models in mice when orally administered at 3~50 mg/kg once daily. Complete tumor regression occurs in most of the tested models at the high dose levels. HMPL-760 shows much stronger antitumor efficacy than LOXO-305 and ARQ 531 at similar dose level, which may be associated with HMPL-760’s higher drug exposures and more sustainable inhibition on BTK phosphorylation in the tumor tissues. Conclusion: HMPL-760 is a reversible, selective, highly potent, BTK inhibitor targeting both BTKWT and BTKC481S. The first-in-human Phase 1 clinical trials of HMPL-760 are under way in patients with r/r B-NHL (NCT05190068, NCT05176691). Citation Format: Linfang Wang, Junqing Liang, Zhihu Gao, Jia Hu, Weigang He, Xianwen Yang, Fangfang Mao, Wei Zhang, Ying Yu, Qihang Zhang, Na Yang, Chun Zhang, Jian Wang, Yu Cai, Xiong Li, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-760 is a highly potent and selective reversible BTK inhibitor, targeting BTK and BTKC481S in B-cell malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4020.

Improved Therapeutic Efficacy of CBD with Good Tolerance in the Treatment of Breast Cancer through Nanoencapsulation and in Combination with 20(S)-Protopanaxadiol (PPD).

Cannabidiol (CBD), a nonpsychoactive major component derived from Cannabis sativa, widely used in neurodegenerative diseases, has now been proven to have growth inhibitory effects on many tumor cell lines, including breast tumors. Meanwhile CBD can effectively alleviate cancer-associated pain, anxiety, and depression, especially tumor cachexia, thus it is very promising as an anti-tumor drug with unique advantages. 20(S)-Protopanaxadiol (PPD) derived from the best-known tonic Chinese herbal medicine Ginseng was designed to be co-loaded with CBD into liposomes to examine their synergistic tumor-inhibitory effect. The CBD-PPD co-loading liposomes (CP-liposomes) presented a mean particle size of 138.8 nm. Further glycosyl-modified CP-liposomes (GMCP-liposomes) were prepared by the incorporation of n-Dodecyl β-D-maltoside (Mal) into the liposomal bilayer with glucose residue anchored on the surface to act as a ligand targeting the GLUT1 receptor highly expressed on tumor cells. In vivo studies on murine breast tumor (4T1 cells)-bearing BALB/c mice demonstrated good dose dependent anti-tumor efficacy of CP-liposomes. A high tumor inhibition rate (TIR) of 82.2% was achieved with good tolerance. However, glycosylation modification failed to significantly enhance TIR of CP-liposomes. In summary, combined therapy with PPD proved to be a promising strategy for CBD to be developed into a novel antitumor drug, with characteristics of effectiveness, good tolerance, and the potential to overcome tumor cachexia.

Abstract 2604: The novel ATR inhibitor ATG-018 is efficacious in preclinical cancer models

Abstract Background: The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DNA damage response (DDR) by activating essential signaling pathways of DNA damage repair. Inhibiting normal ATR function leads to increasing accumulation of double-strand breaks (DSB), particularly in genomically unstable tumor cells with preexisting replication stress, resulting in further genomic instability, mitotic catastrophe, and cell death. Targeting ATR is, therefore, a relevant strategy for the development of novel anticancer agents. This study tested the antitumor effects induced by ATG-018, a novel small molecule ATR inhibitor, in preclinical tumor models. Methods: Firstly, the antiproliferative activity of ATG-018 was evaluated against a panel of 143 tumor cell lines. Cell proliferation was measured after 72 hours of exposure to ATG-018, using CellTiter-Glo Cell Viability Assay. An unbiased genomic analysis of the tested cell lines was used to discover potential predictive biomarkers. The activity of ATR-inhibition by ATG-018 was determined by measuring phospho-Ser345 CHK1 protein level in HT-29 cells, using pCHK1 AlphaScreen assay. A mobility shift-based ATR kinase reaction assay was used to assess the direct inhibition of ATR by ATG-018. The in vivo anti-tumor efficacy of ATG-018 was evaluated in LoVo (colorectal cancer), OE21 (esophageal cancer) and OCI-LY-19 (lymphoma) CDX mouse models, respectively. Results: Potent in vitro and in vivo anti-tumor efficacy has been observed for ATG-018. For cell proliferation inhibition, the IC50 of ATG-018 ranged from 0.22μM to 10μM in 137 out of 143 cell lines, including both solid tumor and hematological malignancies, while ATG-018 did not affect the viability of normal PBMCs. A series of genetic alterations has been discovered to be correlated with the sensitivity to ATG-018, which could be potential predictive biomarkers. For ATR activity inhibition, the IC50 of ATG-018 required for ATR downstream (CHK1) phosphorylation inhibition was 1.4nM in HT-29 cells and the IC50 required for inhibiting ATR kinase reaction was 16nM. Dose-dependent antitumor efficacy was observed with ATG-018 in LoVo, OE21 and OCI-LY-19 CDX models. 10 mg/kg ATG-018 showed a statistically significant antitumor activity in all the three CDX models. In the OCI-LY-19 CDX model, 10 mg/kg ATG-018 showed 73.52% TGI on day 14 after grouping (p value=0.0014), and 50mg/kg ATG-018 induced tumor regression. Conclusions: Single agent ATG-018 exhibited strong monotherapy efficacy in preclinical cancer models with certain homologous recombination deficiencies, suggesting a promising therapeutic strategy for such patient populations. Citation Format: Hui Yuwen, Ya Kong, Lulu Jiang, Min Deng, Jiamei Luo, Bin Jiang, Bing Hou, Bo Shan, Jay Mei. The novel ATR inhibitor ATG-018 is efficacious in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2604.

Abstract 593: Anti-tumor immunity and efficacy of combination treatment of M6223 and bintrafusp alfa versus the combination of M6223 and anti-PD-L1 in preclinical tumor models

Abstract Background: T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) is an inhibitory receptor expressed on lymphocytes and has recently emerged as a target in cancer immunotherapy. M6223 is a fully human antagonistic anti-TIGIT antibody in immunoglobulin (Ig) G1 format with Fc-mediated effector function. Preclinical studies demonstrated that M6223 could induce an anti-tumor immune response by complementary mechanisms including but not limited to direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. M6223 showed dose-dependent anti-tumor efficacy in multiple preclinical tumor models. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β "trap") fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti-PD-L1), was designed to target two key immunosuppressive pathways in the tumor microenvironment. Methods: To attenuate the tumor immunosuppressive microenvironment, the anti-tumor immunity and efficacy of combining M6223 with bintrafusp alfa was investigated in syngeneic models in humanized mice with huTIGIT knock-in sequences. Results: M6223 combined with bintrafusp alfa significantly enhanced anti-tumor efficacy and extended survival compared with either monotherapies or the combination of M6223 with anti-PD-L1. M6223 combined with bintrafusp alfa stimulated higher CD8+ T cells and natural killer cells infiltration, proliferation and cytotoxicity in tumor microenvironment in comparison to the combination of M6223 with anti-PD-L1. The ratio of CD8+ T cells to regulatory T cells and the ratio of CD226 to TIGIT were significantly increased, indicating the conversion of the tumor microenvironment from an immuno-suppressive phenotype to a more immune-permissive phenotype. With the combination therapy, bintrafusp alfa was the main driver promoting CD8+ T cell proliferation, infiltration, and cytotoxicity. Alternatively, adding M6223 to bintrafusp alfa may decrease the risk of immune resistance caused by elevated TIGIT expression triggered by bintrafusp alfa treatment. Conclusion: These complementary mechanisms of M6223 and bintrafusp alfa orchestrate antitumor activity in preclinical tumor models. Currently, M6223 and bintrafusp alfa combination is being investigated in a Phase I clinical trial (NCT04457778) in patients with metastatic or locally advanced solid unresectable tumors. Citation Format: Chunxiao Xu, Sireesha Yalavarthi, Clotilde Bourin, Christie Kelton, Joern-Peter Halle, Jacques Moisan. Anti-tumor immunity and efficacy of combination treatment of M6223 and bintrafusp alfa versus the combination of M6223 and anti-PD-L1 in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 593.

Abstract P2-13-33: Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody

Abstract Background: Many HER2-directed antibody-drug conjugates (ADCs) in early development employ trastuzumab linked to tubulin binding agents as the cytotoxic drug. DHES0815A is an ADC consisting of a THIOMABTM humanized IgG1 anti-HER2 monoclonal antibody (hu7C2), which binds domain 1 of the HER2 ECD, conjugated via a disulfide linker to PBD-monoamide (MA), a DNA mono-alkylating agent. The reduced potency of the PBD-MA payload compared to PBD dimers and the stability of the conjugation site and linker were designed to improve tolerability, whereas the binding of hu7C2 to a HER2 epitope distinct from trastuzumab and pertuzumab allows combination with existing HER2 therapies. . Methods: Modifications at one imine in the PBD (pyrrolobenzodiazepine) dimer produced mono-alkylating PBDs that were assessed for DNA binding, permeability and cell potency. Each modified PBD was conjugated to hu7C2 LC K149C via a disulfide linker and assessed in HER2-positive models in vitro and in vivo. Safety studies were performed in female and male cynomolgus monkey treated every 3 weeks X 5, with a 7-week follow-up. Assessments included hematology, clinical chemistry, anti-drug antibody, standard clinical, neurological and ophthalmic exams and histopathology. Results: Our goal was design of a reduced potency PBD dimer ADC to achieve dosing in the linear PK range for optimal exposure, efficacy and safety. PBD dimers contain 2 reactive imines that bis-alkylate and crosslink DNA. Mono-alkylating PBD dimers were assessed in DNA binding, potency and permeability assays to select the optimal mono-alkylator, PBD-MA, for further investigation. hu7C2-disulfide-PBD-MA, or DHES0815A, was potent across HER2-positive breast and gastric cancer models in vitro and in vivo, with little effect on HER2-negative tumors or normal primary cells in vitro. DHES0815A was more potent than T-DM1 in HER2-positive and HER2-low breast and gastric cancer PDX models and demonstrated combination activity with standard of care agents T-DM1 or docetaxel. Mechanistically, treatment of HER2-positive cells with DHES0815A resulted in late S-phase cell cycle arrest; induction of DNA damage markers γH2AX, phospho-p53, phospho-CHK2, induction of apoptosis, and bystander activity. Pharmacokinetic analysis showed dose-proportional PK, with linear biphasic PK >/= 4mg/kg in cynomolgus monkey. Safety studies in cynomolgus monkey treated with 4, 8 or 12 mg/kg demonstrated that findings were non-adverse, monitorable and manageable. All doses were tolerated with few clinical observations and minimal to mild clinical pathology changes. Target organs were skin, bone marrow, lung and eye. The HNSTD (highest non-severely toxic dose) was 12 mg/kg. This dose enabled a starting dose of 0.6 mg/kg for phase 1 testing. Conclusions: DHES0815A is a HER2-directed ADC comprised of a domain I binding antibody linked to a reduced potency PBD dimer, PBD-MA, via a reducible disulfide linker. The antibody does not interfere with trastuzumab or pertuzumab binding, allowing combination with existing HER2 therapies. The reduced potency payload allows dosing in the linear PK range, supporting our hypothesis. DHES0815A demonstrated dose-dependent anti-tumor efficacy in multiple HER2+ breast and gastric cancer models, and showed efficacy in HER2-low models, where T-DM1 was inactive. Enhanced anti-tumor activity was observed combining DHES0815A with T-DM1, trastuzumab/pertuzumab or docetaxel. Studies in cynomolgus monkey demonstrated a safety profile compatible with initiating a phase 1 clinical trial. The phase 1 data will be presented in a separate presentation. Citation Format: Gail Lewis Phillips, Guangmin Li, Jun Guo, Shang-Fan Yu, Genee Lee, Donglu Zhang, Peter Dragovich, Thomas Pillow, Binqing Wei, M. Violet Lee, Ola Saad, Shab Masih, Douglas Leipold, Lisa Crocker, Melissa Schutten. Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-33.

326 The anti-TIGIT antibody M6223 induces significant anti-tumor efficacy and immune response via multiple mechanisms of action

BackgroundM6223 is a fully human antagonistic anti-T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) antibody in IgG1 format with Fc-mediated effector function.MethodsThe ability of M6223 to block the interaction of TIGIT with its ligands, CD155 and CD112, and the interaction of TIGIT with CD226 was determined by a flow cytometry-based binding assay. The anti-tumor efficacy, immune profile, and effector function of M6223 were investigated in syngeneic tumor models in huTIGIT knock-in mice. M6223 was either formatted with an effector competent mouse IgG2c constant region (M6223-muIgG2c) or formatted with effector null mouse IgG1-D256A constant region (M6223-muIgG1) as two versions of chimeric antibodies for the in vivo studies.ResultsM6223 dose-dependently blocked the binding of TIGIT to its ligands, including CD155 and CD112, thereby inhibiting a TIGIT-mediated immunosuppressive pathway. In addition, M6223 interrupted the interaction of TIGIT with the costimulatory receptor CD226. By blocking the interactions, the chimeric protein M6223-muIgG2c showed anti-tumor efficacy in multiple tumor models, including an MC38 tumor model (figure 1), and generated tumor antigen-specific long-term protective immunity in immunocompetent huTIGIT knock-in mice. M6223 monotherapy dose-dependently elevated the ratio of CD8+ cytotoxic T cells to regulatory T cells and the ratio of CD226 to TIGIT expression in immune cells in the tumor microenvironment. We also found that M6223 selectively depleted suppressive and exhausted TIGIT+ immune cell subsets and the anti-tumor activity of effector null M6223-muIgG1 was significantly lost (p<0.0001), suggesting that Fc-mediated effector function contributes to M6223 anti-tumor activity. Antibody depletion studies demonstrated that CD8+ T cells and natural killer cells contributed to the anti-tumor activity of M6223 in a complementary manner.Abstract 326 Figure 1M6223-muIgG2c displayed dose-dependent anti-tumor efficacy. M6223-muIgG2c displayed dose-dependent anti-tumor efficacy in an MC38 tumor model in hTIGIT knock-in mice.ConclusionsGiven that TIGIT blockade can inhibit an immunosuppressive pathway as well as remove the suppression on a costimulatory pathway, M6223 has the potential to induce an anti-tumor immune response by three complementary mechanisms: direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. Our data demonstrate that these complementary mechanisms orchestrate the anti-tumor activity of M6223. A Phase I, first-in-human clinical trial (NCT04457778) is underway to determine the safety, tolerability, maximum tolerated dose and recommended dose for expansion of M6223 as a single agent (Part 1A) and in combination with bintrafusp alfa (Part 1B) in patients with metastatic or locally advanced solid unresectable tumors.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.