Significant Body Weight Loss Research Articles

Abstract LB329: A potent and selective small molecule inhibitor of CSF-1R ABSK021 demonstrates strong efficacy in preclinical models of osteosarcoma

Abstract Introduction: Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgery combined with multimodal chemotherapy remains as the standard treatment for osteosarcoma patients. However, patients with osteosarcoma metastasis have a five-year survival rate of less than 30%, and their long-term outcomes have not improved over the last 30 years. CSF-1/CSF-1R signaling is crucial for the survival, function, proliferation and differentiation of myeloid lineage cells, including osteoclasts and monocytes/macrophages. Targeting CSF-1R either on tumor cells or tumor-associated macrophages has been reported to limit osteosarcoma progression in preclinical models. ABSK021, an oral, highly potent and selective small molecule inhibitor of CSF-1R discovered by Abbisko, has entered into Phase I trial (NCT04192344) and showed significant anti-tumor activity and favorable safety profile in patients with advanced tenosynovial giant cell tumor. Here, we conducted a series of preclinical in vitro and in vivo experiments, as well as human osteosarcoma profiling, to investigate the treatment potential of ABSK021 for osteosarcoma patients. Methods: Cellular potency of ABSK021 was evaluated by Celltiter-Glo assay in osteosarcoma cell lines and CSF-1R expression was evaluated by western blot. In vivo efficacy studies were conducted in murine osteosarcoma models and pharmacodynamics changes were measured. CSF-1R IHC staining was conducted in tissue microarray samples from osteosarcoma patients. Results: ABSK021 potently inhibited the proliferation of K7M2, a murine osteosarcoma cell line with high expression of CSF-1R. In animal, ABSK021 showed strong anti-tumor activity on K7M2 syngeneic model and SA4094 osteosarcoma PDX model without causing significant body weight loss. Pharmacodynamics analysis displayed robust inhibition of macrophage infiltration and CSF-1R signaling by ABSK021, confirming its effective target engagement in vivo. Parallel IHC analysis of human tissue microarray samples revealed high prevalence of positive CSF-1R staining in osteosarcoma patients. Conclusion: Taken together, these results demonstrate that ABSK021 has strong inhibition of CSF-1R activity and corresponding anti-tumor activity in preclinical osteosarcoma models. High prevalence of CSF-1R expression was also found in osteosarcoma patients, suggesting great potential of utilizing ABSK021 as a novel therapy to treat osteosarcoma patients in clinic. Citation Format: Nannan Zhang, Cheng Dai, Jiacheng Zhang, Shuqun Yang, Jie Wang, Jie Zhang, Manqi Liu, Zhui Chen. A potent and selective small molecule inhibitor of CSF-1R ABSK021 demonstrates strong efficacy in preclinical models of osteosarcoma [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 LB329.

Abstract 5040: Novel HER3 targeting antibody radioconjugates, 225Ac-HER3 ARC and 177Lu-HER3 ARC, exhibit potent antitumor efficacy in HER3-positive solid tumors

Abstract Background: HER3 is a unique member of the EGFR family that collaborates with other EGFR receptors to induce tumorigenesis and drug resistance. Moreover, HER3 expression is linked to poor survival for patients with solid tumors. Despite HER3 being a rational cancer therapeutic target, no HER3-directed therapies have been approved for clinical use. However, new therapeutic strategies such as antibody drug conjugates (ADCs) are being investigated. Targeted radiotherapy, including radiolabeled antibodies (ARCs), is unique mechanistically by inducing cell death independent of biologic pathway inhibition, and can be efficacious with less toxicity relative to other therapeutic modalities. Therefore, we hypothesized that the cytotoxic effects of alpha (225Ac) or beta (177Lu) emitting radionuclides combined with the specificity of anti-HER3 antibody targeting is a compelling therapeutic approach for HER3-expressing tumors. Here we evaluated the antitumor effects of 225Ac or 177Lu armed HER3 ARCs across multiple HER3-expressing cancer models such as ovarian, colorectal, prostate, and renal cancer. Methods: ARCs were prepared by radiolabeling AT-02, an anti-HER3 antibody, with 225Ac or 177Lu using p-SCN-Bn-DOTA to yield 225Ac or 177Lu-HER3 ARC. The binding activity and tumor cell cytotoxicity of HER3 ARCs were assessed by ELISA using human recombinant HER3, flow cytometry on HER3-expressing cells, and colony forming assays. To evaluate the antitumor growth effects of 225Ac-HER3 and 177Lu-HER3 ARCs in vivo, preclinical human tumor xenograft models were developed. Mice bearing HER3-positive tumors were dosed with 225Ac-HER3 ARC (0.2 or 0.4 µCi), or 177Lu-HER3 ARC (200 or 400 µCi) and tumor growth and body weight was monitored. Results: The pharmacological binding properties of HER3 antibody radiolabeled with 225Ac or 177Lu were similar to that of unmodified antibody as demonstrated by HER3 binding ELISA and flow cytometry. HER3 ARCs induced cytotoxicity and inhibited colony formation of HER3-positive tumor cell lines. Significant in vivo human tumor xenograft growth inhibition was observed in response to 225Ac or 177Lu HER3 ARCs compared to control groups (unmodified AT02 or IgG ARCs) in the models studied. No significant loss of body weight was observed in mice treated with HER3 ARCs suggesting that all treatments were well tolerated. Conclusions: In this study, both 225Ac-HER3 ARC and 177Lu-HER3 ARC demonstrated significant antitumor activity against HER3-expressing tumors in a dose-dependent manner. The HER3 targeted radiotherapy approach that we have undertaken could potentially overcome the limitations of current solid tumor therapies in resistance settings and warrants further evaluation in patients with HER3-expressing tumors. Citation Format: Denis Beckford-Vera, Megan McCloskey, Jason Li, Caroline Jennings, Le-Cun Xu, Debbie Lewis, Patrik Brodin, Amanda Chin, Monideepa Roy, Mary Chen, Helen Kotanides. Novel HER3 targeting antibody radioconjugates, 225Ac-HER3 ARC and 177Lu-HER3 ARC, exhibit potent antitumor efficacy in HER3-positive solid tumors. [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 5040.

Abstract 5041: TEM-1 targeted alpha therapeutic [Ac-225]-FPI-1848 induces regression in pre-clinical sarcoma xenograft models

Abstract Background: Tumor endothelial marker 1 (TEM-1 or CD248) is a cell surface receptor belonging to a family of C-type lectin transmembrane proteins. Identified as a fetal antigen, expression peaks during embryogenesis but is minimally detected in adulthood. In pathological conditions TEM-1 has been shown to be overexpressed in cancer and fibrosis. In sarcomas, expression occurs throughout the tumor environment in malignant cells, stromal cells and perivascular cells. The first line of treatment for sarcomas is surgical removal; however, many sarcomas arise in inoperable sites prompting a need for novel therapies. Herein, we describe the development of a novel TEM-1 targeting radiotherapeutic, [225Ac]-FPI-1848, in mouse xenograft models of sarcoma. Methods: [225Ac]-FPI-1848 is a radioimmunoconjugate consisting of a humanized anti-TEM-1 monoclonal antibody conjugated to a proprietary DOTA-based chelate (FPI-1397) and radiolabeled with actinium-225 [225Ac]. [177Lu]-FPI-1835 is the lutetium-177 radiolabeled analogue. The biodistribution and therapeutic efficacy of these radioimmunoconjugates were evaluated in human sarcoma xenograft models using nu/nu mice of Balb/c background. For biodistribution studies, approximately 37 MBq/kg of [177Lu]-FPI-1835 was injected intravenously into mice and tumor/organ radioactivity was quantified ex vivo at timepoints between 4 and 196 h. For therapeutic studies, a single intravenous dose of [225Ac]-FPI-1848 was administered over a dose range of 18.5 to 740 kBq/kg. Tumor size and body weight were monitored for 28 days. Results: Biodistribution studies with [177Lu]-FPI-1835 confirmed robust tumor uptake with minimal normal tissue uptake in three sarcoma models expressing high (SJSA-1), moderate (SK-N-AS) or low (A673) levels of TEM-1. Furthermore, the degree of uptake trended with TEM-1 expression levels across these models (SJSA1> SK-N-AS> A673). In corresponding therapeutic studies, [225Ac]-FPI-1848 demonstrated dose-dependent efficacy consistent with tumor uptake. In the SJSA-1 model, tumor regression was observed at doses of 92.5 kBq/kg or greater. Tumor regression was observed at doses of 185 kBq/kg or greater in the SK-N-AS model. In the low TEM-1 expressing A673 model, significant tumor regression was only observed at the highest radionuclide dose level (740 kBq/kg). Overall, therapy was well tolerated with no significant loss of body weight or clinical signs of poor health. Conclusions: [177Lu]-FPI-1835 demonstrated target-dependent uptake consistent with the degree of TEM-1 expression in human sarcoma xenograft models. In turn, corresponding efficacy studies with [225Ac]-FPI-1848 demonstrated strong dose-dependent, and target level-dependent efficacy with no apparent toxicity. These results suggest [225Ac]-FPI-1848 could be a promising therapy for TEM-1 expressing sarcomas. Citation Format: Sean Collens, Nicole Robinson, Matt Moran, Ryan Simms, Yarina Storozhuk, Meiduo Hu, Natalie Grinshtein, John Forbes, Christopher P. Leamon, John Valliant. TEM-1 targeted alpha therapeutic [Ac-225]-FPI-1848 induces regression in pre-clinical sarcoma xenograft models. [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 5041.

Abstract 2635: N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates

Abstract Introduction: There is a need for ADC payloads with an improved therapeutic index, consequently we have coupled potent and selective inhibitors of NMT to trastuzumab to test their potential in both in vitro and in vivo models. Experimental Procedures: A highly potent NMT inhibitor was conjugated to trastuzumab using two different linkers to produce an ADCs with a DAR of 5. These ADCs were tested in vitro in cell killing studies using Her2+ and Her2- cell lines and subsequently tested in vivo in breast cancer and gastric cancer xenografts. Results: Our first Trastuzumab-NMT inhibitor ADC (MYX2449) caused cell death in Her2+ BT474 cells with an EC50 of 0.2nM and peaking after 8 days of incubation, but was inactive against Her2- cells MCF7 at concentrations up to 100nM. MYX2449 was tested in a BT474 xenograft implanted orthotopically in the mammary gland fat pad and compared to Trastuzumab and vehicle controls. Both antibodies were dosed IV once a week for four weeks at 2.5 and 5 mg/Kg. MYX2449 therapy at 2.5mg/Kg resulted in a partial response (TGI 55% on day 21) and 5mg/Kg resulted in a TGI of 108% on day 21 with 7/10 mice having undetectable tumor by day 33 with no significant body weight loss during the study. Trastuzumab resulted in no response at 2.5mg/Kg a partial response at 5mg/Kg. MYX2449 was subsequently tested in a gastric cancer xenograft, NCI-N87 inoculated subcutaneously and compared to an NMT inhibitor conjugated isotype control and to Trastuzumab and Trastuzumab deruxtecan. All antibodies were dosed IV at 2.5mg/Kg or 5mg/Kg on day 1 and day 8. Day 21 TGI for the 5mg/Kg Trastuzumab group was 193%, for the 5mg/Kg Trastuzumab-deruxtecan group TGI was 154% and for the 5mg/Kg MYX2449 group TGI was 280%. In the MYX2449 5mg/Kg group, 6/10 mice had undetectable tumors on day 21 and 2/10 of the MYX2449 2.5mg/Kg group. Mean body weights of all groups had increased by day 21. Conclusions: Potent small molecule inhibitors of NMT can be readily conjugated to therapeutic monoclonal antibodies to generate novel highly effective and well tolerated ADC payloads. NMT inhibitors represent a totally novel class of ADC payloads that exploit cancer cell dependency on myristloylated proteins. The potential of this novel mechanism to deliver high efficacy with better TI is currently being investigated in preclinical models. Examples of Myricx patented chemical structures will be disclosed. RS, ND, FF and RC are employees of Myricx Pharma.JW, FO and ET are funded by Myricx Pharma.ET is a board member and founder of Myricx Pharma. Citation Format: Robin A. Carr, Roberto Solari, Nikki D'Arcy, Ed Tate, Josephine Walton, Folake Orafidiya. N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates [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 2635.

Abstract 4673: Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is typically treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Although most patients can be cured with R-CHOP, up to one-third of them relapses with a dismal outcome in most cases. Numerous approaches have been attempted to improve the treatment outcomes with R-CHOP. Polatuzumab vedotin is an antibody-drug conjugate targeting CD79b, which is ubiquitously expressed on over 90% of B-cell NHL malignancies, including DLBCL. To determine the potential of polatuzumab vedotin as a therapeutic agent, we evaluate its potency across a series of DLBCL patient derived xenograft (PDX) models. Method We established and characterized a series of DLBCL lymphoma PDX models. Eight DLBCL PDX models were selected for polatuzumab vedotin efficacy study, of which three are GCB DLBCL. Gene expression analysis of these lymphoma PDX models was performed using an Illumina NovaSeq 6000 system following Illumina-provided protocols for 2 × 150 paired-end sequencing. The expression status of CD79b on these DLBCL PDX models was also evaluated by IHC. Conclusion Our data showed that a single dose of polatuzumab vedotin at just 2 mg/kg could reached a comparable effect with R-CHOP and no significant body weight loss was observed. Our result showed that there is no apparent correlation between polatuzumab vedotin responses (TGI) and the CD79b expression (IHC Score). Although polatuzumab vedotin demonstrated encouraging activity in the treatment of DLBCL, some of the PDX models are also resistant to polatuzumab vedotin treatment. To further investigate the molecular parameters of the relative sensitivity of polatuzumab vedotin. Gene expression profiling of those PDX models and their sensitivity to polatuzumab vedotin were conducted, we found that the expression level of BCL-XL was correlated with reduced sensitivity to polatuzumab vedotin. Citation Format: Hui Qi, Fuyang Wang, Bingrui Han, Xiaomin Wang, Xiangnan Qiang, Zhixiang Zhang, Qiangyang Gu. Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma. [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 4673.

Abstract 2627: The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance

Abstract KRAS pathways are frequently upregulated in cancers bearing KRAS mutations or by other signaling modifications. Also, resistance to KRASG12C inhibitors and other KRAS/RAF/MEK pathway drugs is commonly observed in the treated cancer patients. A vital unmet need is the development of agents that potently and safely inhibit the altered KRAS signaling in these settings. A broadly acting KRAS mutation small molecule inhibitor lead series was developed using KRAS domain structure-guided scaffold modifications starting from a group of KRASG12C inhibitors. Optimized properties of these compounds were evaluated by tumor cell selectivity in vitro and pharmacophore elaboration. A novel lead series was identified with robust tumor cell growth inhibition, exemplified with the TEB-17112 lead candidate, having IC50 of inhibition against multiple KRAS-altered cell lines: NCI-H358 (KRAS-G12C, 33.6nM), AGS (KRAS-G12D, 23.5 nM), SW480 (KRAS-G12V, 22.5 nM) and A375 (KRAS-wildtype, 58.9 nM) respectively. TEB-17112 demonstrated inhibition of SW480 and H358 mouse xenografts at 25 or 50 mg/kg PO QD (21 days) without significant body weight loss. On further optimization of the lead series, TEB-17231 had on average a higher potency against KRAS mutant tumor cell lines, including H358 (12 nM), AGS (5.4 nM), SW480 (3.6 nM), and A427 (KRAS-G12D, 9.7 nM). With a candidate group of 7 compounds from this lead series, there was consistently a 5- to 6-fold selectivity of inhibition for KRAS mutant over KRAS wildtype cell lines. The lead candidate TEB-17231 was also observed to inhibit cell lines with other KRAS mutations: NCI-H1734 (KRAS-G13C, 6.5 nM), MDA-MB-231 (KRAS-G13D,11.2 nM), RPMI-8226 (KRAS-G12A, 11.3 nM), Calu-6 (KRAS-Q61K,19.8 nM), KP-2 (KRAS-G12R, 24.1 nM), LS123 (KRAS-G12S, 21 nM), as well as HRAS and NRAS mutant cell lines. TEB-17231 inhibits phospho-ERK downstream signaling in several tumor cell lines. Further, TEB-17231 exhibited tumor growth inhibition (IC50) in KRAS-G12C Ba/F3 (14 nM) and derivative cell lines that acquired resistance to G12C inhibitor MRTX849 by the additional KRAS domain mutations as follows: H95Q (32 nM), H95D (40 nM), Y96C (39 nM), Y96D (20 nM) and R68S (22 nM), compared with greater than 1μM for MRTX849. TEB-17231 induces late apoptosis of NCI-H358 cells in a manner that is time and concentration dependent. In addition to the improved selectivity and potency, TEB-17231 demonstrated satisfactory pharmacokinetics and oral bioavailability in mice (55%). Furthermore, with the SW480 (G12V) in vivo xenograft model, TEB-17231 showed clear tumor growth inhibition with QD dosing for 4-8 mg/kg PO without body weight loss. These properties highlight TEB-17231 as a potential clinically actionable inhibitor for the KRAS-modified tumor population and warrant investigation of TEB-17231 in combination with other agents in the future. Citation Format: Zusheng Xu, David T. Weaver, Robert Drakas, Yangtong Lou. The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance [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 2627.

Abstract 576: ACTM-838, a novel immunotherapy that enriches in solid tumors after IV dosing and comprehensively reverses the immunosuppressive TME to promote durable anti-tumor immunity

Abstract ACTM-838 is an avirulent bacterial immunotherapy that encodes an engineered IL-15 (IL-15plex) and a constitutively active STING variant (eSTING). The ACTM-838 strain is a highly modified, attenuated S. Typhimurium that lacks several major inflammatory and immunogenic components on the surface of the microbe and is designed to naturally and specifically enrich in the tumor microenvironment (TME) via auxotrophic dependency on metabolites of the adenosine pathway and purines, to achieve tumor-specific payload delivery. ACTM-838 was selectively internalized by phagocytic, primary human antigen presenting cells (APCs) but not endothelial cells. ACTM-838 was stable in human whole blood and is not inactivated by complement. Human macrophage uptake of labeled ACTM-838 led to acidic lysosomes and nuclear delivery with a concomitant increase in expression of encoded IL-15plex and eSTING payloads such as IL-15, IRF3 reporter activity and IFN ⍺/β secretion, respectively. Primary human and murine macrophages polarized towards a dual co-stimulatory and phagocytic M1/M2 dual phenotype in response to ACTM-838. ACTM-838 delivered eSTING payload in STING knockout macrophages and induced significantly higher Type I IFN production over a negative control strain. IV delivered ACTM-838 showed dose and payload dependent tumor enrichment and potent single-agent efficacy in two independent syngeneic tumor models, EMT6 (orthotopic) and MC38. Sustained memory anti-tumor immunity upon tumor rechallenge in cured animals was demonstrated. ACTM-838 treated tumors exhibited a significant increase in human IL-15plex protein payload. ACTM-838 as a single agent induced profound immune reprogramming and remodeling of the TME over time through increased infiltration and activation of myeloid, NK, B and cytolytic CD8 T cells, MHC class I and II, antigen presentation as well as decreased Treg, PD1+Lag3+ exhausted T cells, and a remodeled stromal signature, as assessed by bulk RNAseq, and confirmed by flow cytometry. Synergistic anti-tumor activity was observed in multiple tumor models when ACTM-838 was dosed in combination with anti-PD1, safely inducing complete responses. No significant body weight loss or death was observed in any treatment regimens of ACTM-838 alone (single or multiple doses) or in combination with anti-PD1. Analyses of public RNAseq datasets from patients with primary or metastatic disease revealed cancer types with high levels of myeloid infiltration and adenosine enrichment, suggesting that ACTM-838 efficacy could provide benefit in these indications. ACTM-838 is a novel immunotherapy delivering IL-15plex + eSTING payloads to phagocytic APCs, inducing a durable anti-tumor immune response, after IV dosing. It possesses a compelling safety profile, and is currently in IND-enabling, preclinical development. Citation Format: Ping Fang, Kyle R. Cron, Oanh Pham, Julie R. Janes, John Brandenburg, Fauna Yarza, Bret Peterson, Christopher Thanos, Chan Whiting, Akshata R. Udyavar. ACTM-838, a novel immunotherapy that enriches in solid tumors after IV dosing and comprehensively reverses the immunosuppressive TME to promote durable anti-tumor immunity [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 576.

Abstract 1839: CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity

Abstract Despite clinical evidence of anti-tumor activity, cytokine therapies have been hampered by a narrow therapeutic window and limited efficacy. Two cytokines of interest are IL-2 and IL-12, which synergize to proliferate and activate T cells and natural killer cells. However, the only approved human IL-2 product (Proleukin) is rarely used in the clinic due to systemic toxicities, and no IL-12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic designed for intratumoral (IT) injection and retention, that co-delivers IL-2 and IL-12 in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of human IL-2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and human IL-12. LAIR2 and HSA functionally retain CLN-617 in the injected tumor by binding collagen and increasing molecular weight, respectively. A murine surrogate of CLN-617, mCLN-617, was delivered either IV or IT in B16F10 tumor-bearing mice. The concentration of mCLN-617 as measured in serum two hours after IT administration was <5% that of the concentration following IV administration, demonstrating robust retention of mCLN-617 in the tumor. Following mCLN-617 treatment, durable and complete responses with no significant body weight loss were observed in checkpoint-refractory tumor models, including B16F10, CT26, and MC38. mCLN-617 triggered a robust systemic anti-tumor immune response based on three lines of evidence. First, in mice implanted with two MC38 tumors, 70% of injected tumors and 40% of non-injected tumors were eradicated. When combined with IV-administered anti-PD1 antibody, >90% of both injected and non-injected tumors showed a complete response, while anti-PD1 alone exhibited no anti-tumor activity. Second, in mice implanted with an MC38 tumor in the flank and with luciferase-expressing MC38 cells delivered to the liver via intra-splenic injection, the injected flank tumor exhibited ~90% growth inhibition with either mCLN-617 monotherapy or anti-PD1 co-administration. Growth of established metastatic tumors, as measured by luciferase signal, was inhibited by mCLN-617 monotherapy, and metastatic tumor burden significantly regressed in combination with anti-PD1, while anti-PD1 alone exhibited no anti-tumor activity in the metastases. Third, in mice bearing two MC38 flank tumors, tumor-specific CD8+ T cells expanded in both the injected and non-injected tumors and in peripheral blood. We conclude that IT-administered mCLN-617 is efficiently retained in the injected tumor, thereby preventing systemic toxicity of the two cytokines, yet can activate and remodel systemic immunosurveillance for clearance of non-injected distal lesions. A phase I trial of CLN-617 in patients with advanced solid tumors is expected to commence in 2023. Citation Format: Naveen K. Mehta, Kavya Rakhra, Kristan Meetze, K. Dane Wittrup, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity [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 1839.

Abstract 4168: BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy

Abstract Introduction: T cell immune response plays critical roles in cancer immunotherapy. CD3 complex, composed of CD3ε, CD3δ and CD3γ chains, is an essential component of TCR-CD3 complex mediating TCR signaling. Targeting CD3 complex has therefore become a popular strategy for manipulation of T cell function. Clinically relevant animal models for proper evaluation of human CD3 antibodies is in great demand in the field. Currently, adoptive transfer of human CD3+ T cells or transplantation of human hematopoietic stem/precursor cells to immunodeficient mice are usually employed. However, these mouse models are not fully immunocompetent. In this study we have evaluated in vitro CD3 EDG functionality and in vivo therapeutic efficacy, in BALB/c-hCD3EDG knock-in mouse in which all the three components of the CD3 complex — Cd3ε, Cd3δ, and Cd3γ— are replaced by their human counterparts, CD3E, CD3D, and CD3G. Methods: A humanized CD3EDG mouse model (HuGEMM) on BALB/c background was generated by replacing the entire mouse CD3EDG with its human counterpart through ES cell-based gene targeting. The cell surface expression of CD3E and intracellular expression of CD3D and CD3G were confirmed by FACS. Naïve CD3+ T cells were isolated from the splenocytes of homozygous CD3EDG HuGEMM and activated by anti-hCD3 antibody. T cell proliferation was demonstrated by CFSE dilution assay. To validate the TCR functionality of humanized CD3EDG in vivo, we evaluated bispecific antibody anti-hCD3/hEpCAM5 in BALB/c-CD3EDG HuGEMM bearing CT26-hEpCAM5. Results: The expression of human CD3E, CD3D and CD3G were confirmed by flow cytometry. hEpCAM5 antigen expression was confirmed in CT26 cell line in vitro and tumor in vivo by flow cytometry. The TCR functionality was detected by in vitro T cell activation assays using anti-hCD3 antibody. In vivo efficacy was demonstrated using a bispecific antibody that simultaneously binds to human CD3 and human tumor associated antigen EpCAM5 in human CD3 EDG knock-in mice engrafted with CT26-hEpCAM5 tumor. The therapeutic efficacy of the bispecific antibody showed 32% tumor growth inhibition compared to control treatment. No significant body weight loss was recorded in any of the animals. Conclusion: This study showed the successful establishment of humanized CD3 knock-in BALB/c mouse models providing a full immunocompetent model for human CD3-targeted tumor immunotherapies evaluation. Citation Format: Demi Xiaojuan Liu, Kaixia Lian, Xuefei Yan, Jie Lin, Lei Zheng, Annie Xiaoyu An, Xiaoxi Xu, Ludovic Bourre, Jingjing Wang. BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy. [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 4168.

Abstract 4096: Assessing individual variability in efficacy and toxicity of autologous and allogeneic chimeric antigen receptor T-cell immunotherapy using a PBMC-humanized mouse model

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a revolutionary therapeutic to fight cancer. However, side effects such as cytokine-release syndrome and neurotoxicity often accompany and remain unpredictable. While autologous CAR-Ts are FDA-approved, allogeneic CAR-Ts are in active development in pursuit of “off-the-shelf” availability and reduced manufacturing costs. However, the same concern for adverse events applies. Importantly, how the immune characteristics of the patient may predispose towards toxicity is not well modeled. Further, it is unclear how autologous versus allogeneic products interact with the patient immune system. Toxicity does not correlate with in vitro characterization of the CAR-Ts, adding unpredictability to the toxicity and efficacy. These highlight the need for in vivo mouse models to explore biological variation, predictive biomarkers, and therapeutic intervention. Therefore, we developed a humanized mouse model using NSG™-MHC Class I/II double knock-out (DKO) mice engrafted with human PBMCs to access individual variability in CAR-T-induced toxicity and efficacy. DKO mice are optimal for assessing human cytokine induction from CAR-Ts as they show delayed onset of GvHD and low baseline cytokine levels. To test donor variation in autologous CAR-T toxicity, we humanized mice and treated with autologous CD19 CAR-Ts containing the CD28 costimulatory domain and CD3z chain from two human PBMC donors (Donor A and B). CAR-T successfully eliminated human B cells from both PBMC donors in vivo. We observed drastic body weight loss and 83% of Donor A mice (engrafted with 17M PBMCs) reached the humane endpoint. In contrast, Donor B mice did not experience body weight loss. CAR-T induced human cytokines such as IFN-ꝩ, IL-2, IL-4, IL-6, IL-10, TNFα, MIG, MIP-1a, and IP-10, recapturing clinical data. To test donor variation in allogeneic CAR-T toxicity, we engrafted mice with 10M PBMCs from 6 healthy donors and treated with allogeneic CAR-Ts from Donor A or B. Allogenic efficacy was greater in Donor B CAR- than Donor A CAR-Ts. Donor-specific variability was observed; mice humanized with one of the six donors, experienced significant body weight loss only from the Donor B CAR-Ts but not from Donor A CAR-Ts. Principal component analysis of cytokine data revealed that Donor B CAR-Ts induced more distinguished cytokine responses than Donor A CAR-Ts, driven by IFN-ꝩ and IL-10. Lastly, the data indicates that the CAR-T cells show poor expansion in allogeneic treatment, corresponding to the current literature. PBMC-humanized mice provide an in vivo platform to assess the toxicity and efficacy of autologous and allogeneic CAR-T treatment. This platform can be used to preclinically assess cytokine induction from the interaction between the patient immune system and allogeneic CAR-Ts from healthy donors. Citation Format: Won Lee, Destanie Rose, Katelyn Burleigh, Blair Armstrong, Heather Gustafson, Rebecca Gardner, James G. Keck, Jiwon Yang. Assessing individual variability in efficacy and toxicity of autologous and allogeneic chimeric antigen receptor T-cell immunotherapy using a PBMC-humanized mouse model. [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 4096.

Hepatic but not Intestinal FBP1 Is Required for Fructose Metabolism and Tolerance.

Fructose intolerance in mammals is caused by defects in fructose absorption and metabolism. Fructose-1,6-bisphosphatase 1 (FBP1) is a key enzyme in gluconeogenesis, and its deficiency results in hypoglycemia as well as intolerance to fructose. However, the mechanism about fructose intolerance caused by FBP1 deficiency has not been fully elucidated. Here, we demonstrate that hepatic but not intestinal FBP1 is required for fructose metabolism and tolerance. We generated inducible knockout mouse models specifically lacking FBP1 in adult intestine or liver. Intestine-specific deletion of Fbp1 in adult mice does not compromise fructose tolerance, as evidenced by no significant body weight loss, food intake reduction, or morphological changes of the small intestine during 4 weeks of exposure to a high-fructose diet. By contrast, liver-specific deletion of Fbp1 in adult mice leads to fructose intolerance, as manifested by substantial weight loss, hepatomegaly, and liver injury after exposure to a high-fructose diet. Notably, the fructose metabolite fructose-1-phosphate is accumulated in FBP1-deficient liver after fructose challenge, which indicates a defect of fructolysis, probably due to competitive inhibition by fructose-1,6-bisphosphate and may account for the fructose intolerance. In conclusion, these data have clarified the essential role of hepatic but not intestinal FBP1 in fructose metabolism and tolerance.

Antidiabetic activity of mango peel extract and mangiferin in alloxan-induced diabetic rats

BackgroundIn diabetic animals, there is a significant increase in plasma glucose, serum total cholesterol, triglyceride, and low-density lipoprotein levels, and decreased body weight, liver and muscle glycogen, and high-density lipoprotein. Effective treatment of diabetes mellitus is not yet known, even though the management of diabetes mellitus is considered a global concern. Plants and herbs have played an important role in the healthcare of many societies throughout history. Today’s researchers are investigating the potential for using these nonpharmaceutical approaches to treat and control diabetes, either in conjunction with standard treatments or as an alternative to them. Herbal formulations are favored because to lower cost and fewer side effects compared to other methods for alleviating diabetes and its consequences. In ethnomedicinal practices, different parts of Mangifera indica are used to treatment of diabetes. The present investigation was undertaken to evaluate the antidiabetic activity of an ethanolic extract of Mangifera indica and mangiferin in alloxan-induced diabetic rats. This experiment was conducted in a set of two with four groups of animals namely control (Tc), treatment alloxan (Ta), treatment extract (Tae), and treatment mangiferin (Tam). To develop diabetes, Wistar rats treated with 150 mg/kg b.w. of alloxan monohydrate were injected intraperitoneally. Tae and Tam’s groups received a freshly prepared single dose of extract and mangiferin in distilled water via the oral route. All experimental groups received laboratory pallet feed diet and drinking water ad libitum. Diabetic rats were treated for 21 days with an ethanolic extract of mango peel and pure mangiferin orally daily at rates of 200 mg/kg b.w. and 20 mg/kg b.w.ResultsAn alloxan-induced diabetic rat treated with mango peel extract and mangiferin significantly improved the overhead impact due to diabetes. There was a significant (p < 0.05) body weight loss in the alloxan-induced diabetic rats (Ta), whereas animals given mango peel extract and mangiferin showed a significant increase in body weight from 2 weeks onwards in comparison with control. Alloxan-induced rats (Ta) group have higher blood glucose levels and are significantly different (p < 0.01) from the control group. Mango peel extract and mangiferin significantly reduced the levels of fasting glucose after 21 days of treatment in comparison with diabetic animals. Mango peel extract and mangiferin influence the glycogen synthesis pathway in diabetes groups by increasing glycogen levels in muscle and liver. mango peel extract and mangiferin were found to have a nonsignificant impact on plasma cholesterol and HDL levels compared with the control group. Mango peel extract was found to have a significant difference (p < 0.05) in LDL levels compared with the control group. Mangiferin was found to have a significant difference (p < 0.05) in triglyceride and VLDL levels when compared with the control group. Histopathological examination of the pancreas in rats with type I diabetes caused by alloxan found that therapy with an ethanolic extract of mango peel and mangiferin restored beta cell function as well as rejuvenation of Islets of Langerhans.ConclusionsMango peel extract and mangiferin have antidiabetic, glycogenesis, and hypolipidemic properties when administered to alloxan-induced diabetic rats.Graphical abstract

Impact of a Community-Based Weight Management Program in a North Carolina Health Care System.

Current strategies for obesity management in primary care leave many patients inadequately treated or unable to access treatment entirely. We aimed to evaluate a comprehensive, primary care clinic-based weight management program's clinical effectiveness in a community practice setting. Methods: This was an18-month pre/postintervention study. We collected demographic and anthropometric data on patients enrolled in a primary care-based weight management program.The primary outcomes were percent weight loss postintervention and the proportion of patients who achieved a clinically significant total body weight loss (TBWL) of 5% or greater. Results: Our program served 550 patients over 1,952 visits from March 2019 through October 2020.A total of 209 patients had adequate program exposure, defined as four or more completed visits. Among these, all received targeted lifestyle counseling and 78% received antiobesitymedication.Patients who attended at least four visits had an average TBWL of 5.7% compared to an average gain of 1.5% total body weight for those with only one visit. Fifty-three percent of patients (n=111)achieved greater than 5% TBWL, and 20%(n=43)achieved greater than 10% TBWL. We demonstrated that a community-based weight management program delivered by obesity medicine-trained primary care providers effectively produces clinically significant weight loss. Future work will include wider implementation of this model to increase patient access to evidence-based obesity treatments in their communities.

Carborane bearing pullulan nanogel-boron oxide nanoparticle hybrid for boron neutron capture therapy

Boron neutron capture therapy shows is a promising approach to cancer therapy, but the delivery of effective boron agents is challenging. To address the requirements for efficient boron delivery, we used a hybrid nanoparticle comprising a carborane = bearing pullulan nanogel and hydrophobized boron oxide nanoparticle (HBNGs) enabling the preparation of highly concentrated boron agents for efficient delivery. The HBNGs showed better anti-cancer effects on Colon26 cells than a clinically boron agent, L-BPA/fructose complex, by enhancing the accumulation and retention amount of the boron agent within cells in vitro. The accumulation of HBNGs in tumors, due to the enhanced permeation and retention effect, enabled the delivery of boron agents with high tumor selectivity, meeting clinical demands. Intravenous injection of boron neutron capture therapy (BNCT) using HBNGs decreased tumor volume without significant body weight loss, and no regrowth of tumor was observed three months after complete regression. The therapeutic efficacy of HBNGs was better than that of L-BPA/fructose complex. BNCT with HBNGs is a promising approach to cancer therapeutics.

Effects of Unilateral Vagotomy on LPS-Induced Aspiration Pneumonia in Mice.

Respiratory-related dysphagia and aspiration pneumonia can be attributed to multiple causes. However, reproduction of multiple factor-related respiratory distress and aspiration pneumonia in a single animal model is challenging. To validate animals with vagal nerve palsy as novel models for severe aspiration pneumonia associated with respiratory distress, we investigated the effects of unilateral vagotomy on the swallowing function and severity of pneumonia after forced aspiration in mice. Unilateral vagotomy was performed in C57BL6 male mice that subsequently underwent evaluation of swallowing function by videofluoroscopic swallow study (VFSS) and histological assessments for aspiration pneumonia induced by lipopolysaccharide (LPS). VFSS examinations demonstrated that unilateral vagotomy did not cause apparent aspiration in mice, but it resulted in a significant loss of body weight (BW) due to decreased oral intake. In addition, when aspiration pneumonia was induced by forced administration of LPS, significantly prolonged BW loss and severe infiltration of inflammatory cells associated with aspiration pneumonia were observed in the mice that underwent unilateral vagotomy. In conclusion, the vagotomized mice showed appropriate characteristics as a model of aspiration pneumonia caused by multiple factors, including the paralysis of vocal fold movement and respiratory distress. This model can help elucidate the pathogenesis of aspiration pneumonia and the treatment methods for the respiration-compromised model.

Surf4 (Surfeit Locus Protein 4) Deficiency Reduces Intestinal Lipid Absorption and Secretion and Decreases Metabolism in Mice.

Postprandial dyslipidemia is a causative risk factor for cardiovascular disease. The majority of absorbed dietary lipids are packaged into chylomicron and then delivered to circulation. Previous studies showed that Surf4 (surfeit locus protein 4) mediates very low-density lipoprotein secretion from hepatocytes. Silencing hepatic Surf4 markedly reduces the development of atherosclerosis in different mouse models of atherosclerosis without causing hepatic steatosis. However, the role of Surf4 in chylomicron secretion is unknown. We developed inducible intestinal-specific Surf4 knockdown mice (Surf4IKO) using Vil1Cre-ERT2 and Surf4flox mice. Metabolic cages were used to monitor mouse metabolism. Enzymatic kits were employed to measure serum and tissue lipid levels. The expression of target genes was detected by qRT-PCR and Western Blot. Transmission electron microscopy and radiolabeled oleic acid were used to assess the structure of enterocytes and intestinal lipid absorption and secretion, respectively. Proteomics was performed to determine changes in protein expression in serum and jejunum. Surf4IKO mice, especially male Surf4IKO mice, displayed significant body weight loss, increased mortality, and reduced metabolism. Surf4IKO mice exhibited lipid accumulation in enterocytes and impaired fat absorption and secretion. Lipid droplets and small lipid vacuoles were accumulated in the cytosol and the endoplasmic reticulum lumen of the enterocytes of Surf4IKO mice, respectively. Surf4 colocalized with apoB and co-immunoprecipitated with apoB48 in differentiated Caco-2 cells. Intestinal Surf4 deficiency also significantly reduced serum triglyceride, cholesterol, and free fatty acid levels in mice. Proteomics data revealed that diverse pathways were altered in Surf4IKO mice. In addition, Surf4IKO mice had mild liver damage, decreased liver size and weight, and reduced hepatic triglyceride levels. Our findings demonstrate that intestinal Surf4 plays an essential role in lipid absorption and chylomicron secretion and suggest that the therapeutic use of Surf4 inhibition requires highly cell/tissue-specific targeting.

Antitumor activity of intraperitoneal (IP) paclitaxel to mucinous appendiceal adenocarcinoma in orthotopic patient-derived xenograft model.

151 Background: Appendiceal adenocarcinomas (AAs) are a rare and heterogeneous mix of tumors for which only few preclinical models exist for drug discovery. These tumors are commonly treated with chemotherapy like those for colorectal cancer despite clear evidence that the two cancers are distinctly different in their clinical behavior and molecular profiles. Taxanes such as paclitaxel (PTX) and docetaxel have been used in the systemic treatment of gastric cancer with peritoneal metastases. IP administration of PTX has been considered a promising treatment for eliminating peritoneal metastasis, as IP delivery of PTX can attain a higher drug exposure in the peritoneal cavity with reduced systemic toxicity because of high local concentrations over a long period of time due to its hydrophobic property. The purpose of this study was to test the efficacy of IP PTX treatment in orthotopic AA PDX models. Methods: AA tumors TM00351, PMP-2 and PMCA-3 were implanted in the peritoneal cavity of NSG mice. PDX models were treated with PTX (6.25, 12.5 or 25.0 mg/kg, IP) with weekly (3 weekly treatments and 1 week off, for two cycles) or biweekly (biweekly treatments for up to 12 weeks) schedule. The peritoneal tumor burden was monitored by MRI. Solid tumor size was evaluated by modified peritoneal Response Evaluation Criteria In Solid Tumors (mpRECIST) method using ImageJ software and mucinous tumor volume was quantified by OsiriX Lite software. Results: TM00351, taken from a high-grade tumor (Grade 3), formed mucin-rich solid tumors with mucinous ascites. PDX models PMP-2 (Grade 2) and PMCA-3 (Grade 3), both with GNASR201C mutation, had a mucinous phenotype similar to GNAS mutant appendiceal tumors in humans. Weekly 25.0 mg/kg of IP PTX treatment reduced AA tumor growth of TM00351 (77.9% reduction vs. control), PMP-2 (98.6% reduction vs. control), and PMCA-3 (85.6% reduction vs. control). Similarly, biweekly 25.0 mg/kg of IP PTX treatment was effective on PMCA-3 (66.2% reduction vs. control). Survival of PMCA-3 PDX mice was significantly improved by 12.5 mg/kg (p=0.0254, log-rank test) and 25.0 mg/kg (p=0.0038) of IP PTX. At 25.0 mg/kg IP PTX treatment induced 12.0% body weight loss of PMCA-3 PDX mice 1 week after the treatment. Lower doses of IP PTX treatment, 6.25 and 12.5 mg/kg, did not induce significant body weight loss or any noticeable adverse effects on mice. Comparing the efficacy of IV to IP administration, neither 6.25 or 12.5 mg/kg of IV PTX reduced growth of PMCA-3, 25.0 mg/kg of IV PTX was lethal to mice shortly after administration. Conclusions: IP PTX is a therapeutically active for mucinous AA tumors.

Observações da infecção coccidiana, Eimeria labbeana-like, em pombos domésticos (Columba livia domestica) experimentalmente infectados, associada a efeitos patológicos

ABSTRACT Genus Eimeria is a group of obligate Protista parasites within phylum Apicomplexa that causes a major threat to animal production. Little information is available about the coccidia of pigeons. This study was undertaken to study experimental infection in domesticated pigeons, its clinical signs, and pathological interpretation due to Eimeria species. Eimeria labbeana-like oocysts were used as a model parasite for experimental infection of pigeons. Twenty pigeons were brought and examined for 7 days to ensure they were coccidia-free. Five pigeons were separated into parasite passage. The remaining pigeons were divided into a control group of five pigeons, and ten pigeons were infected with 3×104 E. labbeana-like oocysts. Infected pigeons showed ruffled feathers, greenish watery diarrhea, weakness, blood-tinged droppings, etc. Pre-patent and patent durations were five and fourteen days of oral inoculation of Eimeria. Oocysts shedding began on the 5th-day p.i. and peaked on the 8th-day p.i. There is a significant loss in body weight due to infection. Histopathological examination showed damage to intestinal mucosa with various Eimeria developmental stages in the duodenal of the small intestine. This study is considered basic information on the first detection of biology of Eimeria species infecting pigeons in Saudi Arabia.

Characteristics of behavioral reactions and the profile of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism

The neurotoxins rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (МPTP) are used for modeling Parkinson's disease in animals (PD). They induce the mitochondrial respiratory chain dysfunction, which leads to the dopaminergic (DA) neuron degeneration. The advantage of the rotenone model consists in ability of rotenone to cause neurodegeneration showing symptoms and molecular biological characteristics similar to those of PD. Isatin (indoldione-2,3) is an endogenous regulator found in tissues and biological fluids of humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. In this work we have investigated behavioral reactions and profiles of brain isatin-binding proteins of rats with Parkinson's syndrome (PS) in comparison with the corresponding parameters of MPTP-induced Parkinsonism in mice. Systemic injection of rotenone caused severe PS comparable with the effect of MPTP injection. It was accompanied by significant body weight loss, death, oligokinesia, muscular rigidity, and postural instability of animals. In spite of the same pathogenic basis of PS caused by rotenone and MPTP, the molecular mechanisms of their action differ. In the case of rotenone-induced PS, the pool of isatin-binding proteins common of the control rats and the rats with PS (146) significantly exceeded the pool of the common proteins of control mice and mice with PS induced by MPTP, whether right after neurotoxin injection (27), or (all the more) in a week after the MPTP injection (14). The comparison of isatin-binding proteins specific of the animals with MPTP-induced PS and with the rotenone-induced PS (as compared with the control animals) revealed total absence of proteins common of these two models of PD. It is to be noted that both neurotoxins particularly affected the proteins participating in the signal transmission and enzyme activity regulation. The changes of the profile of isatin-binding proteins in response to the injection of rotenone suggest that the neuroprotector isatin could also influence positively in the case of the rotenone model of PD.

Discovery of DS-9300: A Highly Potent, Selective, and Once-Daily Oral EP300/CBP Histone Acetyltransferase Inhibitor

Histone acetylation is a post-translational modification of histones that is catalyzed by histone acetyltransferases (HATs) and plays an essential role in cellular processes. The HAT domain of EP300/CBP has recently emerged as a potential drug target for cancer therapy. Here, we describe the identification of the novel, highly potent, and selective EP300/CBP HAT inhibitor DS-9300. Our optimization efforts using a structure-based drug design approach based on the cocrystal structures of the EP300 HAT domain in complex with compounds 2 and 3 led to the identification of compounds possessing low-nanomolar EP300 HAT inhibitory potency and the ability to inhibit cellular acetylation of histone H3K27. Optimization of the pharmacokinetic properties in this series resulted in compounds with excellent oral systemic exposure, and once-daily oral administration of 16 (DS-9300) demonstrated potent antitumor effects in a castrated VCaP xenograft mouse model without significant body weight loss.