Preclinical Toxicology Research Articles

Characterization of anti-CD79b/CD3 bispecific antibody, a potential therapy for B cell malignancies.

High rates of relapse and poor prognosis confer an urgent need for novel therapeutic agents for B cell non-Hodgkin lymphomas (B-NHLs). Herein, we describe a human IgG-like anti-CD79b/CD3 bispecific antibody (IBI38D9-L) that selectively depletes antigen-positive malignant B cells as an alternative treatment option for relapsed or refractory NHL patients. The antitumor activity and mechanism of action of IBI38D9-L were investigated in vitro using B-NHL cell lines and human primary effector cells and in vivo using xenograft models reconstituted with human PBMCs (peripheral blood mononuclear cells). Pharmacokinetic (PK) properties and preclinical toxicology were evaluated in cynomolgus monkeys and HSC-NPG mice. IBI38D9-L exerted potent B cell killing as well as T cell activation and proliferation in a tumor cell-dependent manner in vitro and was active against B-NHL cell lines with various CD79b expression levels. Subcutaneous xenograft tumors in NOG mice engrafted with human PBMCs were eradicated by IBI38D9-L treatment. Moreover, IBI38D9-L-treated mice showed a strong infiltration of activated T cells. In HSC-NPG mice, IBI38D9-L resulted in potent B cell depletion in peripheral blood and induced only slight body weight loss and cytokine release syndrome without significant toxicological findings. In cynomolgus monkeys, IBI38D9-L was well tolerated with good pharmacokinetic profiles. Collectively, these preclinical efficacy and safety data provide strong scientific rationales for using anti-CD79b/CD3 bispecific antibody as a promising therapeutic agent for B cell malignancies.

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

Application of Pharmacokinetic/Pharmacodynamic Modeling to Bridge Mouse Antitumor Efficacy and Monkey Toxicology Data for Determining the Therapeutic Index of an Interleukin-10 Fc Fusion Protein

Pharmacokinetic/pharmacodynamic (PK/PD) modeling was performed to quantitatively integrate preclinical pharmacology and toxicology data for determining the therapeutic index (TI) of an interleukin-10 (IL-10) fragment crystallizable (Fc) fusion protein. Mouse Fc fused with mouse IL-10 (mFc-mIL-10) was studied in mice for antitumor efficacy, and the elevation of interleukin-18 (IL-18) was examined as a PD biomarker. The in vivo mFc-mIL-10 EC50 for the IL-18 induction was estimated to be 2.4 nM, similar to the in vitro receptor binding affinity (Kd) of 3.2 nM. The IL-18 induction was further evaluated in cynomolgus monkeys, where the in vivo induction EC50 by a human IL-10 human Fc-fusion protein (hFc-hIL-10) was 0.08 nM vs. 0.3 nM measured as the in vitro Kd. The extent of the IL-18 induction correlated with mouse antitumor efficacy and was used to connect mouse efficacy to that in monkeys. The PD-based efficacious dose projected in monkeys was comparable to the results obtained using a PK-based method in which mouse efficacious exposure was targeted and corrected for affinity differences between the species. Furthermore, PK/PD relationships were developed for anemia and thrombocytopenia in monkeys treated with hFc-hIL-10, with thrombocytopenia predicted to be dose-limiting toxicity. Using quantitative pharmacology and toxicology information obtained through modeling work in the same species, the TI of hFc-hIL-10 in monkeys was determined to be 2.4 (vs. PD-based efficacy) and 1.2–3 (vs. PK-based efficacy), indicating a narrow safety margin. The model-based approaches were proven valuable to the developability assessment of the IL-10 Fc-fusion protein.

Abstract 1358: The selective HDAC6 inhibitor ITF3756 stimulates an antitumor immune response and leads to tumor regression in combination with anti CTLA-4 antibody in a colon carcinoma murine model

Abstract Stimulation of tumor immune response with antibodies blocking the repressive activity of immune checkpoints gives impressive results with long-lasting tumor regression that is achieved regrettably only in a fraction of patients. To overcome these suboptimal results, different targets and combination therapies are the aim of active non-clinical and clinical research. Importantly, clinical trials of combination therapies have demonstrated better efficacy but also an increase of adverse events. HDAC6 is a member of the Zn-dependent histone deacetylase family of enzymes with peculiar structure and functions. In contrast to what is observed with many other HDAC family members, HDAC6 KO mice are viable and have no peculiar phenotype indicating that selective HDAC6 inhibitors should be well tolerated. Since HDAC6 is an obligate regulator of cytokine-induced PD-L1 expression, HDAC6 inhibitors might have a role in modulating tumor immune responses. We here report the in vivo efficacy of ITF3756, a potent and selective HDAC6 inhibitor that reduces in vitro the expression of PD-L1 on human monocytes and on CD8 T cells, counters immune exhaustion in CD8 T cells but shows no direct cytotoxic effects on a panel of murine tumor cell lines. In syngeneic tumor models ITF3756 showed anti-tumor activity that was comparable to the efficacy of an anti PD1 antibody and that went along with increased immune cell infiltration and the generation of tumor-reactive CD8 and CD4 cells. ITF3756 was inactive in immune-deficient animals and selective depletion of either CD8 or CD4 cells severely blunted the antitumor activity of ITF3756. In combination with an anti CTLA-4 antibody, ITF3756 lead to a complete tumor eradication in 50% of the animals. Re-challenge of these animals did not result in tumor growth indicating that the combination therapy had elicited tumor immunity. Blocking the PD-1/PD-L1 axis either alone or in combination with inhibition of CTLA4 in NOD mice strongly accelerated the development of auto-immune diabetes that was lethal in some treatment groups. In contrast neither ITF3756 alone nor the combination with anti CTLA4 lead to any significant induction of diabetes despite the activity of ITF3756 on the PD-1/PD-L1 axis. We conclude that ITF3756 induces an in vivo antitumor immune response and a durable antitumor activity when combined with an anti CTLA-4 antibody. Neither ITF3756 monotherapy nor the combination with anti CTLA4 accelerated the induction of autoimmune diabetes in NOD mice suggesting a favorable safety profile that was confirmed in preclinical GLP toxicology studies. Phase I clinical trials with ITF3756 will be initiated this year. Citation Format: Gianluca Fossati, Chiara Ripamonti, Gianluca Caprini, Pietro Pozzi, Elisabetta Galbiati, Paola Cordella, Mattia Marchini, Barbara Vergani, Giovanni Sandrone, Andrea Stevenazzi, Christian Steinkuhler. The selective HDAC6 inhibitor ITF3756 stimulates an antitumor immune response and leads to tumor regression in combination with anti CTLA-4 antibody in a colon carcinoma murine model [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 1358.

Abstract ND14: Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers

Abstract Pharmacological profile and anti-tumor properties of FHD-286, a novel BAF inhibitor for the treatment of transcription factor-driven cancersMartin Hentemann, Rishi G. Vaswani, Lan Xu, Richard C. Centore, Luis M. M. Soares, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Sarah Reilly, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Liyue Huang, Jessica Piel, Johannes Voigt, David S. Millan, Ho Man Chan, Ryan G. Kruger, Carl P. Decicco, Samuel Agresta, Steven F. BellonFoghorn Therapeutics, 500 Technology Square, Suite 700, Cambridge, MA 02139The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a compound that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that this SMARCA4/SMARCA2 inhibitor acts through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. FHD-286 is orally bioavailable and demonstrates robust efficacy in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. In preclinical efficacy and toxicology studies, this first in class molecule demonstrates an acceptable therapeutic index which should allow for flexibility in clinical designs. FHD-286 is currently in two Phase I trials for both relapsed refractory AML and MDS, and metastatic uveal melanoma. Citation Format: Murphy Hentemann. Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers [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 ND14.

Abstract 572: Pre-clinical evaluation of Claudin 18.2 TAC T cells for the treatment of gastric cancer

Abstract Background: The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the re-direction of T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the goal to elicit a safe and durable anti-tumor response. Based on this preclinical pharmacology and toxicology data, TAC01-HER2, a first-in-class TAC T product targeting HER2 (ERBB2), has entered a phase I/II clinical trial in patients with HER2-positive solid tumors. Here, we present the development of a new TAC T product targeting Claudin 18.2 (CLDN18.2) to treat gastric cancer. CLDN18.2 belongs to a family of Claudin tight junction proteins whose expression is naturally exclusive to normal stomach. In gastric cancer cells, however, CLDN18.2 surface expression is upregulated and perturbed, leading to tumor-selective surface expression of CLDN18.2. Thus, CLDN18.2 is a preferred antigen for the specific targeting of tumor cells via TAC T cells. Materials and Methods: CLDN18.2-TAC receptor functionality was characterized using a variety of in vitro and in vivo assays. In vitro assays were based on flow cytometric analysis of TAC surface staining and cytokine release. Cytotoxicity was assessed via luminescence-based co-culture assays and real-time microscopy. In vivo studies examined the anti-tumor effect of TAC-engineered T-cells against established solid CLDN18.2 expressing tumor xenografts. Results: The CLDN18.2-TAC receptor showed strong surface expression and specific activation when co-cultured with a variety of cancer cells expressing CLDN18.2 in vitro. Secretion of IL2, IFNg and TNFa were comparable with cytokine levels produced by activated control TAC T cells. In vitro cytotoxicity assays demonstrated a strong anti-CLDN18.2 response and killing of CLDN18.2 expressing target cell lines. No increases in cytokine levels and no cytotoxicity were observed in non-transduced T cells and CLDN18.2-TAC T cells co-cultured with CLDN18.2-negative target cells, indicating that the T cell response is specific to the CLDN18.2 antigen. Intravenous administration of CLDN18.2-TAC T cells in mice carrying CLDN18.2-positive solid tumor xenografts led to a sustained anti-tumor response. Conclusion: The in vitro and in vivo data confirm strong and specific activity of CLDN18.2-targeted TAC T cells against CLDN18.2-expressing solid tumor models and highlight the versatility of the TAC platform for therapeutic applications in solid tumors. Citation Format: Tania Benatar, Ling Wang, Thanyashanthi Nitya-Nootan, Heather MacGregor, Suzanna Prosser, Philbert Ip, Prabha Lal, Laura Shaver, Sadhak Sengupta, Christopher W. Helsen, Andreas G. Bader. Pre-clinical evaluation of Claudin 18.2 TAC T cells for the treatment of gastric cancer [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 572.

Abstract 3203: Cutting edge biomarkers strategy to provide early insights into activity of EVT-801, a novel selective VEGFR-3 inhibitor that targets tumor angiogenesis during the FIH clinical trial

Abstract Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 and acts by inhibiting lymphangiogenesis and tumor angiogenesis in and around the tumor. It has shown compelling activity in a wide range of cancer models and is well-tolerated in preclinical toxicology studies. A phase I study is underway and will focus primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses. Methods: The first stage of the study is designed to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801, with a second stage aiming to confirm RP2D in selected cancer patient populations. Clinical samples from these patients will be used to explore preliminary signals of clinical efficacy and investigate the biological activity of the drug using several biomarkers. Efficacy biomarkers will include imaging approaches (DCE-MRI and CEUS) to characterize tumor vascularization, as well as analysis of protein and mRNA quantification in on-treatment biopsies vs pre-treatment biopsies. Moreover, the relationship between activity of EVT801 and expression of key markers at protein and mRNA level will be investigated to potentially establish biomarkers for patient stratification and selection. Lastly, target engagement and pharmacodynamics effects will be investigated by immuno-monitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. We expect that these analyses will help to better understand the effects of the drug in human subjects and may also help to select the most responsive patients and provide early signs of clinical efficacy. Citation Format: Michael R. Paillasse, James Garner, Michael Fitzgerald, Lise Davenne, Pierre-Benoit Ancey, celine Poussereau-Pomie, Michael Esquerre, Gaelle Badet, Joel Tuyaret, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Ménétrier-Caux, Chrsitophe CAUX, Philippe Cassier, Jean-Pierre Delord, Carlos Gomez Roca, Pierre Fons. Cutting edge biomarkers strategy to provide early insights into activity of EVT-801, a novel selective VEGFR-3 inhibitor that targets tumor angiogenesis during the FIH clinical trial [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 3203.

Abstract CT248: Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors

Abstract Background: BRAFV600 alterations (BRAF Class (Cl) I alterations) encode a highly active monomeric oncogenic kinase that can be targeted by approved RAF kinase inhibitors (RAFi) in patients (pts) with melanoma (MEL), NSCLC, colorectal & anaplastic thyroid cancers. Cl II and III represent molecularly distinct BRAF alterations driving dimer-dependent signaling. Cl II alterations include single nucleotide variants (SNVs), indels and gene fusions that induce BRAF homodimer formation, leading to oncogenic kinase activation. Cl III alterations, typically SNVs, are low activity alterations that promote RAF dimerization and RAS-dependent oncogenic MAPK pathway signaling. Approved BRAFi have limited clinical activity in solid tumors driven by BRAF Cl II or III alterations, highlighting the urgency to develop effective targeted therapies for these patients. Combinations of a RAFi and a MEK inhibitor (MEKi) show promising results in early clinical trials. KIN-2787 is a potent, highly selective type II pan-RAFi designed to target both monomeric & dimeric forms of the altered BRAF kinase and minimize paradoxical activation, a liability often observed with other RAFi that can adversely impact tolerability & require addition of a MEKi to suppress pathway activation. KIN-2787 has favorable pharmaceutical properties, achieves substantial systemic exposures in pre-clinical toxicology studies, & induces regressions in human cancer xenograft models driven by BRAF Cl I, II, or III alterations. Combinations of KIN-2787 with a MEKi show promising activity in preclinical models. Methods: This is a FIH, multicenter, non-randomized Ph1 study (NCT04913285) of KIN-2787 in adult pts with BRAF mutant advanced & metastatic solid tumors (AMST). KIN-2787 will be given orally bid continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 155 pts in 2 parts: Part A is a dose-escalation to MTD open to pts with AMST driven by either BRAF genomic alterations (Cl I, II or III ) or by NRAS. Part A1 assesses single agent KIN-2787; Part A2 assesses KIN-2787 in combination w/binimetinib. Part B will evaluate a selected dose of KIN-2787 in 3 cohorts of pts (MEL, NSCLC, or other AMST), each driven by BRAF Cl II or III alterations. Standard Ph1 enrollment criteria are required (ECOG PS 0-2, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases, prior BRAF-, MEK-, or MAPK inhibitor therapy (unless for FDA approved indications), HBV/HCV seropositivity. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, & duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of MAPK signaling inhibition. Enrollment began in August 2022. Trial is open globally. Citation Format: Meredith McKean, Cesar A. Perez, Shumei Kato, Alexander I. Spira, Yan Xing, Hao Xie, Michael Millward, Anthony Olszanski, Ken Kobayashi, Nichol Miller, Paul Severson, Betty Tam, Caro Unger. Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors [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 CT248.

Abstract 5443: BPI-421286: A highly potent small molecule inhibitor targeting KRASG12C mutation

Abstract KRAS is a GTP-binding protein that links receptor tyrosine kinase activation to intracellular signaling. Mutation of KRAS favors the GTP-bound active state and constitutive activation of downstream effects. KRASG12C mutation has been identified as an oncogenic driver of tumorigenesis, which is found in approximately 14% of lung cancer, 4% of colorectal (CRC), and 1-3% of other solid tumors. Herein, we report BPI-421286, a highly potent and selective covalent small molecular KRASG12C inhibitor. In vitro, BPI-421286 showed a high Kinact/Ki value (256,000 M-1 s-1), and inhibited KRAS-GTP/GDP exchange with a lower IC50 (55 nM). In cell-based assays, BPI-421286 suppressed proliferation of KRASG12C mutant cell lines with sub-nanomolar potency, but did not affect cell lines with wide type or non-G12C KRAS mutations. BPI-421286 also inhibited phospho-ERK in MIA PaCa-2 cell with a low IC50 value (2.9 nM). In vivo, daily oral administration of 3-100 mg/kg BPI-421286 led to tumor growth suppression or regression in multiple KRASG12C mutant xenograft or PDX models of NSCLC, CRC and PDAC. The in vivo PKPD in MIA PaCa-2 xenograft model showed that durable pERK inhibition can be achieved without continuous drug exposure, featuring BPI-421286 as a covalent, non-reversible KRAS inhibitor. Combining BPI-421286 with several targeted agents, including a SHP2 inhibitor BPI-442096 and a CDK4/6 inhibitor BPI-16350, demonstrated enhanced tumor growth inhibition compared to either agent alone. Notably, for the acquired resistance mutations that are clinically observed after KRASG12C inhibitor treatment, such as G12C/H95D, G12C/H95Q, G12C/R68S and G12C/Y96C, BPI-421286 also exhibited strong inhibitory activities, suggesting that BPI-421286 could overcome the acquired resistance of currently approved KRASG12C inhibitors. Taken together, BPI-421286 is a highly potent and selective KRASG12C inhibitor, with favorable ADME properties and wide therapeutic index in pre-clinical toxicology studies. BPI-421286 is currently in Phase 1 clinical study. Citation Format: Xiaoguan Zhu, Yuan Lu, Jing Guo, Dan Yan, Boyan Li, Xiaoping Chen, Haibo Chen, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-421286: A highly potent small molecule inhibitor targeting KRASG12C mutation [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 5443.

Organoid-derived intestinal epithelial cells are a suitable model for preclinical toxicology and pharmacokinetic studies

SummaryIntestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.

A DNA vaccine candidate delivered by an electroacupuncture machine provides protective immunity against SARS-CoV-2 infection

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3–5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261–285) and receptor-binding domain (a.a. 352–363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

Preclinical nerve conduction: Nerve battery options for primate studies

Electrophysiological neurodiagnostic tests of nerve conduction (NC) are key assays included in preclinical safety and toxicology programs to assess the peripheral neuropathy (PN) liability of a new drug. Despite their increased use, standardization of nerve conduction studies (NCS) is lacking in the preclinical space, with limited regulatory guidelines stipulating type and number of nerves or minimum combinations appropriate for each stage of drug development or indication. Detection of subtle peripheral toxicities depends on choosing appropriate nerve targets for testing, especially when functional changes remain above the lower limit of normal values. To support robust preclinical toxicology study designs, the current short communication provides options and recommendations for selecting peripheral nerves for clinically translatable nerve conduction batteries applicable to toxicology and gene therapy, with a focus on primate models. A comprehensive compilation of accessible nerve locations is offered including lower and upper extremity motor nerves, and sensory nerves with origin at multiple DRG levels. Rankings of technique difficulty and repeatability across serial collections are presented for each assay informed by serial nerve conduction from 500 adult primates. The goal of this communication is to support the standardization and preclinical implementation of this important assay.

Discovery and Preclinical Profiling of GSK3839919, a Potent HIV-1 Allosteric Integrase Inhibitor.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have been of interest recently because of their novel mechanism of action. Strategic modifications to the C5 moiety of a class of 4-(4,4-dimethylpiperidinyl)-2,6-dimethylpyridinyl ALLINIs led to the identification of a tetrahydroisoquinoline heterocycle as a suitable spacer element to project the distal hydrophobic aryl ring. Subsequent optimization of the aryl substitutions identified 12 as an ALLINI with single-digit nanomolar inhibitory potency and low clearance across preclinical species. In preclinical toxicology studies with 12 in rats, lipid hepatocellular vacuolation was observed. Removal of the C6 methyl group resulted in GSK3839919 (22), which exhibited a reduced incidence and severity of lipid vacuolation in both in vitro assays and in vivo studies while maintaining the potency and pharmacokinetic (PK) properties of the prototype. The virology, PK, and toxicology profiles of 22 are discussed.

Preclinical Pharmacology and Toxicology of intrathecally infused AAV9-hABCD1, a gene therapy candidate for AMN, in Non-Human Primates (S29.002)

Preclinical Pharmacology and Toxicology of intrathecally infused AAV9-hABCD1, a gene therapy candidate for AMN, in Non-Human Primates (S29.002)

Pre-clinical safety and toxicology profile of a candidate vaccine to treat oxycodone use disorder

Opioid use disorders (OUD) and overdose represent a public health threat, resulting in thousands of deaths annually worldwide. Vaccines offer a promising treatment for OUD and potentially the prevention of fatal overdoses. The Oxy(Gly)4-sKLH Conjugate Vaccine, Adsorbed (Oxy(Gly)4-sKLH) has shown promising pre-clinical efficacy at reducing the behavioral and pharmacological effects of oxycodone. To support its clinical evaluation, a GLP toxicology study was performed to address the safety of Oxy(Gly)4-sKLH. Sprague Dawley rats were vaccinated with either aluminum adjuvant (alum) or vaccine adsorbed on alum. Low and high doses of Oxy(Gly)4-sKLH, equivalent to a 1X or 47X human dose, respectively, were administered every two weeks for a total of four vaccinations. Both vaccine doses induced high antibody titers. Vaccine-related toxicity was assessed postmortem in one experimental group after receiving the fourth immunization of the vaccine’s high dose. For the remaining experimental groups, rats were challenged with 1.5 mg/kg/day s.c. oxycodone for 7 days after the fourth vaccination to assess whether concurrent exposure to oxycodone in vaccinated animals resulted in toxicity. All rats, except a subset of the aluminum control and the high dose vaccine groups, were sacrificed following oxycodone exposure. These subsets were allowed a four weeks recovery period prior to euthanasia. In this study, no Oxy(Gly)4-sKLH-related hematology, clinical chemistry, urinalysis, body weight, organ weight, or anatomic pathology toxicological findings were observed. These results demonstrate that the Oxy(Gly)4-sKLH vaccine is well tolerated, is immunogenic even at low doses, and does not produce undesired side effects in rats.

Preclinical safety evaluation of levonadifloxacin, a novel anti-methicillin-resistant Staphyloccocus aureus benzoquinolizine fluoroquinolone by intravenous and oral administration.

Fluoroquinolone (FQ) antibacterials have drawn heightened attention from various international regulatory agencies due to their class-specific side effects. Levonadifloxacin is a novel broad spectrum benzoquinolizine FQ active against methicillin-resistant Staphyloccocus aureus (MRSA). Owing to FQ-associated safety concerns, extensive preclinical safety pharmacology (central nervous system and cardiac safety) and toxicology studies (subacute repeat-dose toxicity, genotoxicity, phototoxicity and chondrotoxicity) of levonadifloxacin were performed at relatively high doses. Intravenous (IV) and oral studies were conducted using WCK 771 (l-arginine salt of levonadifloxacin) and WCK 2349 (l-alanine ester prodrug of levonadifloxacin), respectively. Safety pharmacology studies following single dose revealed no adverse effects on central nervous system (including seizure) in mice and cardiovascular system (hERG and monkey telemetry). In repeat-dose toxicity studies, except for IV bolus dosing related effects in rat (hyperactivity, mild convulsion, polypnoea and injection site irritation) and dog (emesis and salivation), no other adverse findings limiting the dosing duration were observed. No major biochemical, haematological, gross or histopathological changes suggestive of damage to vital organs were observed in either WCK 771- or WCK 2349-treated groups. WCK 771 and WCK 2349 were found to be nongenotoxic; however, they showed weak phototoxicity that was comparable with levofloxacin. WCK 771 showed chondrotoxicity in the Beagle dog pups on repeat-dose administration; however, the severity level was lower than ofloxacin. Overall, preclinical safety studies helped establish wider safety margin for WCK 771 and WCK 2349 that supports administration of higher therapeutic doses in humans by both IV and oral routes, thereby enabling safe anti-MRSA treatment.

Abstract P1-18-17: Phase I study of cfi-402257, an oral ttk inhibitor, in patients with advanced solid tumors with breast cancer expansion cohorts

Abstract Background: TTK (also known as Mps1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint (SAC), chromosome alignment and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, resulting in chromosomal missegregation, aneuploidy and cell death. CFI-402257 is a potent (Ki = 0.09 nM, IC50 = 1.2 nM), highly selective and orally active inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- and triple negative breast cancer (TNBC) cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. Methods: This multi-center Phase I dose escalation study (3+3 design) was designed to determine the safety, tolerability and maximum tolerated dose (MTD) of CFI-402257 and evaluate anti-tumor activity at the RP2D. CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5mg based on preclinical toxicology. Dose escalation included patients with advanced solid tumors and dose expansion at the RP2D into three expansion cohorts - Cohort A advanced solid tumors, Cohort B advanced ER+ or TNBC with 1-4 prior lines of chemotherapy for metastatic disease and Cohort C ER+/HER2- breast cancer in combination with Fulvestrant (500mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (>= 3 months) and =<1 prior chemotherapy for metastatic disease. Results: As of May 25, 2021, 66 patients had been enrolled, majority (76%) of patients received >3 prior therapies before study entry and 37% of patients’ (23/66) primary malignancy was breast cancer. The maximum administered dose was 294 mg and the study has continued enrolling at the recommended phase 2 dose of 168 mg with grade 3 Neutropenia, grade 3 Febrile Neutropenia and grade 3 Colitis as the dose limiting toxicities. Only 1/6 patients experienced a DLT at 168 mg (grade 3 Neutropenia, >7 days) The most common treatment emergent AEs (Gd3, >5%) were Neutropenia (15.6%), Anaemia (7.8%), Hypophosphataemia and Febrile Neutropenia (6.3%). PR’s confirmed by the Investigator have been seen to date in 33% of the cohort C subjects (2/6 patients), with an additional two cPR’s seen in the breast cancer population (ER+/HER2-) from the expansion cohorts. Conclusion: CFI-402257 is generally well tolerated and continues to enroll at 168mg daily with a manageable AE profile and early signs of anti-tumor activity. Enrollment in the expansion cohorts is ongoing and updated safety and efficacy data for the previously treated ER+/HER2- population will be presented at the time of the meeting. A multi-center phase II clinical trial is planned. Citation Format: John Hilton, Daniel J Renouf, David W Cescon, Aaron R Hansen, Albiruni RA Razak, Lee-Anne Stayner, Trisha Denny, Graham Fletcher, Tak W Mak, Mark Bray, Philippe L Bedard. Phase I study of cfi-402257, an oral ttk inhibitor, in patients with advanced solid tumors with breast cancer expansion cohorts [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 P1-18-17.

A novel device for serial venous blood sampling in a canine model

Diagnostic serial venous blood sampling has the potential to introduce stress-related artifactual elements into the analytical results. We have evaluated the use of a wearable automated serial blood sampling device in a canine model that obviates a need for multiple manual venipuncture. Efficacy in measuring cortisol levels during prolonged serial blood sampling is described. Automated serial blood sampling from the jugular vein was performed in two Beagle dogs. Over two separate 22-h periods, serial blood samples were withdrawn for routine cell counting and blood chemistries from each animal, whilst animal behavior along with durability of catheter placement and tolerability by the vascular system were assessed.During the study, no behavioral changes attributed to the serial blood sampling device were seen and no stress-related deviations from normal blood cell counts and cortisol levels were detected. Minor erythema was eident at the sites of catheter placement, which was consistent with the presence of an indwelling catheter. The wearable, serial blood sampling device performed well and was tolerated by the animals without stress-related changes in blood analysis or behavioral observation. This study has demonstrated the potential significant benefit of the Fluispotter® device for serial blood sampling in veterinary and clinical settings. The samples collected are free from stress-related signatures in blood chemistry otherwise common to serial blood sampling protocols requiring repeated venipuncture. This device has valuable implications for use in routine preclinical pharmacology and toxicology studies.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Towards More Human and Humane Testing: The Role of the Device Supplier Industry.

In vivo testing has been the gold standard for preclinical drug development and toxicology. However, animal-based methods often lack human relevance and have a low predictability rate, not to mention the enormous ethical and financial concerns associated with their use. For instance, according to the US Congressional Budget Office (cbo.gov), it takes an average of 10.5years to take a compound from the preclinical phase to the market, with a cost of US$1-US$2 billion. 90% of drugs that are tested in animals and enter clinical trials fail due to lack of safety and efficacy - this fact questions the significance of in vivo testing. Cells in culture can recapitulate certain aspects of physiology and disease, as well as indicate drug responses and toxicity. Thus, they represent a sophisticated human-relevant and humane alternative. With advances in the in vitro field, it is anticipated that confidence will be gained towards a move away from traditional in vivo models. Specialised supplier industries have been a driving force in the transition to non-animal research, by translating new approach methodologies into scalable products that have been adopted by the regulatory and testing industries - but we are still at the beginning. In this article, we introduce the perspective of the device supplier industry on the current challenges and opportunities surrounding the adoption of new in vitro methods, with the goal of promoting effective co-operation with scientists and other stakeholders. In addition, we highlight some examples of where non-animal approaches have been used in regulatory submissions, as well as listing some educational and training resources that can help when selecting the most appropriate assay.