IND-enabling Toxicology Studies Research Articles

Abstract C152: Preclinical and emerging Phase 1 study data indicates that novel deuterated MET kinase inhibitor DO-2 mitigates the side effects seen with current approved MET kinase inhibitors: Preventing deleterious ‘de-hinging’ to improve tolerability

Abstract Introduction. MNNG HOS transforming gene (MET) kinase inhibitors have demonstrated clinical activity in patients with non-small cell lung cancer (NSCLC) harboring exon14 (METex14) skipping mutations, validating this as an actionable drug target. High daily doses (500-800mg daily) are needed to reach sufficient plasma exposure of active drug to achieve sufficient target engagement to drive efficacy. However, these high doses are associated with toxicities that frequently lead to dose interruptions and reductions which negatively impact efficacy. Background. The 2nd generation of selective Type 1b MET inhibitors (capmatinib, tepotinib, savolitinib), with more soluble metabolites than the 1st generation inhibitors (SGX-523, JNJ-38877605, PF-04217903), have shown efficacy in the clinic. However, they still carry the metabolic liability of the 1st generation agents translating into high levels of circulating metabolites that are not active against the MET kinase, necessitating high daily doses to achieve sufficient active drug exposures. These inactive ‘de-hinged’ metabolites (which are unable to interact with a key recognition site located in the ‘hinge region’ of the MET kinase) remain in the circulation, leading to ‘off-target’ toxicities which are not linked to efficacy, but often necessitates dose reductions and/or-interruptions. Experimental procedures. Based on a detailed understanding of the metabolic liabilities of type 1b MET kinase inhibitors and using site specific deuteration of the key metabolic hotspot, we have used the Kinetic Deuterium Isotope Effect (KDIE) to overcome a metabolic liability that negatively impacts current agents. Summary of the new, unpublished data. The resulting deuterated compound DO-2, is well tolerated and highly potent in pre-clinical xenograft studies (MED ~0.2mg/kg: MTD >50mg/kg), brain penetrant (Kp, uu, brain ; 0.3 in rats), with low once daily doses (3mg/kg) causing complete and sustained cures of all animals (8/8) carrying well established ~300mm3 MET exon14 xenografts. IND enabling toxicology studies in rabbit (most relevant species for metabolism) have shown that the renal effects of the predecessor JNJ-38877605 were no longer apparent with DO-2 at efficacious exposure levels. Based on the pre-clinical efficacy and toxicology data, DO-2 has entered the clinic in a Phase 1 study (NCT05752552), where early data indicates that the improved preclinical profile translates into the clinic with increased active drug exposure when compared to the non-deuterated JNJ-38877605. Conclusion. Preclinical data and emerging clinical data from highly MET inhibitor sensitive MET exon14 tumours as well as in other less sensitive MET driven tumors indicate that DO-2 has the potential to mitigate the side effects seen with current approved MET kinase inhibitors, leading to better tolerability and lower inactive metabolite levels and at lower daily dose levels. Supporting preclinical data and emerging clinical data will be presented. Citation Format: Timothy P.S. Perera, Laurence Mevellec, Hans Prenen, Bernd Dekeyser, Debbie Robbrecht, Sander Bins, Barend Sikkema, Peter de Bruijn, Jean-Pascal Machiels, Rachel Galot, Damien Briol, Richard Knight, Irena Loryan, Yang Hu, Florence Wastelin, Jaap Verweij. Preclinical and emerging Phase 1 study data indicates that novel deuterated MET kinase inhibitor DO-2 mitigates the side effects seen with current approved MET kinase inhibitors: Preventing deleterious ‘de-hinging’ to improve tolerability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C152.

Abstract B167: Preclinical characterization of THE-349, a mutant-selective, CNS-active, fourth-generation EGFR inhibitor to overcome T790M- and C797S-mediated resistance in NSCLC

Abstract Background: EGFR activating mutations are observed in 10-50% of NSCLC patients. Although the common mutations (L858R [L] and exon 19 deletions [D]) are initially sensitive to first-, second-, and third-generation EGFR inhibitors (e.g., erlotinib [1G], afatinib [2G], and osimertinib [3G], respectively), on-target resistance is observed in a substantial percentage of patients, with T790M (T) and C797S (C) observed most frequently (post-1G/2G and post-3G, respectively). Furthermore, EGFR mutational heterogeneity can increase during treatment with existing inhibitors, inhibition of wild type (WT) EGFR leads to dose-limiting toxicities, and tumors commonly metastasize to the brain. We have designed THE-349 to be a fourth-generation EGFR inhibitor with potent activity against all 8 major single- (L, D), double- (LT, DT, LC, DC), and triple- (LTC, DTC) mutant variants, selectivity over WT EGFR, and activity in the CNS. Methods: Kinome selectivity was evaluated biochemically against a panel of 412 kinases. In vitro potency was determined by assessing effects (IC50s) on human and BaF3 cell lines expressing WT or mutant forms of EGFR. Efficacy studies were conducted using engineered BaF3 cell lines, a luciferase-tagged human NSCLC cell line, and a patient-derived xenograft (PDX) model dosed orally, once-daily at levels that did not exceed the maximum tolerated dose (40 mg/kg THE-349 and 25 mg/kg osimertinib). Results:THE-349 potently inhibited the biochemical activity of all major EGFR mutant variants (IC50s <2 nM) with a high degree of kinome selectivity. In engineered BaF3 cells in vitro, THE-349 potently inhibited viability of all 8 major EGFR mutant variants (L, LT, LC, LTC, D, DT, DC, and DTC), with IC50s <6 nM and with ≥10-fold selectivity over WT EGFR. Similarly, THE-349 demonstrated robust anti-tumor activity against the same 8 EGFR variants in vivo, in all cases inducing deep tumor regressions (>80% TR) in mice implanted with engineered BaF3 cells. In a PDX model from an osimertinib-resistant tumor containing D and DTC variants, THE-349 induced deep and sustained regressions (99% TR) through 56 days of dosing, and tumor regrowth that occurred during a subsequent 5-week dosing holiday was reversed (88% TR) upon redosing with THE-349. In an intracranial tumor model using a triple-mutant (LTC) NSCLC cell line, THE-349 significantly prolonged median survival (from 42.5 to 71.5 days), while osimertinib had no effect. THE-349 has a favorable ADME profile and promising safety profile in IND-enabling toxicology studies. Conclusion: THE-349 is a fourth-generation, CNS-active, mutant-selective EGFR inhibitor that has potent, single-agent activity against all major classes of EGFR activating and resistance mutations. A phase 1 clinical trial of THE-349 is planned. Citation Format: Sen Zhang, Sara Nadworny, Wei-Sheng Huang, Adam Talbot, Emily Ye, Narayana Narasimhan, William C Shakespeare, Victor M Rivera. Preclinical characterization of THE-349, a mutant-selective, CNS-active, fourth-generation EGFR inhibitor to overcome T790M- and C797S-mediated resistance in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B167.

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

Abstract ND12: TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers

Abstract STK11 loss of function mutations occur in 12-15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade in patients and preclinical models. STK11-deficient syngeneic mouse tumor models reflect this biology and are insensitive to anti-PD1 treatment. Informed by in vivo CRISPR-based screens, histone deacetylase 1 (HDAC1) was identified as a target gene, which when knocked out in tumor cells, reverses anti-PD1 resistance driven by STK11 tumor suppressor gene loss. Here, we describe the discovery and development of TNG260 as a potent and selective inhibitor of the HDAC1-containing complex CoREST. TNG260 inhibits CoREST deacetylase activity with 500-fold selectivity over the other HDAC1-containing complexes, NuRD and Sin3, and the HDAC3-containing complex NCoR. In preclinical studies, selective CoREST inhibition by TNG260 results in transcriptional reprogramming of STK11-mutant tumor cells, altering tumor cell cytokine secretion and markedly reducing recruitment of suppressive Treg cells to STK11-mutant tumors. Moreover, TNG260, in combination with anti-PD1 treatment, drives durable tumor regressions in multiple syngeneic STK11-mutant xenograft models. TNG260 has no anti-tumor efficacy in athymic mice, indicating the responses with TNG260 are mediated by T cells. Unlike previously developed pan-HDAC inhibitors, which are directly cytotoxic to cancer (and immune) cells, IND-enabling toxicology studies in rat and dog showed that TNG260 was well-tolerated at exposures predicted to be efficacious in humans, with bone marrow suppression only detectable at doses where TNG260 is no longer selective for CoREST inhibition. TNG260 clinical development will be among the first to combine the power of genetic patient selection and immunotherapy, evaluating patients with STK11 mutant cancers in a trial combining TNG260 and a checkpoint inhibitor. Citation Format: Leanne G. Ahronian, Minjie Zhang, Chengyin Min, Alice W. Tsai, Jacques Ermolieff, Patrick McCarren, Margaret Wyman, David Guerin, Ye Wang, Alborz Bejnood, Kenjie Amemiya, Brian McMillan, Nikitha Das, Preksha Shahagadkar, Brian Doyon, Andre Mignault, Colin Liang, Vassil Elitzin, Samuel R. Meier, Ashley Choi, Yi Yu, John P. Maxwell, Brian B. Haines, Jannik N. Andersen, Heather DiBenedetto, Aaron Weitzman, Alan Huang, Xinyuan Wu. TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers [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 ND12.

Abstract IA007: PREVENT agent development pipeline

Abstract The NCI’s PREVENT Cancer Preclinical Drug Development Program is a peer-reviewed program designed to support the preclinical development of promising agents and biomarkers for cancer interception/prevention towards clinical applications. PREVENT is not a grant program but allocates NCI contract resources to advance approved projects in a milestone-driven manner. Results obtained through NCI contract resources are returned to the applicant PIs and used to support further development by the applicants or in partnership with NCI. Resources available to PREVENT Program applicants include preclinical efficacy testing, CGMP manufacturing, GLP pharmacokinetic and IND-enabling toxicology studies, and IND filings. The PREVENT Program is focused on preventive agent development in the areas of Immunoprevention (cancer vaccines and immunomodulatory agents), Chemoprevention (novel mechanisms, anti-inflammatory agents, drug repurposing, toxicity reduction via alternative dosing regimens and agent combinations) and clinically translatable mechanistic biomarkers (pharmacodynamics, immune correlates, and tumor preventive efficacy). Submission deadlines for PREVENT Concept Applications occur twice per year on the second Monday in January and July. Further information can be obtained at the PREVENT Program website: https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical Citation Format: Mark Stevn Miller, Brian Cholewa, John Clifford, Vignesh Gunasekharan, Altaf Mohammed, Shanker Gupta, Robert Shoemaker, Shizuko Sei. PREVENT agent development pipeline [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA007.

Fedratinib Does Not Inhibit Thiamine Uptake or Induce Experimental Wernicke's Encephalopathy in Nonclinical Studies

Introduction:Despite compelling efficacy data from Phase II and Phase III trials, clinical development of fedratinib was halted in 2013 after 8 of 670 treated patients presented with neurological symptoms suggestive of Wernicke's encephalopathy (WE). WE is a neurological disorder that develops due to thiamine deficiency that is treated or prevented simply by using thiamine supplementation. Autopsy studies indicate WE has a prevalence between 0.8-2.8% in the general population with a higher rate in MPN and cancer patients. Confirming a diagnosis of WE is complicated by comorbidities in patients with myeloproliferative neoplasms (MPN) including a high incidence of stroke, hepatic encephalopathy secondary to hepatic extramedullary hematopoiesis or sepsis related to marrow failure in an elderly population. Analysis of patients in fedratinib trials by a panel of neurology and neuroradiology experts indicates that between 1-5 patients of 670 treated experienced WE, less than what might be expected based on historical norms.WE is commonly caused by either poor nutritional status or chronic alcohol use. The most common non-hematological adverse events for fedratinib are nausea, vomiting and diarrhea, suggesting they may be exacerbating factors for malnutrition in patients. Nonetheless, inhibition of thiamine transporter (THTR) has been proposed as a putative mechanism based on Caco-2 studies. Interpretation of data from these studies is complicated based on use of protein-free culture medias that don't replicate protein binding of drug in patients (96% protein bound) and for which the drug is sparingly soluble. No CNS lesions that would be predictive of WE were reported in the IND-enabling toxicology studies with fedratinib, but subtle neurological signs or histology signs may not be clear to non-experts.To evaluate the potential of fedratinib to inhibit thiamine uptake, thiamine transporter studies were run using previously published methods with Caco-2 cells that overexpress thiamine transporters with the significant exception that they were performed using human FBS rather than cell culture media. This addresses concerns about protein binding, solubility and replicates conditions experienced by patients. Further, to evaluate the potential of fedratinib to induce WE non-clinically, rats were dosed orally with 40 mg/kg fedratinib (140% of 500 mg patient dose by allometric scaling) daily for 28 days with careful monitoring of neurological signs of disease followed by evaluation of brains for any signs of lesions consistent with WE.Results:Fedratinib had no effect on THTR-1 at concentrations up to 30 µM (6-12X higher than Cmax at highest dose of drug). Fedratinib IC50 against THTR-2 was >30 µM. This indicates that fedratinib does not inhibit either thiamine transporter at plasma concentration achievable in patients. This is consistent with patient data in which plasma thiamine levels were normal for the 161 patients sampled at end of treatment in the fedratinib clinical studies.In rats dosed orally with 40 mg/kg fedratinib for 28 days, there was no observed ataxia, opisthotonus, nystagmus, loss of righting reflex or any other overt neurological signs of thiamine deficiency. Examination of the brains of these animals found no lesions consistent with focal damage to vulnerable areas of brain, e.g. thalamus, inferior colliculus or brainstem. There was no difference in weight gain in these animals compared to control animals. This data indicates that at doses comparable or higher than the highest fedratinib dose used in phase III studies, there is no nonclinical evidence that fedratinib induces thiamine deficient disease.Conclusion:Transporter studies performed in the presence of human serum indicate that fedratinib does not inhibit thiamine transport at clinically achievable concentrations. This is consistent with plasma thiamine data available from patients. Rodent studies indicate that fedratinib does not induce thiamine deficient disease when administered orally at clinically relevant concentrations. Notably, rats are incapable of emesis, so this model doesn't account for effects of gastric distress. In total, these data suggest that fedratinib does not increase the risk of thiamine deficiency, beyond its potential to exacerbate malnutrition through poor management of preventable GI adverse events. DisclosuresHood:Impact Biomedicines, Inc.: Employment, Equity Ownership. Hazell:Impact Biomedicines, Inc.: Research Funding; University of Montreal Neurosciences: Employment.

Abstract PR014: A novel immunotherapy for relapsed/refractory pediatric T-cell acute lymphoblastic leukemia

Abstract With the recent addition of nelarabine to first-line therapy, the overall 5-year survival for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients approaches 90%. Patient prognosis for those who relapse is heavily dependent on their specific subtype of ALL: those with B-cell ALL (B-ALL) have several antibody therapies available, including blinatumomab and inotuzumab, which specifically target B-cells for degradation. However, no effective standard therapy is available for patients with relapsed T-cell ALL (T-ALL) and few new therapies are in development; as a result, these patients have a much poorer 5-year survival rate, on the order of 20-30%. Therefore, the development of a targeted immunotherapy for T-cell ALL is critical towards improving patient outcome. The IL-7Ralpha protein is part of the IL-7 receptor complex expressed on the surface of T-cells, and the thymic stromal lymphopoietin (TSLP) receptor complex on the surface of B-cells. The IL-7Ralpha protein is critical for the proliferation and maintenance of T-cells and T-ALL cells, and is overexpressed or constitutively activated in up to 70% of pediatric T-ALL patients. Furthermore, the IL-7Ralpha appears to be overexpressed in chemotherapy-resistant T-ALL cells, highlighting the potential for targeting this protein in relapsed T-ALL patients. Here we describe a novel monoclonal antibody (4A10) raised against the IL-7Ralpha protein which effectively labels T-ALL cells for degradation via antibody-dependent cell-mediated cytotoxicity (ADCC) and independent mechanisms and results in significant control of T-ALL xenografts in animal models, diminished infiltration of T-ALL cells into other organs, and prolonged survival time. The 4A10 antibody is effective against relapsing disease in animal models, and may therefore be effective for relapsed T-ALL patients, or in combination with first-line therapy to further improve initial 5-year survival rates. The 4A10 antibody has undergone extensive optimization and formulation for clinical-grade manufacture and is completing IND-enabling toxicology studies with plans to enter Phase I clinical trials in mid-2021. We are developing a novel clinical trial schema, in collaboration with the Therapeutics Advances in Childhood Leukemia and Lymphoma consortium, to assess tolerability and efficacy via a primarily pediatric Phase I trial, to advance relapsed leukemia patients to curative therapies, including bone marrow transplantation or cell therapies. This abstract is also being presented as PO004. Citation Format: Christopher Foley, Julie Hixon, Wengqing Li, Eric Schafer, Susan Rheingold, Atul Varadhachary, Scott Durum. A novel immunotherapy for relapsed/refractory pediatric T-cell acute lymphoblastic leukemia [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR014.

1920-P: First Oral MC4R Agonist for the Treatment of Rare Genetic Obesity

Melanocyte-stimulating hormones, neuropeptides induced by Pro-opiomelanocortin (POMC) in the hypothalamus, act as the endogenous ligands of Melanocortin 4 receptor (MC4R). They decrease appetite by MC4R activation, leading to weight loss. Dysfunction of genes in MC4R pathway is known to cause rare genetic obesity such as POMC deficiency, leptin receptor deficiency, Bardet-Biedl syndrome and Alström syndrome. Therefore, MC4R agonists have the potential to be therapeutic agents for such genetic obesities. Compound LB(LB) is an orally available, small molecule MC4R agonist under clinical development. In vitro activity of LB toward MC4R and selectivity over MC1R, MC3R and MC5R were measured by radio-ligand binding, luciferase, cAMP and β-arrestin assays. LB showed a high potency and selectivity toward MC4R. In vivo efficacy of LB was tested in high fat diet-induced obese mice, rats and KK-Ay mice. Following repeat-dose to obese rodents, LB led to appetite suppression and weight loss comparable to or higher than setmelanotide. To evaluate in vivo pharmacokinetic properties, a single dose of LB was administered to mice, rats, dogs and monkeys. The GLP toxicology program of LB was comprised of general toxicity studies in rats and monkeys up to 4 weeks, genotoxicity and safety pharmacology studies. The IND-enabling toxicology studies demonstrated good safety profiles in the rats and monkeys. LB showed no significant safety issue in the genotoxicity and safety pharmacology studies. Comprehensive in vitro and in vivo studies demonstrated that LB is potent and selective MC4R agonist. LB is safe and well-tolerated compound, as demonstrated in GLP toxicity studies. In addition, the observed efficacy and safety profiles of LB are well described by the preclinical pharmacokinetic properties of LB such as a great penetration to brain tissue. In conclusion, these nonclinical studies suggested that LB can be a safe and effective drug for the patients with genetic obesity. Disclosure H. Park: None. S. Yeo: None. H. Park: Employee; Self; LG Chem. J. Park: None. P.S. Hong: None.

Abstract NT-090: PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER

Abstract OBJECTIVES: Folate receptor alpha (FolRα) is a cell-surface glycoprotein, highly expressed in ovarian and endometrial adenocarcinoma, and thus a promising target for cancer therapy using antibody drug conjugates (ADCs). Most ADCs currently in development are generated by random attachment of the cytotoxic payload to the antibody and result in a heterogeneous mixture, comprised of many different forms that are likely to vary in stability and activity, and therefore may be suboptimal therapeutic agents. We have employed an E. coli cell-free expression system (XpressCFTM) and site-specific conjugation technology, to generate STRO-002, a novel homogenous FolRα-targeting ADC. STRO-002 was optimized by selection of the antibody, drug-linker, conjugation site and drug-antibody ratio (DAR) that conferred the best pharmacological properties. We have conducted preclinical studies to evaluate the stability of STRO-002 and characterize the pharmacological properties of the cytotoxic metabolite SC209. In vitro cytotoxicity assays and in vivo efficacy studies were conducted to evaluate the activity of STRO-002 in multiple ovarian cancer cell lines and xenografts. IND enabling toxicology studies were conducted to determine the safety profiles for STRO-002 and its metabolite SC209 in cynomolgous monkeys and rats, respectively. RESULTS: Based on optimization studies, the anti-FolRα human IgG1 antibody (H01/SP8166) conjugated to a proprietary cleavable drug-linker (SC239) was selected for the lead ADC STRO-002. SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity. Based on most favorable anti-tumor activity, positions 180 and 404 on each heavy chain were selected for conjugation of SC239 to SP8166 to yield an ADC with DAR of ~ 4. The drug-linkage in STRO-002 is highly stable and the released warhead, SC209, is a very weak substrate for cellular drug-resistance efflux pumps and is cleared rapidly from plasma. STRO-002 has potent but highly specific cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and anti-tumor efficacy in ovarian xenograft models. STRO-002 exhibits dose-dependent tumor growth inhibition in Igrov-1 tumor xenografts at a single dose and complete regression is achieved in Igrov-1 and OVCAR-3 tumors with a single dose at 10 and 5 mg/kg, respectively. In addition, administration of STRO-002 in combination with carboplatin confers added benefit in efficacy in Igrov-1 tumors. Toxicology studies show favorable safety profiles for STRO-002 and SC209. The main toxicity finding in monkeys dosed up to 9 mg/kg consists of reversible hematopoietic/lymphoid tissue toxicity, which is considered antigen-independent and is consistent with the anti-proliferative effects of SC209 observed in single-dose toxicology studies in rats. No evidence of ocular toxicity due to SC209 were observed in either species. CONCLUSIONS: STRO-002 is a highly specific FolRα targeting ADC with minimal drug moiety release in circulation and the potential for an improved safety and activity profile, and a reduced risk of tumor drug resistance. Our data supports the advancement of STRO-002 to the clinic as a potential treatment of FolRα expressing malignancies such as ovarian cancer. Citation Format: Cristina Abrahams, Stellanie Krimm, Xiaofan Li, Sihong Zhou, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Dharti Kothari, Abigail Yu, Robert Henningsen, Cuong Tran, Gang Yin, James Zawada, Julie Hang, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Jr., Trevor Hallam. PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-090.

Assessing the risk of drug crystallization in vivo

IntroductionLow intrinsic solubility leading to poor oral bioavailability is a common challenge in drug discovery that can often be overcome by formulation strategies, however, it remains a potential limitation that can pose challenges for early risk assessment and represent a significant obstacle to drug development. We identified a selective inhibitor (BMS-986126) of the IL-1 receptor–associated kinase 4 (IRAK4) with favorable properties as a lead candidate, but with unusually low intrinsic solubility of <1 μg/mL. MethodsConventional histopathology identified the issue of crystal formation in vivo. Subsequent investigative work included confocal Raman micro-spectroscopy, MALDI-MS, polarized light microscopy of fresh wet-mount tissue scrapings and transmission electron microscopy. ResultsBMS-986126 was advanced into a 2-week toxicology study in rats. The main finding in this study was minimal granulomatous inflammation in the duodenum, associated with the presence of birefringent crystals at the highest dosage of 100 mg/kg/day. Considering the safety margin, and the single location of the lesion, BMS-986126 was further progressed into IND-enabling toxicology studies where tolerability deteriorated with increasing dosing duration. Birefringent crystals and granulomatous inflammation were detected in multiple organs at dosages ≥20 mg/kg/day. Raman spectroscopy confirmed the identity of the crystals as BMS-986126. Therefore, follow up investigations were conducted to further characterize drug crystallization and to evaluate detection methods for their potential to reliably detect in vivo crystallization early. DiscussionThe purpose of our efforts was to identify critical factors influencing in vivo drug crystallization and to provide a preliminary assessment (based on one compound) which method would be best suited for identifying crystals. Results indicated a combination of methods was required to provide a complete assessment of drug crystallization and that a simple technique, scraping of freshly collected tissue followed by evaluation under polarizing light was suitable for detecting crystals. However, dosing for 2 weeks was required for crystals to grow to a clearly detectable size.

Discovery of VU2957 (Valiglurax): An mGlu4 Positive Allosteric Modulator Evaluated as a Preclinical Candidate for the Treatment of Parkinson's Disease.

Herein, we report the discovery of a novel potent, selective, CNS penetrant, and orally bioavailable mGlu4 PAM, VU0652957 (VU2957, Valiglurax). VU2957 possessed attractive in vitro and in vivo pharmacological and DMPK properties across species. To advance toward the clinic, a spray-dried dispersion (SDD) formulation of VU2957 was developed to support IND-enabling toxicology studies. Based on its overall profile, VU2957 was evaluated as a preclinical development candidate for the treatment of Parkinson's disease.

Abstract 43: ASN004, a novel 5T4-targeted Dolaflexin ADC, achieves complete regressions and tumor-free survivors in a broad variety of solid tumor models

Abstract ASN004 is an Antibody Drug Conjugate (ADC) that targets the 5T4 oncofetal antigen (trophoblast glycoprotein). High expression of 5T4 is observed in a wide range of malignant tumors, while very limited expression is found in normal tissues. ASN004 incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and several cytotoxic dolastatin (auristatin) analog warheads per ADC molecule with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd &amp;lt; 30 pM) and 5T4-expressing tumor cells; rapid cellular internalization; and potent, selective cytotoxicity. ASN004 provides complete tumor regressions and tumor-free survivors in multiple tumor xenograft models, derived from human tumor cell lines having both high and low 5T4 expression levels. Tumor-free survivors were achieved at doses that are well-tolerated, based on xenograft body weight measures and exploratory toxicology studies in pharmacologically-relevant non-human primates. As well, tumor-free survivors were achieved in xenograft tumor models following administration of a single dose of ASN004, as low as 1 mg/kg iv. The broad activity of ASN004 has been demonstrated by its superior efficacy in a head-to-head study against trastuzumab-DM1, in a low-5T4, high-HER2 expressing tumor xenograft model. Finally, tumor xenografts that initially responded to ASN004 and later showed tumor regrowth, had not developed resistance and responded well to subsequent treatment with ASN004. Overall, ASN004 is demonstrated to be an ADC with high therapeutic potential in multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling toxicology studies are ongoing, in preparation for advancement to clinical studies in patients with solid tumors. Citation Format: Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. ASN004, a novel 5T4-targeted Dolaflexin ADC, achieves complete regressions and tumor-free survivors in a broad variety of solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2017-43

Development and validation of an ICP-MS method for the determination of elemental impurities in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and international conference on harmonization (ICH) regulating elemental impurities limits in pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉 method implementation, validation and quality control during the analytical process are described. We herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr, Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-enabling toxicology studies has led to the initiation of a phase 1 clinical trial.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4months at Genentech), leading to a potential acceleration of 4months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400L scale and three GMP batches at 2000L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Abstract 1734: In vitro characterization of EGF816, a third-generation mutant-selective EGFR inhibitor

Abstract EGFR is a major oncogene in NSCLC. Patients with the oncogenic mutations L858R and Ex19Del are responsive to the first generation pan-EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. The associated dose-limiting toxicities (DLTs) are severe rash and GI tolerability due to WT EGFR inhibition. However, therapy is universally limited by the development of acquired drug resistance where EGFR gatekeeper mutation T790M accounts for 50% of incidence. Second generation irreversible pan-EGFR TKI afatinib was developed to overcome T790M resistance. Though effective in animal models, the efficacy of afatinib on T790M patients is largely limited by its DLTs due to potent inhibition of WT EGFR. A strong medical need still exists for better tolerated therapy to treat NSCLC patients harboring EGFR mutations. Here we report the discovery and development of a potent third generation, irreversible mutant-selective EGFR TKI that is expected to improve/maintain efficacy on oncogenic EGFR mutant patients while demonstrating reduced side effects. EGF816 potently inhibits both activating (L858R and Ex19Del) and T790M resistant mutations in various cellular assays; it is selective against a large panel of kinases in both Ambit and BaF3 profiling, and more importantly is selective against WT EGFR. EGF816 is efficacious in mutant EGFR-driven xenograft models, is well tolerated in IND-enabling toxicology studies, and is entering phase 1 trials. Citation Format: Yong Jia, Jose Juarez, Mari Manuia, Gerald Lelais, Shailaja Kasibhatla, Oliver Long, Matthew McNeill, Michael DiDonato, Badry Bursulaya, Debbie Liao, Eric Murphy, Robert Epple, Thomas Marsilje, Nuzhat Pathan, Pierre-Yves Michellys, Steven Bender, Jennifer Harris. In vitro characterization of EGF816, a third-generation mutant-selective EGFR inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2014-1734

Abstract LB-250: Development of a miR34-based cancer therapy.

Abstract miR-34 is a tumor suppressor miRNA that functions within the p53 pathway and regulates the expression of more than twenty oncogenes, including MET, MYC, WNT, and MYB. We have created a mimic of miR-34 called miR-Rx34 that induces cell cycle arrest, senescence, and apoptosis in cancer cells. Transient transfection of miR-Rx34 significantly reduces the proliferation of cancer cells derived from patients with liver, lung, breast, colon, and pancreatic cancers as well as melanoma, lymphoma, and multiple myeloma. miR-Rx34 has been encapsulated in an anionic liposome to produce a particle called MRX34 that has a diameter of ~120 nm. Intravenous injections of MRX34 causes dose-dependent increases in miR-34 levels in liver tumor, lung tumor, liver, lung, spleen, and other highly vascularized tissues in mice without inducing an immune response. Systemic delivery of MRX34 causes complete tumor regression in two orthotopic models of liver cancer. The exceptional potency of MRX34 likely results from its capacity to stimulate the p53 pathway and down-regulate genes in the wnt/B-catenin, MET, MAPK, and VegF pathways in liver tumors. The no adverse effects level observed for MRX34 in IND-enabling toxicology studies in monkeys and rats are 10-100-fold higher than the doses that produce complete tumor regression in mice, suggesting that the drug candidate will have a broad therapeutic window in cancer patients. Clinical studies with MRX34 that are expected to begin in early 2013 will provide the first measure of the therapeutic potential of a tumor suppressor miRNA. Citation Format: Chris Daige, Jason Wiggins, Leslie Priddy, Kevin Kelnar, Jane Zhao, Andreas Bader, David Brown. Development of a miR34-based cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-250. doi:10.1158/1538-7445.AM2013-LB-250

A sensitive LC–MS/MS method for the determination of free maytansinoid DM4 concentrations—Method development, validation, and application to the nonclinical studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4) in rats and monkeys

A sensitive, specific, and high throughput method was developed and validated for the quantitation of free maytansinoid DM4 in cynomolgus monkey and Sprague-Dawley rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The Sciex API 5000 mass spectrometer was operated in multiple-reaction monitoring (MRM) and electrospray positive ionization mode. An MS/MS transition of 780→216 was used to monitor the analyte DM4 and 635→547 for the internal standard (IS) ansamitocin P-3. A liquid–liquid extraction was utilized for sample pre-treatment with a volume of 100μL plasma. A Thermo Hypersil Gold PFP column was used for chromatographic separation with a 2.0min HPLC gradient. The quantitation range of the method was 0.500–100ng/mL with a lower limit of quantitation of 0.500ng/mL. The method was validated in monkey and rat plasma according to FDA guidance on Bioanalytical Method Validation (2001). The intra- and inter-day precision and accuracy were found to be within the FDA recommended acceptance criteria. The validated method was employed to monitor the free DM4 levels in plasma in the IND-enabling toxicology studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4).

A biopharmaceutical classification-based Right-First-Time formulation approach to reduce human pharmacokinetic variability and project cycle time from First-In-Human to clinical Proof-Of-Concept

A Right-First-Time approach is described for developing bona fide formulations for First-In-Human (FIH) to Proof-Of-Concept (POC) studies to meet an overarching goal of reduced project cycle time from IND to NDA (as short as four years). Bona fide formulations are tailor-made according to the drug’s biopharmaceutical properties including solubility, permeability and stability. Solubilization techniques are used extensively to reduce oral absorption variability for most compounds. Bona fide formulations contain all necessary functional excipients such as diluent, solubilizer, stabilizer, pH adjuster, disintegrant and lubricant so formulation changes are minimized to avoid significant PK bridging studies. Cycle time of FIH formulation development is aligned with IND-enabling toxicology studies, generally 4–6 months. Resources range from 0.5 full time equivalents (FTE) for a BCS-1 compound to 3 FTE for a BCS-4 compound with high drug delivery hurdles. We have achieved our goal by taking the same formulation from FIH to POC 90% of the time and maintaining the same formulation platform from POC to commercial manufacturing 80% of the time in the past eight years. This strategy enables cycle time reduction from 7 to 4 years for IND to NDA by overlapping clinical study phases and eliminating clinical downtime due to PK bridging studies.