Efficacy In Xenograft Models Research Articles

Species-dependent hepatic and intestinal metabolism of selective estrogen receptor degrader LSZ102 by sulfation and glucuronidation

1. LSZ102 is an orally bioavailable selective estrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass. 2. To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase enzymes potentially involved in metabolism of LSZ102 were characterized. 3. Sulfate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolized by several human sulfotransferases expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulfation in human but glucuronidation in rat. 4. As LSZ102 is metabolized by a number of different sulfotransferases, drug-drug interactions resulting from the inhibition of one sulfotransferase are unlikely. 5. Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulfate M5, glucuronide M4, and secondary glucuronide/sulfate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Preliminary Results from a Phase I Study of HMPL-523, a Selective Oral Syk Inhibitor, in Patients with Relapsed or Refractory Lymphoma

Background: Spleen tyrosine kinase (Syk) plays an integral role in B-cell receptor signaling critical in the development and survival of several subtypes of lymphoma. HMPL-523 is a selective, oral Syk inhibitor that has shown strong anti-tumor efficacy in xenograft models of B-cell and T-cell lymphoma. HMPL-523 had a manageable safety profile and demonstrated anti-tumor activity in a phase I study of lymphoma patients in China (NCT02857998). Here, we report the safety and preliminary anti-tumor activity of HMPL-523 in the dose escalation phase of a phase 1 study of relapsed/refractory lymphoma patients in the United States and Europe (NCT03779113).Methods: The primary objectives of the phase I study were to evaluate the safety and tolerability of HMPL-523 and to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D). Secondary objectives were to assess the pharmacokinetics (PK) and evaluate the preliminary efficacy of HMPL-523. Eligible patients had histologically confirmed lymphoma, exhausted all approved therapy options, and had good organ function, including creatinine clearance ≥ 40 ml/min by Cockcroft-Gault, absolute neutrophil count ≥ 1000/µL, platelet count ≥ 50,000/µL, and hemoglobin ≥ 8.0 g/dL. Dose escalation was performed according to a 3+3 study design. Treatment emergent adverse events (AEs) were assessed per NCI CTCAE v5.0. Treatment responses were assessed by Lugano criteria at weeks 8, 16, and 24, and then every 12 weeks. Patients received HMPL-523 treatment daily in 28-day cycles until disease progression or unacceptable toxicity.Results: As of July 15, 2021, 21 patients had been enrolled and dosed with HMPL-523 at one of six dose levels (100 to 800 mg once daily). Baseline tumor subtypes included Hodgkin lymphoma (HL; n=5); diffuse large B-cell lymphoma (DLBCL; n=4); follicular lymphoma (FL; n=4); marginal zone lymphoma (MZL; n=2); and 1 patient each with mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), mixed HL/DLBCL, and Richter's transformation. Patients were predominantly Caucasian (90.5%) and male (71.4%). The median age was 61 years (range 27 to 89 years) and 71.4% had an ECOG performance status of 1. The median lines of prior therapy was 4 (range 2 to 17). The majority of patients had prior anti-CD20 antibody exposure (71.4%), and four patients (19%) received prior Bruton tyrosine kinase inhibitors.Five patients continue to receive study treatment. The most frequently reported treatment emergent AEs were aspartate aminotransferase increase (23.8%), anemia (23.8%), neutropenia (19%), hyponatremia (19%), creatinine increase (19%), and nausea (19%). The most common grade ≥ 3 AEs were neutropenia (14.3%), hyponatremia (14.3%), and anemia (9.5%). Three dose limiting toxicities were observed: 1 in the 100 mg cohort (grade 3 confusion) and 2 in the 800 mg cohort (grade 3 fever and grade 3 alanine aminotransferase increase). The dose was deescalated to 700 mg, which was determined to be the MTD and RP2D.Among 17 efficacy evaluable patients, 2 patients (1 HL, 1 FL) dosed at 600 mg and 800 mg (reduced to 600 mg due to toxicity) achieved complete response, and 1 patient (dose increased from 400 to 600 mg) achieved partial response (FL). Stable disease was observed in 5 (29.4%) patients (2 DLBCL, 1 MCL, 1 SLL, 1 PTCL). At steady state, HMPL-523 showed approximately dose proportional PK over the daily dose range of 100 to 700 mg.Conclusions: HMPL-523 was well tolerated at all dose levels within the range of 100 mg to 700 mg and demonstrated proof of activity at dose levels of 400 mg or higher in heavily pre-treated patients. The dose expansion phase of the study will evaluate safety and efficacy in patients with multiple subtypes of B-cell and T-cell lymphoma at the RP2D of 700 mg. Updated safety, PK, and anti-tumor activity will be presented. DisclosuresStrati: Astrazeneca-Acerta: Research Funding; Roche-Genentech: Consultancy. González-Barca: Roche: Honoraria, Other: Travel; Kyowa Kirin: Consultancy; EUSA Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel; Takeda. Abbvie: Honoraria. Taszner: Roche, Takeda: Consultancy, Other: Travel. Pillai: HUTCHMED: Current Employment. Chien: HUTCHMED: Current Employment, Current equity holder in publicly-traded company. Nanda: HUTCHMED: Current Employment, Current equity holder in publicly-traded company, Other: Travel. Rudinski: HUTCHMED: Current Employment. Jayaprakash: HUTCHMED, Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months; Astrazeneca: Current equity holder in publicly-traded company. Hahka-Kemppinen: HUTCHMED: Current Employment, Current holder of individual stocks in a privately-held company; Eli Lilly: Current holder of individual stocks in a privately-held company. Kania: HUTCHMED: Current Employment, Current equity holder in publicly-traded company.

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αβ T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

43P Pre-clinical evaluation of potent and orally bioavailable next-generation inhibitors targeting the family of mutants that drive oncogenic BRAF dimer formation

The canonical BRAF V600E (class I) mutation is a potent oncogene which is uniquely active as a RAS-independent monomer, and which has been successfully targeted by several FDA-approved inhibitors. While active against monomeric BRAF V600E, these first generation BRAF inhibitors induce paradoxical activation of RAS-driven BRAF dimers in cells expressing wild-type RAF, and this can lead to secondary malignancies. More recently, numerous non-canonical BRAF oncogenic mutations including BRAF-fusions have been described as oncogenes that drive RAS-independent (class II) or RAS-dependent (class III) dimers. These non-canonical dimeric BRAF oncogenes are resistant to the first-generation drugs, effective only against the monomeric BRAF V600E mutation. Discovery of an inhibitor directed against the family of dimeric BRAF oncogenic mutations which avoids paradoxical activation is a major unmet need. We applied our proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic class II and class III BRAF mutation clusters. We further demonstrate that this ensemble of both novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a series of small molecule inhibitors with potent anti-proliferative activity directed against tumor cells harboring dimer-inducing BRAF oncogenic mutations and which are devoid of paradoxical RAF activation. Leading exemplars of BDTX compounds are orally available inhibitors that achieve target engagement of BRAF dimer oncogenes in vivo and robust anti-tumor efficacy in xenograft models in mice. These data support continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients with BRAF mutant tumors.

Abstract PS17-11: Her2-xpat, a novel protease-activatable prodrug t-cell engager (tce), exhibits potent t-cell activation and efficacy in her2 tumors, yielding large predicted safety margins based on non-human primate (nhp)

Abstract Background TCEs are effective at inducing remissions in hematologic cancers but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include fractionated or step-up dosing and/or complex molecular designs, but these have been unsuccessful due to toxicity and/or enhanced immunogenicity. Amunix has developed a conditionally active TCE, XPAT or XTENylated Protease-Activated bispecific T-Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFVs targeting CD3 and HER2. Two unstructured polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a highly potent TCE with a short T1/2 and further improving the TI. Although checkpoint inhibitors have been ineffective in HER2+ or HR+ breast cancer, TCEs can co-opt T-cells regardless of their antigenic specificity, providing the potential to introduce effective T-cell immunity against tumors expressing HER2. HER2-XPAT, as a tumor protease-activatable prodrug with a wide safety margin, offers the potential to induce effective T-cell cytotoxicity against HER2hi tumors and HR+/HER2 1-2+ tumors. Methods Preclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytolytic activity in vitro, for anti-tumor efficacy in xenograft models, and for stability and safety in NHPs. Results HER2-PAT (cleaved XPAT) demonstrated potent in vitro tumor-directed T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and production of cytokines by PBMCs. HER2-PAT also exhibited cytotoxicity in HR+, HER2 1+ MCF7s at higher doses (EC50 0.13nM). HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and exhibited no cytotoxicity against cardiomyocytes at concentrations up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumor models with equimolar dosing to HER2-PAT, whereas non-cleavable HER2-NonClv had no efficacy, supporting tumor protease cleavage requirement for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD) but was not tolerated at 50mg/kg. HER2-XPAT demonstrated early T-cell margination at 2mg/kg but largely spared CRS, cytokine production, and tissue toxicity at doses up to 42mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable in NHP, indicating minimal systemic cleavage of XPAT, consistent with its ex vivo stability when incubated in the plasma of cancer pts for 7 days at 37°C. Given by continuous infusion, HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d, but was tolerated at 0.2mg/kg/d, providing HER2-XPAT with >3000-fold protection in tolerated Cmax versus HER2-PAT, >4 logs over cytotoxicity EC50s for HER2 and HR+ cell lines, and a 20-fold margin of safety over the dose required for pharmacodynamic activity. Conclusions HER2-XPAT is a potent prodrug T-cell engager with promising evidence of activity at low doses while exhibiting minimal CRS and a potentially wide TI based on NHP tolerability at doses up to 42mg/kg. With XTEN’s prior clinical data demonstrating low immunogenicity, the XPAT TCEs provide a promising solution. IND studies are currently ongoing. Additional PK, PD, cytokines, safety, and efficacy data will be presented. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Caitlin Koski, Mikhail Hammond, Angela Henkensiefken, Trang Dao-Pick, Mika K Derynck, Bryan Irving, Volker Schellenberger. Her2-xpat, a novel protease-activatable prodrug t-cell engager (tce), exhibits potent t-cell activation and efficacy in her2 tumors, yielding large predicted safety margins based on non-human primate (nhp) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-11.

Regulatory T cells promote glioma cell stemness through TGF-\u03b2\u2013NF-\u03baB\u2013IL6\u2013STAT3 signaling

Glioma stem cells (GSCs) contribute to the malignant growth of glioma, but little is known about the interaction between GSCs and tumor microenvironment. Here, we found that intense infiltration of regulatory T cells (Tregs) facilitated the qualities of GSCs through TGF-β secretion that helped coordinately tumor growth. Mechanistic investigations indicated that TGF-β acted on cancer cells to induce the core cancer stem cell-related genes CD133, SOX2, NESTIN, MUSASHI1 and ALDH1A expression and spheres formation via NF-κB–IL6–STAT3 signaling pathway, resulting in the increased cancer stemness and tumorigenic potential. Furthermore, Tregs promoted glioma tumor growth, and this effect could be abrogated with blockade of IL6 receptor by tocilizumab which also demonstrated certain level of therapeutic efficacy in xenograft model. Additionally, expression levels of CD133, IL6 and TGF-β were found to serve as prognosis markers of glioma patients. Collectively, our findings reveal a new immune-associated mechanism underlying Tregs-induced GSCs. Moreover, efforts to target this network may be an effective strategy for treating glioma.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Targeting the myeloid checkpoint receptor SIRP\u03b1 potentiates innate and adaptive immune responses to promote anti-tumor activity

BackgroundSignal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47–SIRPα interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47–SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47.MethodsHuman macrophages derived from donors with various SIRPα v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys.ResultsSIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg.ConclusionsThe in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47–SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.

51 Poster Discussion - Vertical pathway inhibition with a SOS1::KRAS inhibitor enhances the efficacy of KRAS G12C inhibitors, delays feedback resistance and demonstrates durable response

BI 1701963 is the first SOS1::KRAS inhibitor, which has entered Phase I clinical trials both as a mono-therapy as well as in combination with MEK inhibitors in patients with KRAS mutated advanced solid tumors. Both, BI 1701963 as well as the previously reported probe compound BI-3406 are orally bioavailable small molecules, designed to bind to the catalytic domain of SOS1, thereby preventing the interaction with GDP-loaded KRAS. In KRAS-dependent cancers, both SOS1::KRAS inhibitors potently reduce the formation of GTP-loaded KRAS and inhibit MAPK pathway signaling. SOS1:: KRAS inhibitors exhibit activity on a broad spectrum of KRAS alleles, including all major G12D/V/C and G13D oncoproteins, while sparing the interaction of KRAS with SOS2. This increases the targetable patient population beyond tumors with a KRAS G12C mutation and suggests a favorable therapeutic window to enable rational combinations. We previously showed that combining the SOS1::KRAS inhibitor BI-3406 with a MEK inhibitor blocks the negative feedback relief induced by MAPK inhibition. Combining SOS1::KRAS inhibition with MEK inhibitor treatment enhances pathway blockade leading to stronger anti-tumor efficacy in xenograft models driven by mutant KRAS such as G12D/V/C, G13D as well as Q61 K.

Abstract 5315: BPI-28592, a next-generation TRK kinase inhibitor that potently inhibits primary and resistant NTRK mutations

Abstract Oncogenic NTRK gene fusions occur at very low frequency in common cancers but high frequency in rare tumors including childhood malignances. The 1st-generation TRK inhibitors, e.g., larotectinib and entrectinib, have been used successfully to treat cancers carrying the NTRK fusions with excellent response rate and long duration. Nonetheless, duration of response may eventually be limited by acquired resistance mutations, such as TRKA-G595R or TRKC-G623R. We aimed at identifying a 2nd-generation inhibitor which can cover the primary TRK fusions and overcome the common resistant mutants induced by the 1st -generation TRK inhibitors. Using structural modeling to guide rational drug design, we discovered BPI-28592 as a 2nd-generation TRK inhibitor with desired potency and good drug-like properties. BPI-28592 is of a novel structure and possessed sub-nanomolar potency against a variety of primary TRK fusions. Importantly, the molecule exhibits low nanomolar inhibitory activity against acquired mutants commonly identified in patients who are refractory to 1st-generation TRK inhibitors, including TRKA-G595R TRKA-F589L, TRKA-G667C, and TRKC-G623R, etc. BPI-28592 showed a highly selective profile in a Kinome scan of 468 individual kinases. In cell-based assays, BPI-28592 dose-dependently inhibited proliferation of primary as well as resistant TRK mutant cell lines, blocked TRK protein phosphorylation and downstream signaling. Furthermore, BPI-29592 demonstrated in vivo efficacy in xenograft models derived from primary and resistant TRK mutant cells. BPI-28592 exhibited a profile of high oral exposure across multiple pre-clinical species, favorable ADME properties, and wide therapeutic window in pre-clinical toxicology studies. In conclusion, we discovered BPI-28592 as a novel, potent and selective next-generation TRK kinase inhibitor. The molecule has completed IND-enabling studies and is planned to enter Phase 1 clinical development in early 2020. Citation Format: Bang Fu, Dan Yan, Lingling Liu, Jing Guo, Jisheng Huang, Tianyi Ma, Yinlong Li, Wei Ren, Jie Chen, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-28592, a next-generation TRK kinase inhibitor that potently inhibits primary and resistant NTRK mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5315.

Abstract 1753: Targeting cancer with selective cbp/p300 bromodomain inhibitors

Abstract Background: The Bromodomain (BRD) and Extra-Terminal domain (BET) family of proteins are key regulators of epigenetic control. Although pan BET Inhibitors show good clinical activity, progressive disease was seen after several months of treatment in clinical responders, likely due to secondary resistance mechanisms. The plausible resistance mechanisms are increased expression of TCF7L2, c-Myc, Survivin and PIM1. Cyclic AMP response element binding protein (CREB)-binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or p300) are two closely related histone acetyl transferases with oncogenic roles in a variety of cancers. They are known to be co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Studies have indicated that p300 is also upregulated in SPOP-mutated prostate cancer. Thus, targeting CBP/p300 represents an attractive approach for developing novel therapies. Methods and Results: Multiple potent and selective CBP/p300 bromodomain inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The compounds were optimized towards attaining good physicochemical properties and DMPK profile. The anti-proliferative activity of the lead compounds was studied across multiple tumor types in a 3-day assay. The lead compounds potently inhibited viability of a wide range of hematological and solid tumor cell lines including prostate cancer cell lines VCaP and 22Rv1. In H929 cell line the lead compounds showed dose-dependent inhibition of cMYC and increase in cPARP. In a single dose PK-PD study in MV4-11 xenograft model, the compounds showed modulation of cMYC and Survivin. Conclusions: In summary, our studies demonstrate that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in-vitro. Profiling of efficacy in xenograft models, and further toxicological evaluation are in progress. Citation Format: Mahaboobi Jaleel, Ramesh S. Senaiar, Chandrasekhar Abbineni, Girish A. Renukappa, Subhendu Mukherjee, Sivapriya Marappan, DS Samiulla, AB Aravind, Naveen R. Kumar, Venkata Siva N. Reddy, Asha Babu, Akhila P. Srinivas, Prasad Yadlapalli, Suraj Tgore, Raghavendra NR, Chandranath D. Naik, Sanjeev Giri, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, laura Ravanti, Mikko Myllymäki, Gerd Wohlfahrt, Elina Mattila, Stefan Karlsson, Mari Björkman, Reetta Riikonen, Tarja Ikonen, Laura Leimu, Chira Mälmström, Timo Korjamo, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. Targeting cancer with selective cbp/p300 bromodomain inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1753.

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Abstract C061: BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models

Abstract Small molecule toxin conjugates offer a novel approach to tumor antigen specific targeting, allowing reduced systemic exposures and efficient tumor penetration of cytotoxic payloads, compared to large antibody toxin conjugates. BT8009 is a Bicycle Toxin Conjugate (BTC®) in which a Nectin-4 binding Bicycle® (bicyclic peptide) is conjugated through an inert sarcosine spacer chain, and a cleavable linker, to the antimitotic toxin MMAE. Increased Nectin-4 expression has been reported in multiple tumor types (including bladder, breast, esophageal, colorectal, lung, ovarian and pancreatic cancers) and so represents an appropriate tumor binding target. Bicycles are small (1.5KDa) fully synthetic, structurally constrained peptides with the specificities and affinities akin to antibodies, and pharmacokinetic properties more like small molecules. These features make Bicycles very attractive novel drug modalities for therapeutic approaches in which low systemic exposures coupled with high local drug concentrations are desired. We have previously described the identification and optimization process leading to selection of BT8009 as a candidate for IND enabling studies. BT8009 has low nanomolar (3nM) affinity for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3 and the 5 nectin-like family members (Necl1-5). BT8009 is active in a broad range of Nectin-4 positive cell line and patient derived xenograft tumors in vivo, leading to stable diseases and tumor regressions with durable responses. This abstract describes recent work characterizing this molecule. Selectivity of the Bicycle has been further demonstrated by assessing the binding of the biotinylated version of the Nectin-4 Bicycle on a human, plasma membrane protein cell array expressing 5528 full length human plasma membrane and secreted proteins. In this assay the Bicycle bound only Nectin-4 itself, again demonstrating the excellent target specificity. Efficacy data for BT8009 has now been extended to demonstrate the anti-tumor activity of BT8009 in large (1000mm3) xenograft tumors, in which rapid and near complete responses are observed. We have further characterized the efficacy profile of BT8009 in a panel of patient-derived non-small cell lung cancer (NSCLC) xenograft models, as well as in a panel of patient-derived pancreatic ductal (PDAC) xenograft models demonstrating a high frequency of stable diseases and partial responses. We will also describe the ongoing work for identification and validation of biomarkers for treatment response. BT8009 shows excellent efficacy in xenograft models expressing Nectin-4 target and its pharmacokinetic profile enables it to provide a rapid attainment of high tumor exposure levels with reduced systemic exposure. IND-enabling studies are currently ongoing with BT8009. Citation Format: Michael Rigby, Gavin Bennett, Liuhong Chen, Gemma Mudd, Paul Beswick, Helen Harrison, Sophie Watcham, Heather Allen, Amy Brown, Katerine Van Rietschoten, Philip Jeffrey, Peter U Park, Eric Haines, Nicholas Keen, Johanna Lahdenranta. BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C061. doi:10.1158/1535-7163.TARG-19-C061

Single Chain Variable Fragment Linker Length Regulates CAR Biology and T Cell Efficacy

Single Chain Variable Fragment Linker Length Regulates CAR Biology and T Cell Efficacy

Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.

Binary and ternary solid dispersions of an anticancer preclinical lead, IIIM-290: In vitro and in vivo studies

The objective of this study was to formulate an anticancer preclinical lead, IIIM-290, loaded in solid dispersions to enhance its solubility, dissolution, and oral pharmacokinetics. IIIM-290 is an in-house preclinical anticancer lead prepared by semisynthetic modification of the natural product rohitukine. It is an orally bioavailable Cdk inhibitor showing efficacy in xenograft models of pancreatic, colon and leukemia cancer. It demonstrated in vivo efficacy at a relatively higher dose owing to its poor aqueous solubility (~8.6 µg/mL). Binary and ternary solid dispersions containing PVP K-30, xanthan gum, and PEG-PPG-PEG were selected after solubility screening of various hydrophilic polymers. Several formulations with varying ratios of polymers, alone and in combination, were prepared and investigated for their effects on the solubility enhancement of IIIM-290. The binary solid dispersion VKB-SD75, prepared with PVP K-30 at the ratio of 1:4 w/w, was identified as the optimized composition that displayed 17-fold improvement in the aqueous solubility of IIIM-290. VKB-SD75 was scaled up to a 100-g scale. IIIM-290 and VKB-SD75 were evaluated for DSC, p-XRD, FTIR, 1H NMR, SEM, in vitro dissolution, and oral pharmacokinetics, as well as for in vivo anticancer activity in the Ehrlich solid tumor model. The oral administration of VKB-SD75 in BALB/c mice resulted in a 1.9-fold improvement in plasma exposure. These findings also correlated well when the formulation was administered to mice in the Ehrlich solid tumor model. The newly developed solid dispersion is expected to reduce the dose of IIIM-290 by ~40-50% in preclinical and clinical studies.

Abstract 2694: A first-in-class small-molecule modulator of the p300/CBP interactome exhibits broad efficacy in xenograft models of solid tumors and hematologic malignancies

Abstract Background: Dysregulation of the cellular transcription machinery is a fundamental feature of cancer. E1A binding protein (p300) and CREB binding protein (CBP) are two closely related paralog transcription co-activators involved in the expression of oncogenic drivers in cancer cells. Methods: Small molecule ligands of the CH1/TAZ1 domain of p300/CBP were rationally designed and optimized for their anti-proliferative activity in vitro. Subsequent characterization focused on their ability to modulate downstream biological processes controlled by p300/CBP, including hormone-dependent androgen receptor signalling, the HIF-1 alpha/VEGF pathway and reactivation of p53 in HPV-positive carcinomas. The in vivo efficacy of this novel compound class was assessed in a variety of patient and cell line derived xenograft models, and pharmacodynamic effects were investigated. Results: Several modulators of p300/CBP exhibited anti-proliferative effects at low nanomolar concentrations in more than 60 cancer cell lines inducing both senescence and apoptosis in a highly context dependent manner. Transcriptome analyses revealed the downregulation of various drivers of cell cycle progression. Once daily oral administration triggered strong growth-inhibitory effects in several xenograft models up to complete tumor eradication at well tolerated doses. p300/CBP dependent biomarkers like VEGF (colon cancer) and PSA (prostate cancer) were significantly downregulated in the tumors of treated animals. In an orthotopic model of acute myeloid leukemia, the growth of primary tumors and metastasis was almost completely inhibited. Conclusions: Reprogramming the transcriptional profile of cancer cells by modulation of p300/CBP activity by targeting the CH1/TAZ1 domain represents a novel and broadly applicable approach for the treatment of cancer. The presented data illustrate the clinical potential of this new class of therapeutics against a broad variety of tumor types, including prostate and colon cancer as well as hematological malignancies. Citation Format: Bernd Hentsch, Valentino Cattori, Marc Labelle, Ulrich Kessler. A first-in-class small-molecule modulator of the p300/CBP interactome exhibits broad efficacy in xenograft models of solid tumors and hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2694.

Abstract 4477: MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic antitumor activity in a range of hematologic malignancies

Abstract One of the hallmarks of cancer is evasion of apoptosis. The B-cell lymphoma-2 (Bcl-2) family of proteins represents a crucial point of control of apoptosis. The Bcl-2 family comprises both pro- and anti-apoptotic members, the latter of which (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-2A1) are often overexpressed in cancer cells, supporting their aberrant survival. Thus, these anti-apoptotic proteins have become an attractive target for cancer therapy. BH3 mimetics have been shown to bind to the BH3 binding groove of anti-apoptotic Bcl-2 family members and inhibit their function, resulting in apoptotic cell death, and one such BH3 mimetic, ABT-199 (venetoclax), has recently been approved for treatment of relapsed or refractory Chronic Lymphocytic Leukemia. We have developed two novel and potent BH3 mimetics: MIK665/S64315, a highly selective inhibitor of Mcl-1 and BCL201/S55746, a selective Bcl-2 inhibitor. Both compounds, individually induce apoptosis in hematological cancer cell lines, primary patient samples and demonstrate anti-tumor efficacy in xenograft models. MIK665/S64315 is currently in phase 1 clinical development in AML and MDS (NCT 02979366) and in MM and lymphoma (NCT02992483). Here, we describe the activity of the combination of MIK665/S64315 with BCL201/S55746 or venetoclax, both in vitro and in vivo, across a range of hematological indications (AML, MM and DLBCL). In vitro, a strong synergy was observed with these combinations, resulting in a remarkable induction of cell death in majority of cell lines tested. In vivo, MIK665/S64315 and BCL201/S55746 combinations lead to complete and durable antitumor responses in many different xenograft models in mice and rats. Taken together, these data demonstrate that a combination of MIK665/S64315 and BCL201/S55746 provide strong therapeutic benefit over either monotherapy, and support a rationale for testing Mcl-1 and Bcl-2 inhibitor combinations in patients with hematological malignancies. Citation Format: Ensar Halilovic, Maïa Chanrion, Prakash Mistry, Markus Wartmann, Shumei Qiu, Sneha Sanghavi, Yan Chen, Gaëlle Lysiak, Ana Leticia Maragno, Ulrike Pfaar, Felix Huth, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Youzhen Wang. MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic antitumor activity in a range of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4477.

Combining the DNA Repair Inhibitor Dbait With Radiotherapy for the Treatment of High Grade Glioma: Efficacy and Protein Biomarkers of Resistance in Preclinical Models.

High grade glioma relapses occur often within the irradiated volume mostly due to a high resistance to radiation therapy (RT). Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by RT. Here we evaluate the potential of Dbait to sensitize high grade glioma to RT. First, we demonstrated the radiosensitizer properties of Dbait in 6/9 tested cell lines. Then, we performed animal studies using six cell derived xenograft and five patient derived xenograft models, to show the clinical potential and applicability of combined Dbait+RT treatment for human high grade glioma. Using a RPPA approach, we showed that Phospho-H2AX/H2AX and Phospho-NBS1/NBS1 were predictive of Dbait efficacy in xenograft models. Our results provide the preclinical proof of concept that combining RT with Dbait inhibition of DNA repair could be of benefit to patients with high grade glioma.

Activity of RX-3117, an oral antimetabolite nucleoside, in subjects with advanced urothelial cancer: Preliminary results of a phase IIa study.

455 Background: RX-3117 is an oral small molecule antimetabolite that is activated by uridine cytidine kinase 2 (UCK2) which is predominantly expressed in cancer cells. RX-3117 has shown efficacy in xenograft models of gemcitabine resistant pancreatic, bladder and colorectal cancer. Preliminary data from an analysis of a Phase 2a clinical study of RX3117 in advanced urothelial cancer is described. Methods: In the Phase 2a study designed to evaluate safety, tolerability and efficacy, subjects were treated with oral RX-3117 (700 mg) once-daily for 5 consecutive days on and 2 days off for 3 of 4 weeks or all 4 weeks in a 28-day cycle. Eligible subjects (aged ≥ 18 years) had relapsed/refractory metastatic urothelial cancer, ECOG PS of 0 to 1, normal organ function (hepatic, renal and hematology) with no limit on the number of prior therapies. The primary Phase 2a endpoints are progression free survival (PFS) and/or objective clinical response with secondary endpoints of safety, TTP, DOR and ORR. Results: As of October 5, 2018; 33 subjects were treated (23 males and 10 females, median age 67.5 years); 29 subjects were evaluable having completed more than 1 cycle of therapy or discontinued due to a related adverse event. Twenty subjects had received 3 or more prior therapies; 30 received gemcitabine/cisplatin and 25 received a checkpoint inhibitor. The most common related adverse events were anemia (G1-2%, G2-3%, G3-3%), fatigue (G1-6%, G2-3%), neutropenia (G2-2%, G3-5%, G4-2%), diarrhea (G1-4%, G2-2%), and thrombocytopenia (G2-2%, G3-3%, G4-1%). One subject had a complete response after 4 cycles of therapy and continues therapy beyond 10 cycles; 5 subjects had PFS ranging from 133 to 315 days. Conclusions: RX-3117 appears to be safe and well-tolerated in chemotherapy and immunotherapy refractory advanced urothelial cancer with acceptable toxicities. Preliminary results show anti-tumor activity in heavily pre-treated patients. Clinical trial information: NCT02030067.