Picomolar Potency Research Articles

Abstract 6703: A fully synthetic EphA2/4-1BB tumor-targeted immune cell agonist (TICATM) induces tumor localized 4-1BB agonism

Abstract 4-1BB (CD137) is a member of the TNFR superfamily involved in stimulation of several immune cell types, including T cells and NK cells. CD137 is well validated pre-clinically, as agonism with anti-CD137 antibodies is effective in vivo [1], however, clinical utility to date has been limited by dose dependent hepatotoxicity. Bicycles® are a new therapeutic modality - fully synthetic, constrained bicyclic peptides with high affinity and excellent target selectivity. We hypothesized that Bicycle CD137 agonists that lack Fc domains and exhibit rapid renal elimination may induce CD137 mediated anti-tumor activity while avoiding liver toxicity. Initially, we identified and optimized CD137 specific Bicycle agonists which, when multimerized together, induced CD137 mediated anti-tumor activity [2]. We have built on this initial approach and developed second generation molecules to enable potent stimulation of immune cells exclusively at the tumor site. These tumor-targeted immune cell agonists (TICAsTM) comprise CD137 binding Bicycles coupled to tumor antigen binding Bicycles. Erythropoietin-producing hepatocellular A2 receptor (EphA2) is a tumor target overexpressed in several human cancers and its overexpression correlates with poor clinical prognosis. Here, we present new preclinical data demonstrating the potent immunomodulatory activity of dual targeting EphA2/CD137 TICAs. EphA2/CD137 TICAs engage EphA2 and CD137 with high affinity resulting in picomolar potency in co-culture assays consisting of cancer cell lines endogenously expressing EphA2, and CD137 Jurkat NF-kB/luciferase reporter cells. Moreover, EphA2/CD137 TICAs potentiate cytokine secretion (e.g. IFNγ) in immune cell co-culture experiments and promote caspase activity in T cell mediated cell killing assays. In vivo, EphA2/CD137 TICA (BCY9173), when dosed at 15 mg/kg (BID) to PBMC-humanized mice bearing HT29 xenografts, increased CD8+ T cells in tumors but not in plasma, suggesting local tumor target specific stimulation of T cells without systemic CD137 agonism. Intermittent dosing with various regimens of BCY12491, an EphA2/CD137 TICA, demonstrated robust anti-tumor activity including complete responses (CR) in 10/12 animals in a syngeneic humanized CD137 MC38 mouse model. Importantly, CR mice are resistant to re-challenge with MC38 tumor cells demonstrating a memory response, a phenomenon which has been previously reported for CD137 agonists [1]. Our EphA2/CD137 TICAs have shown the potential to precisely and potently stimulate immune cells in tumors without systemic immune activation. This provides a strong rationale to further develop first-in-class Bicycle TICAs to potentially treat EphA2 expressing cancers.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

Frequency of capsid substitutions associated with GS-6207 in vitro resistance in HIV-1 from antiretroviral-naive and -experienced patients.

GS-6207 is a first-in-class HIV capsid inhibitor, targeting several functions of the HIV capsid in the viral cycle, including viral particle assembly, capsid formation and nuclear entry. GS-6207 has demonstrated picomolar potency in vitro, activity confirmed by high potency in a Phase 1 clinical study, with a long-acting antiretroviral profile with potential dosing every 6 months. In vitro resistance selections previously conducted with increasing doses of GS-6207 have identified capsid variants with reduced susceptibility to GS-6207. To study the prevalence of capsid mutations associated with in vitro resistance to GS-6207 in people living with HIV (PLWH). Plasma samples from ART-naive or -experienced PLWH, including PI-experienced people, were sequenced and analysed for the presence of capsid variants identified during in vitro resistance selection: L56I, M66I, Q67H, K70N, N74D, N74S and T107N. Among the samples from the 1500 patients studied, none of the seven GS-6207 resistance mutations identified during in vitro selection experiments was detected, regardless of HIV subtype or PLWH treatment history. Out of the seven HIV capsid substitutions previously selected in vitro and shown to confer phenotypic resistance to GS-6207, none of these seven mutations was observed in this large dataset, suggesting that neither PLWH with previous PI failure nor PLWH with emergence of PI resistance mutations are anticipated to impact GS-6207 activity in these diverse HIV-infected populations.

Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel.

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

Discovery of Reversible Inhibitors of KDM1A Efficacious in Acute Myeloid Leukemia Models.

Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.

A Versatile Chemoenzymatic Synthesis for the Discovery of Potent Cryptophycin Analogs.

The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent antiproliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocyclization and epoxidation of over 20 natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl group. N-Acetyl cysteamine activated forms of each intermediate were assessed for conversion to macrocyclic products using wild type CrpTE, which demonstrated the exceptional flexibility of this enzyme. Semipreparative scale reactions were conducted for isolation and structural characterization of new cryptophycins. Each was then evaluated as a substrate for CrpE P450 and its ability to generate the epoxidized products from these substrates that possess altered electronics at the unit A styrenyl double bond position. Finally, biological evaluation of the new cryptophycins revealed a des-β-epoxy analog with low picomolar potency, previously limited to cryptophycins bearing epoxide functionality.

An Optimized Full-Length FLT3/CD3 Bispecific Antibody Demonstrates Potent Anti-leukemia Activity and Reversible Hematological Toxicity

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines invitro and invivo. 7370 was also capable of activating Tcells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

Reinvestigation of Mycothiazole Reveals the Penta-2,4-dien-1-ol Residue Imparts Picomolar Potency and 8S Configuration.

Reinvestigation of mycothiazole (1) revealed picomolar potency (IC50 = 0.00016, 0.00027, 0.00035 μM) against pancreatic, (PANC-1), liver (HepG2), and colon (HCT-116) tumor cell lines. Reevaluation of 1 provided [α]D data indicating Vanuatu specimens of C. mycofijiensis contain the 8S enantiomer of 1 and not the 8R configuration previously reported. Semisynthesis provided 8-O-acetylmycothiazole (2), 8-oxomycothiazole (8), mycothiazole nitrosobenzene derivatives (MND1, MND2: 9a, 9b), and MND3 (10) with IC50 = 0.00129, >1.0, >1.0, >1.0, >1.0 μM, respectively, against PANC-1 cell lines. These results highlight the significance of the penta-2,4-dien-1-ol residue as a key structural feature of 1 required for its cytotoxicty against tumor cell lines.

Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

Phase 1 First-in-Human Trial of AMV564, a Bivalent Bispecific (2:2) CD33/CD3 T-Cell Engager, in Patients with Relapsed/Refractory Acute Myeloid Leukemia (AML)

Background: AMV564 is a novel bivalent, bispecific (2x2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3e on T-cell receptors with strong avidity, thus creating an immune synapse between CD33-expressing cells and T cells, initiating T-cell directed lysis of CD33 expressing cells, and inducing expansion, differentiation and proliferation of T cells. By design, AMV564 has reduced clearance and therefore has a longer half-life (t1/2) than monovalent, bispecific T-cell engagers. In preclinical investigations using both leukemic cell lines and primary cells from AML patients, AMV564 eliminated myeloid blasts with picomolar potency and broad activity independent of cytogenetic or molecular abnormalities, CD33 expression level, and disease stage, with no nonspecific activation of T cells (Reusch U et al. Clin Cancer Res 2016;22:5829-38). Methods: This is an ongoing Phase 1 study with a 3+3 dose-escalation design (NCT03144245). The primary objectives of this study are to characterize the safety, tolerability, and preliminary anti-leukemic activity of AMV564. Evaluation of pharmacokinetics (PK), cytokine changes, and immunophenotyping are secondary objectives. Key inclusion/exclusion criteria are: adults with relapsed and/or refractory AML after 1-2 prior induction regimens (with a standard anthracycline-based regimen or hypomethylating agent) and no more than 2 prior salvage regimens. AMV564 is administered by continuous intravenous infusion (CIV) for 14 consecutive days for up to 2 induction cycles. AMV564 and cytokine (IL2, IL4, IL6, IL8, IL10, TNF-α, and IFN-γ) concentrations were measured by validated immunoassays. T-cell activation was measured using flow cytometry to quantify T cells expressing CD25, CD38, CD69, or HLA-DR. Results: To date, 19 patients (10 male/9 female) with a median age of 72 years (range 24-84) have been enrolled in 6 dosing cohorts: 0.5, 1.5, 5.0, 15, 50, and 100 mcg/day. Thirteen patients (68%) had secondary AML and/or adverse cytogenetics, including 6 patients (32%) with a p53 mutation. Fifteen patients (79%) had received at least 1 prior salvage regimen and 11 (58%) had received prior intensive chemotherapy, including 6 patients (32%) who had received a high-dose (≥ 1 gm/m2) cytarabine-based regimen. Overall, 18 patients were evaluable for toxicity and response. No dose-limiting toxicity or treatment-related grade ≥ 3 adverse events (AE) were reported. Grade 2 CRS was observed in 1 patient (treated at 50 mcg/day) without a lead-in dose and was managed with drug interruption and 1 dose of tocilizumab. The patient was able to resume dosing and completed the full 14-day scheduled therapy without recurrence of CRS. Subsequent patients treated at 50 mcg/day and above were given a 15 mcg/day lead-in dose for 3 days followed by 11 days at the assigned dose level. The most common grade ≥ 3 treatment-emergent AE has been febrile neutropenia, reported in 39% (7/18) of patients and all considered unrelated to study drug. No patient has died within 30 days of treatment initiation. AMV564 PK was linear with a terminal t1/2 of 2-3 days. Plasma concentrations increased gradually, with times to steady-state concentration of 3-7 days. Marked increases in IL6 (peak concentration, 1.1 ng/mL), IL8 (1.5 ng/mL), and IL10 (0.3 ng/mL) cytokines were observed and increased numbers of activated T-cells were detected post-treatment. Reductions in bone marrow blasts, ranging from 13% to 91%, were observed in 12 of 18 evaluable patients including a partial response after cycle 1 in 1 patient at the 100 mcg/day dose level. Conclusions: AMV564 is well-tolerated and demonstrates anti-leukemic activity through T-cell engagement. AMV564 has a unique PK profile with a gradual increase in drug concentration and thus the potential for controlled T-cell activation. Disclosures Roboz:Daiichi Sankyo: Consultancy; Argenx: Consultancy; Sandoz: Consultancy; Aphivena Therapeutics: Consultancy; Cellectis: Research Funding; Argenx: Consultancy; Eisai: Consultancy; Celgene Corporation: Consultancy; Roche/Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka: Consultancy; Roche/Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka: Consultancy; AbbVie: Consultancy; Astex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; Janssen Pharmaceuticals: Consultancy; AbbVie: Consultancy; Astex Pharmaceuticals: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Sandoz: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Aphivena Therapeutics: Consultancy; Pfizer: Consultancy; Cellectis: Research Funding; Eisai: Consultancy; Orsenix: Consultancy; Celltrion: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Janssen Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Orsenix: Consultancy. Cortes:Novartis: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Lee:AstraZeneca: Consultancy; Clinipace: Consultancy; Karyopharm Therapeutics Inc: Consultancy; LAM Therapeutics: Research Funding; Amgen: Consultancy. Rettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Han:Amphivena Therapeutics, Inc: Employment. Guenot:Amphivena Therapeutics, Inc: Employment. Feldman:Amphivena Therapeutics, Inc: Employment.

DDIS-17. DEVELOPMENT OF BRAIN-PENETRANT EGFR INHIBITORS FOR CNS MALIGNANCIES

Abstract The epidermal growth factor receptor (EGFR) is altered in nearly 60% of glioblastoma (GBM) tumors, however, EGFR tyrosine kinase inhibitors (TKIs) have failed to improve outcomes for patients with GBM. This can be attributed to the inability of clinically available EGFR TKIs (e.g., erlotinib, gefitinib, lapatinib, afatinib, cetuximab) to effectively cross the blood-brain-barrier (BBB) and reach adequate pharmacological levels for a tumor response. Herein, we performed a structure-activity relationship (SAR) to obtain EGFR TKIs with both high brain penetrance and potency against EGFR-activated GBM cells. From over 80 novel compounds synthesized, our lead EGFR TKI—JCN068—exhibited exceptional BBB penetration (350% brain to plasma) while also having optimal ADME properties (60% oral bioavailability, ~5 hr half-life, >100 µg/mL solubility, etc). Moreover, JCN068 demonstrated picomolar potency against purified EGFR kinase, 1000-fold selectivity for EGFR relative to other kinases, and nanomolar activity against EGFR-altered, GBM patient-derived cells in culture. Importantly, JCN068 demonstrated superior efficacy—with negligible toxicity—compared to clinically available small molecule EGFR TKIs (erlotinib and lapatinib) against multiple EGFR-altered patient-derived orthotopic GBM xenografts. Due to these excellent drug-like properties, JCN068 is currently progressing towards clinical development for EGFR-activated GBM patients.

Kinetic Tuning of HDAC Inhibitors Affords Potent Inducers of Progranulin Expression.

Histone deacetylases (HDACs) are enzymes involved in the epigenetic control of gene expression. A handful of HDAC inhibitors have been approved for the treatment of cancer, and HDAC inhibition has also been proposed as a novel therapeutic strategy for neurodegenerative disorders. These disorders include progranulin (PGRN)-deficient forms of frontotemporal dementia caused by mutations in the GRN gene that lead to haploinsufficiency. Hydroxamic-acid-based inhibitors of HDACs 1-3, reported to have fast-on/fast-off binding kinetics, induce increased expression of PGRN in human neuronal models, while the benzamide class of slow-binding HDAC inhibitors does not produce this effect. These observations indicate that the kinetics of HDAC inhibitor binding can be tuned for optimal induction of human PGRN expression in neurons. Here, we further expand on these findings using human cortical-like, glutamatergic neurons. We provide evidence that two prototypical, potent hydroxamic acid HDAC inhibitors that induce PGRN (panobinostat and trichostatin A) exhibit an initial fast-binding step followed by a second, slower step, referred to as mechanism B of slow binding, rather than simpler fast-on/fast-off binding kinetics. In addition, we show that trapoxin A, a macrocyclic, epoxyketone-containing class I HDAC inhibitor, exhibits slow binding with high, picomolar potency and also induces PGRN expression in human neurons. Finally, we demonstrate induction of PGRN expression by fast-on/fast-off, highly potent, macrocyclic HDAC inhibitors with ethyl ketone or ethyl ester Zn2+ binding groups. Taken together, these data expand our understanding of HDAC1-3 inhibitor binding kinetics, and further delineate the specific combinations of structural and kinetic features of HDAC inhibitors that are optimal for upregulating PGRN expression in human neurons and thus may have translational relevance in neurodegenerative disease.

Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent, selective and long acting DPP-4 inhibitors: Scaffold-hopping and prodrug study

A series of novel xanthine derivatives 2a-l incorporating benzoic acid moieties were rapidly generated by using strategy of scaffold-hopping from our previously reported scaffold uracil to xanthine, a scaffold of approved drug linagliptin. After systematic structure-activity relationship (SAR) study around benzoic acid moieties, 5 novel DPP-4 inhibitors with low picomolar potency range (IC50 < 1 nM) and excellent selectivity against various DPP-4 homologues were identified, in which the best one, compound 2f, with the IC50 value of 0.1 nM for DPP-4, showed 22-fold improvement in inhibitory activity compared to lead compound uracil 1, its activity was 45-fold more potent than alogliptin. 2e, 2f, 2i and 2k were selected for pharmacokinetic evaluation, and 2f and 2i showed the better pharmacokinetic profiles after iv administration, but poor oral bioavailability. To improve the oral pharmacokinetic profile, prodrug design approach was performed around 2f and 2i. Esters of 2f and 2i were synthesized and evaluated for stability, toxicity and pharmacokinetics. Compound 3e, the methyl ester of compound 2f, was identified to demonstrate good stability, low toxicity and improved oral bioavailability, with 3-fold higher blood concentration compared to 2f in rats. The following in vivo evaluations revealed 3e provided a sustained pharmacodynamics effect for 48h, and robustly improved glucose tolerance in normal ICR and db/db mice in dose-dependent manner. Chronic treatments investigations demonstrated that 3e achieved more beneficial effects on fasting blood glucose levels and glucose tolerance than alogliptin in type 2 diabetic db/db mice. The overall results have shown that compound 3e has the potential to efficacious, safety and long-acting treatment for T2DM.

Abstract 216: pro-Pyrrolobenzodiazepine (pro-PBD) bioconjugates, the legacy: Design and synthesis of pro-PBD conjugates containing self-immolative substituted disulfide linkers

Abstract Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of highly potent antitumor antibiotics, isolated from various streptomycin species. PBDs covalently bond and cross-link with discrete sequences of base pairs in the minor groove of DNA. A wide range of synthetic PBDs and PBD dimers have been shown to be highly potent cytotoxic agents, and have been used as payloads in antibody drug conjugates (ADCs). However, all PBDs as stand-alone agents or as delivered via small molecule drug conjugates (SMDCs), cause undesired off-target toxicity. Recently, we designed folate conjugated pro-PBDs, masking the imine moiety with its synthetic precursors: namely, 1,3-oxazolidine carbamate and an aromatic amine. The 1,3-oxazolidine carbamate provided a conjugation site for attachment of pro-PBD to a targeting ligand. After delivery to the targeted cell and internalization, reductive cleavage of the linker system results in the generation of a 1,3-oxazolidine. The oxazolidine reacts with water to form an aldehyde which subsequently reacts intramolecularly to ultimately form the PBD diazepine ring. Herein, we report the design and synthesis of folate conjugates of pro-PBDs and self-immolative sterically hindered disulfide linker systems, which pave the way toward novel, highly-specific therapeutics which have the potential to minimize off-target toxicities. One of the so designed conjugates, EC2629, satisfied all of our criteria for selection for pre-clinical development: • EC2629 shows high affinity towards FR mediated binding. • Cross-links DNA only following self-immolative release of free drug. • Picomolar potency against FR-expressing cells. • Curative activity with no weight loss in animal models. • Active against drug resistant tumor xenograft models in-vivo. • EC2629 is active against patient derived xenograft models in-vivo. • Based on high cytotoxicity and minimal off target toxicity (in-vitro &amp; in-vivo), EC2629 is selected for pre-clinical development. • Biological evaluation of EC2629 was presented at World ADC, Berlin, Germany (March, 2018). Citation Format: Iontcho Vlahov, Longwu Qi, Hari K. Santhapuram, Garth Parham, Kevin Wang, Jeremy F. Vaughn, Spencer J. Hahn, Marilynn Vetzel, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. pro-Pyrrolobenzodiazepine (pro-PBD) bioconjugates, the legacy: Design and synthesis of pro-PBD conjugates containing self-immolative substituted disulfide linkers [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 216.

S877 SAFETY AND CLINICAL ACTIVITY OF AMV564, A CD33/CD3 T‐CELL ENGAGER, IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA (AML): UPDATED RESULTS FROM THE PHASE 1 FIRST‐IN‐HUMAN TRIAL

Background:AMV564 is a novel bivalent, bispecific (2:2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3ε on T‐cell receptors. AMV564 creates an immune synapse between CD33‐expressing cells and T cells, initiating T‐cell directed lysis of CD33 expressing cells, and inducing activation, differentiation and proliferation of T cells. In preclinical studies, AMV564 eliminated myeloid blasts with picomolar potency and demonstrated broad activity independent of cytogenetic or molecular abnormalities, CD33 expression level, and disease stage, with no nonspecific activation of T cells (Reusch U et al. Clin Cancer Res 2016; 22:5829–38).Aims:The primary objectives of this study are to characterize the safety, tolerability, and preliminary anti‐leukemic activity of AMV564. Evaluation of pharmacokinetics (PK), assessment of cytokine changes, and immunophenotyping of T cells and myeloid cells are secondary objectives.Methods:This is an ongoing Phase 1 study with a 3+3 dose‐escalation followed by a dose expansion (NCT03144245). The key inclusion/exclusion criteria are: adults with relapsed and/or refractory AML after 1–2 prior induction regimens (with a standard anthracycline‐based regimen or hypomethylating agent) and no more than 2 prior salvage regimens. AMV564 is administered by continuous intravenous infusion (CIV) for 14 consecutive days in a 28 day cycle. Responding patients are eligible to receive additional cycles until disease progression or unacceptable toxicity. AMV564 and cytokine concentrations are measured by validated immunoassays and immunophenotyping is performed by flow cytometry.Results:To date, 30 patients (16 male/14 female) with a median age of 72 years (range 24–84 years) have been enrolled in 8 dose cohorts from 0.5 to 200 mcg/day. Twenty‐two patients (73%) had secondary AML and/or adverse cytogenetics, including 9 patients (30%) with a p53 mutation. Nineteen patients (63%) had received at least 1 prior salvage regimen and 18 (60%) had received prior intensive chemotherapy, including 9 patients (30%) who had received a high‐dose (≥ 1 g/m2) cytarabine‐based regimen. At the time of this analysis, 28 patients were evaluable for safety and 27 patients were evaluable for activity. No dose‐limiting toxicity or treatment‐related Grade ≥ 3 adverse events (AE) were reported. Using a lead‐in dose regimen, Grade 2 CRS was observed in 1 patient (treated at 200 mcg/day) and no Grade 3 or higher CRS has been observed. The most common Grade ≥3 treatment‐emergent AE has been febrile neutropenia, reported in 9 (30%) of 30 patients. No patient has died within 30 days of treatment initiation. Bone marrow blast reductions were observed in 17 (63%) of 27 efficacy evaluable patients. At doses of 100 mcg or higher, objective responses (1 complete response [CR], 1 CR with incomplete hematologic recovery, and 1 partial response) were observed in 3 of 9 evaluable patients. AMV564 PK was dose proportional with a terminal half‐life of 2–3 days. Serum concentrations increased gradually, with times to steady‐state concentration of 3–7 days. Marked increases in serum IL6, IL8, and IL10 were observed with evidence of T‐cell activation in both blood and bone marrow.Summary/Conclusion:AMV564 is well‐tolerated and demonstrates anti‐leukemic activity through T‐cell engagement using a 14‐day administration schedule. AMV564 has a unique PK profile with a gradual increase in drug concentrations and thus the potential for better controlled T‐cell activation.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvβ3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)–cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvβ3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

A2B Adenosine Receptor Antagonists with Picomolar Potency.

The A2B adenosine receptor (A2BAR) was proposed as a novel target for the (immuno)therapy of cancer since A2BAR blockade results in antiproliferative, antiangiogenic, antimetastatic, and immunostimulatory effects. In this study, we explored the structure-activity relationships of xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents attached to the sulfonamide residue. A new, convergent strategy was established, which facilitated the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar affinity for the A2BAR combined with high selectivity. Functional groups were introduced, which will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-1-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A2B-antagonist ( Ki 0.0835 nM, KB 0.0598 nM, human A2BAR) with >10 000-fold selectivity versus all other AR subtypes. It was similarly potent and selective at the mouse A2BAR, making it a promising tool for preclinical studies. Computational studies predicted halogen bonding to contribute to the outstanding potency of 34.

Phase 1 First-in-Human Trial of AMV564, a Bivalent Bispecific (2x2) CD33/CD3 T-Cell Engager, in Patients with Relapsed/Refractory Acute Myeloid Leukemia (AML)

▪Background: AMV564 is a novel bivalent, bispecific (2x2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3ε on T-cell receptors with strong avidity, thus creating an immune synapse between CD33-expressing cells and T cells, initiating T-cell directed lysis of CD33 expressing cells, and inducing expansion, differentiation and proliferation of T cells. By design, AMV564 has reduced clearance and therefore has a longer half-life (t1/2) than monovalent, bispecific T-cell engagers. In preclinical investigations using both leukemic cell lines and primary cells from AML patients, AMV564 eliminated myeloid blasts with picomolar potency and broad activity independent of cytogenetic or molecular abnormalities, CD33 expression level, and disease stage, with no nonspecific activation of T cells (Reusch U et al. Clin Cancer Res 2016;22:5829-38).Methods: This is an ongoing Phase 1 study with a 3+3 dose-escalation design (NCT03144245). The primary objectives of this study are to characterize the safety, tolerability, and preliminary anti-leukemic activity of AMV564. Evaluation of pharmacokinetics (PK), cytokine changes, and immunophenotyping are secondary objectives. Key inclusion/exclusion criteria are: adults with relapsed and/or refractory AML after 1-2 prior induction regimens (with a standard anthracycline-based regimen or hypomethylating agent) and no more than 2 prior salvage regimens. AMV564 is administered by continuous intravenous infusion (CIV) for 14 consecutive days for up to 2 induction cycles. AMV564 and cytokine (IL2, IL4, IL6, IL8, IL10, TNF-α, and IFN-γ) concentrations were measured by validated immunoassays. T-cell activation was measured using flow cytometry to quantify T cells expressing CD25, CD38, CD69, or HLA-DR.Results: To date, 19 patients (10 male/9 female) with a median age of 72 years (range 24-84) have been enrolled in 6 dosing cohorts: 0.5, 1.5, 5.0, 15, 50, and 100 mcg/day. Thirteen patients (68%) had secondary AML and/or adverse cytogenetics, including 6 patients (32%) with a p53 mutation. Fifteen patients (79%) had received at least 1 prior salvage regimen and 11 (58%) had received prior intensive chemotherapy, including 6 patients (32%) who had received a high-dose (≥ 1 gm/m2) cytarabine-based regimen. Overall, 18 patients were evaluable for toxicity and response. No dose-limiting toxicity or treatment-related grade ≥ 3 adverse events (AE) were reported. Grade 2 CRS was observed in 1 patient (treated at 50 mcg/day) without a lead-in dose and was managed with drug interruption and 1 dose of tocilizumab. The patient was able to resume dosing and completed the full 14-day scheduled therapy without recurrence of CRS. Subsequent patients treated at 50 mcg/day and above were given a 15 mcg/day lead-in dose for 3 days followed by 11 days at the assigned dose level. The most common grade ≥ 3 treatment-emergent AE has been febrile neutropenia, reported in 39% (7/18) of patients and all considered unrelated to study drug. No patient has died within 30 days of treatment initiation. AMV564 PK was linear with a terminal t1/2 of 2-3 days. Plasma concentrations increased gradually, with times to steady-state concentration of 3-7 days. Marked increases in IL6 (peak concentration, 1.1 ng/mL), IL8 (1.5 ng/mL), and IL10 (0.3 ng/mL) cytokines were observed and increased numbers of activated T-cells were detected post-treatment. Reductions in bone marrow blasts, ranging from 13% to 91%, were observed in 12 of 18 evaluable patients including a partial response after cycle 1 in 1 patient at the 100 mcg/day dose level.Conclusions: AMV564 is well-tolerated and demonstrates anti-leukemic activity through T-cell engagement. AMV564 has a unique PK profile with a gradual increase in drug concentration and thus the potential for controlled T-cell activation. DisclosuresRoboz:Daiichi Sankyo: Consultancy; Argenx: Consultancy; Sandoz: Consultancy; Aphivena Therapeutics: Consultancy; Cellectis: Research Funding; Argenx: Consultancy; Eisai: Consultancy; Celgene Corporation: Consultancy; Roche/Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka: Consultancy; Roche/Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka: Consultancy; AbbVie: Consultancy; Astex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; Janssen Pharmaceuticals: Consultancy; AbbVie: Consultancy; Astex Pharmaceuticals: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Sandoz: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Aphivena Therapeutics: Consultancy; Pfizer: Consultancy; Cellectis: Research Funding; Eisai: Consultancy; Orsenix: Consultancy; Celltrion: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Janssen Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Orsenix: Consultancy. Cortes:Novartis: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Lee:AstraZeneca: Consultancy; Clinipace: Consultancy; Karyopharm Therapeutics Inc: Consultancy; LAM Therapeutics: Research Funding; Amgen: Consultancy. Rettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Han:Amphivena Therapeutics, Inc: Employment. Guenot:Amphivena Therapeutics, Inc: Employment. Feldman:Amphivena Therapeutics, Inc: Employment.

Design and 22-step synthesis of highly potent D-ring modified and linker-equipped analogs of spongistatin 1

Spongistatin 1 is among the most potent anti-proliferative agents ever discovered rendering it an attractive candidate for development as a payload for antibody–drug conjugates and other targeted delivery approaches. Unfortunately, it is unavailable from natural sources and its size and complex stereostructure render chemical synthesis highly time- and resource-intensive. As a result, the design and synthesis of more acid-stable and linker functional group-equipped analogs that retain the low picomolar potency of the parent natural product requires more efficient and step-economical synthetic access. Using uniquely enabling direct complex fragment coupling crotyl- and alkallylsilylation reactions, we report a 22-step synthesis of a rationally designed D-ring modified analog of spongistatin 1 that is characterized by GI50 values in the low picomolar range, and a proof-of-concept result that the C(15) acetate may be replaced with linker functional group-bearing esters with only minimal reductions in potency.

Abstract 5769: Targeted Engineered Toxin Bodies provide a novel mechanism of action against HER2 positive cancers

Abstract Molecular Templates develops highly potent, specific, next-generation immunotoxins that are proprietarily de-immunized to avoid both innate and adaptive immune recognition. These Engineered Toxin Bodies (ETBs) destroy cancer cells by enzymatic destruction of ribosomes, a mechanism of action (MOA) distinct from that of other therapeutics. Molecular Templates' lead compound, MT-3724, has seen clinical activity in heavily pre-treated lymphoma patients, underscoring the potential for this class of agents to work in the refractory/relapsed setting. Current modalities for the targeted treatment of HER2 positive breast cancer include monoclonal antibodies, antibody-drug conjugates, and tyrosine kinase inhibitors. Although all these modalities have demonstrated clinical benefit, the majority of patients will unfortunately relapse due to a variety of resistance mechanisms. Upon relapse, most of these patients will still express HER2, allowing for the development of new HER2-targeted modalities. MT-5111, a HER2-targeted ETB with picomolar potency against HER2 expressing cells, was designed to overcome mechanisms of resistance to current HER2 modalities such as: escape from antibody dependent cell-mediated cytotoxicity (ADCC), alterations in signal transduction, epitope masking, and enhanced small molecule efflux. Additionally, MT-5111 was genetically engineered to reduce the anti-drug antibody response and signalling through innate receptors, allowing for repeat dosing. MT-5111 binds to an epitope on HER2 distinct from trastuzumab and ado-trastuzumab emtansine (T-DM1), and exhibits effective cytotoxicity on both T-DM1 sensitive and T-DM1 resistant HER2 positive cell lines. The targeting of MT-5111 to a distinct epitope allows for activity of the ETB on cell lines where the trastuzumab epitope is masked, and allows for combination with targeted agents trastuzumab or T-DM1 without competition for binding to HER2. Previous reports have shown that resistance to T-DM1 may be due to increased drug efflux; however, the MT-5111 cytotoxic payload is a large molecule not subject to this mechanism of resistance. Additionally, the novel MOA for MT-5111 should allow for cell kill independent of changes to the tumor microenvironment or alterations in HER2-mediated signal transduction. Pre-clinical data demonstrate the potent activity of MT-5111, including in T-DM1-resistant cell lines, as well as the potential for co-administering MT-5111 and trastuzumab or T-DM1, allowing for concurrent treatment of two HER2-targeted agents with distinct and non-interfering mechanisms of action. In vitro and in vivo data will be presented, highlighting the potential for MT-5111 as a novel agent under development for treatment of breast carcinomas, and other malignancies overexpressing the HER2 receptor. Molecular Templates intends to initiate clinical studies with MT-5111 in 2018. Citation Format: Brigitte Brieschke, Garrett L. Robinson, Sangeetha Rajagopalan, Hilario J. Ramos, Jensing Liu, Jack P. Higgins, Erin K. Willert. Targeted Engineered Toxin Bodies provide a novel mechanism of action against HER2 positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5769.