HPN328, a Trispecific T Cell-activating Protein Construct Targeting DLL3-Expressing Solid Tumors.

Delta-like ligand 3 (DLL3) is expressed in more than 70% of small cell lung cancers (SCLCs) and other neuroendocrine-derived tumor types. SCLC is highly aggressive and limited therapeutic options lead to poor prognosis for patients. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. In vitro assays, rodent models and non-human primates were used to assess the activity of HPN328. HPN328 induces potent dose-dependent killing of DLL3-expressing SCLC cell lines in vitro concomitant with T cell activation and cytokine release. In an NCI-H82 xenograft model with established tumors, HPN328 treatment led to T cell recruitment and anti-tumor activity. In an immunocompetent mouse model expressing a human CD3ε epitope, mice previously treated with HPN328 withstood tumor rechallenge, demonstrating long-term anti-tumor immunity. When repeat doses were administered to cynomolgus monkeys, HPN328 was well tolerated up to 10 mg/kg. Pharmacodynamic changes, such as transient cytokine elevation, were observed, consistent with the expected mechanism of action of T cell engagers. HPN328 exhibited linear pharmacokinetic in the given dose range with a serum half-life of 78 to 187 hours, supporting weekly or less frequent administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A phase 1/2 clinical trial is currently underway testing safety and efficacy in patients with DLL3 expressing malignancies.

TriTACs, a Novel Class of T-Cell-Engaging Protein Constructs Designed for the Treatment of Solid Tumors.

T cells have a unique capability to eliminate cancer cells and fight malignancies. Cancer cells have adopted multiple immune evasion mechanisms aimed at inhibiting T cells. Dramatically improved patient outcomes have been achieved with therapies genetically reprogramming T cells, blocking T-cell inhibition by cancer cells, or transiently connecting T cells with cancer cells for redirected lysis. This last modality is based on antibody constructs that bind a surface antigen on cancer cells and an invariant component of the T-cell receptor. Although high response rates were observed with T-cell engagers specific for CD19, CD20, or BCMA in patients with hematologic cancers, the treatment of solid tumors has been less successful. Here, we developed and characterized a novel T-cell engager format, called TriTAC (for Trispecific T-cell Activating Construct). TriTACs are engineered with features to improve patient safety and solid tumor activity, including high stability, small size, flexible linkers, long serum half-life, and highly specific and potent redirected lysis. The present study establishes the structure/activity relationship of TriTACs and describes the development of HPN424, a PSMA- (FOLH1-) targeting TriTAC in clinical development for patients with metastatic castration-resistant prostate cancer.

Preclinical Characterization of HPN536, a Trispecific, T-Cell-Activating Protein Construct for the Treatment of Mesothelin-Expressing Solid Tumors.

PURPOSE: Mesothelin (MSLN) is a glycophosphatidylinositol-linked tumor antigen overexpressed in a variety of malignancies, including ovarian, pancreatic, lung, and triple-negative breast cancer. Early signs of clinical efficacy with MSLN-targeting agents have validated MSLN as a promising target for therapeutic intervention, but therapies with improved efficacy are still needed to address the significant unmet medical need posed by MSLN-expressing cancers. EXPERIMENTAL DESIGN: We designed HPN536, a 53-kDa, trispecific, T-cell-activating protein-based construct, which binds to MSLN-expressing tumor cells, CD3ε on T cells, and to serum albumin. Experiments were conducted to assess the potency, activity, and half-life of HPN536 in in vitro assays, rodent models, and in nonhuman primates (NHP). RESULTS: HPN536 binds to MSLN-expressing tumor cells and to CD3ε on T cells, leading to T-cell activation and potent redirected target cell lysis. A third domain of HPN536 binds to serum albumin for extension of plasma half-life. In cynomolgus monkeys, HPN536 at doses ranging from 0.1 to 10 mg/kg demonstrated MSLN-dependent pharmacologic activity, was well tolerated, and showed pharmacokinetics in support of weekly dosing in humans. CONCLUSIONS: HPN536 is potent, is well tolerated, and exhibits extended half-life in NHPs. It is currently in phase I clinical testing in patients with MSLN-expressing malignancies (NCT03872206).

Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit.

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists.

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers.

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Interaction of the S-phase cyclin Clb5 with an "RXL" docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch.

Cyclin-dependent kinases are critical regulators of eukaryotic DNA replication. We show that the S-phase cyclin Clb5 binds stably and directly to the origin recognition complex (ORC). This interaction is mediated by an "RXL" target sequence, or "Cy" motif, in the Orc6 subunit that is recognized by the "hydrophobic patch" region on Clb5. The Clb5-Orc6 interaction requires replication initiation, and is maintained throughout the remainder of S phase and into M phase. Eliminating the Clb5-Orc6 interaction has no effect on initiation of replication but instead sensitizes cells to lethal overreplication. We propose that Clb5 binding to ORC provides an origin-localized replication control switch that specifically prevents reinitiation at replicated origins.

Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23.

We found that PPM1D, encoding a serine/threonine protein phosphatase, lies within an epicenter of the region at 17q23 that is amplified in breast cancer. We show that overexpression of this gene confers two oncogenic phenotypes on cells in culture: attenuation of apoptosis induced by serum starvation and transformation of primary cells in cooperation with RAS.