figures legends, tables, and references. There are six figures and one table. The supplemental information contains an additional seven figures and seven tables. Research.
In the past we have reported significant cognitive deficits in mice receiving 5-fluorouracil in combination with low-dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio-analytical method was therefore developed and validated for 5-fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB-C18 column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 μL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio-analytical method was used for the pharmacokinetic characterization of 5-fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5-fluorouracil over a period of 24 h.
HPN328: An Anti-DLL3 T Cell Engager for Treatment of Small Cell Lung Cancer Delta-like protein 3 (DLL3) is a Notch inhibitory ligand is expressed in more than 70% of small cell lung cancers (SCLCs), while there is little to no surface expression in normal adult tissues outside of the CNS *1. SCLC is an aggressive neuroendocrine tumor that represents about 15 percent of all lung cancers. Although often responsive to standard of care treatment, relapse is common, with a median progression-free survival of 2–3 months and median overall survival (OS) of 8–13 months and 5-year OS rate of <5%*2. DLL3 thus represents an attractive potential target for T cell–redirecting immunotherapy in SCLC. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a N-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR), a middle single domain antibody (sdAb) that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal sdAb that binds to human DLL3. HPN328 is designed to simultaneously engage DLL3 on a target SCLC cell and CD3 on a T cell resulting in T cell activation, proliferation, and the eventual lysis of the target lung cancer cell. HPN328 is a highly stable single polypeptide of ~ 53 kDa expressed in CHO cells. It binds to human and cynomolgus monkey DLL3, albumin, and CD3ϵ with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of DLL3 positive and DLL3 negative tumor cell lines confirmed binding of HPN328 to its native targets expressed on the cell surface. HPN328 induces potent killing of DLL3 expressing SCLC cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, HPN328 mediated dose-dependent and DLL3-dependent cytotoxicity. Concomitant with target tumor cell killing, HPN328 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when DLL3 positive tumor cells were present. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, including TNFα, IL-2 and IFNγ, were detected. Nonclinical in vivo properties of HPN328 were evaluated in an NCI-H82 SCLC established tumor model. When administered to mice bearing human SCLC xenografts and human T cells, HPN328 eradicated subcutaneous tumors. In a single dose pilot toxicity study in cynomolgus monkeys, HPN328 was well tolerated at 1 and 10 mg/kg. Pharmacodynamic changes such as transient cytokine elevation mainly at the high dose were observed, consistent with the expected mechanism of action of T cell engagers. There were no clinically significant or adverse test article-related changes in hematology or clinical chemistry, and no apparent adverse findings at terminal and recovery necropsy. HPN328 exhibited linear PK properties in the given dose range with a serum half-life of 64 to 85 hours. Pharmacokinetic analysis supports weekly administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A first-in-human phase 1 clinical trial is planned to evaluate HPN328 in SCLC. *1. Saunders, L et al. (2015) Sci Transl Med. 7(302): 302ra136. *2. Navarro, A et al. (2017) Transl Lung Cancer Res. 6(1): S78–S83. Citation Format: Wade H Aaron, Richard Austin, Manasi Barath, Evan Callihan, Michael Cremin, Thomas Evans, Maria Gamez, Vaishnavi Ganti, Golzar Hemmati, Kathryn Kwant, Che-Leung Law, Bryan Lemon, Llewelyn Lao, Mary Ellen Molloy, Jessica O’Rear, Laura Sun, Holger Wesche, Stephen Yu, Timothy Yu. HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer [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 C033. doi:10.1158/1535-7163.TARG-19-C033
5552 Background: HPN424 is a first-in-class, prostate-specific membrane antigen (PSMA)-targeting T-cell engager designed as a small, globular protein to enable efficient solid-tumor penetration with prolonged half-life. HPN424 is derived from the TriTAC platform (Tri-specific T-Cell-Activating Construct) and engineered with three binding domains: anti-PSMA for tumor cell engagement, anti-albumin for half-life extension and anti-CD3 for T-cell engagement. Methods: This Ph I study is evaluating HPN424 in progressing mCRPC patients (pts) who have received >2 prior systemic therapies. Primary endpoints are safety, tolerability and determination of MTD/RP2D. Secondary objectives are pharmacokinetics (PK), pharmacodynamics, immunogenicity, and preliminary anti-tumor activity. HPN424 is administered IV once weekly. Tumor assessments include PSA, CT, and bone scans every 9 weeks. Results: As of 1/17/20, 27 pts were dosed in 8 cohorts ranging from 1.3 to 72ng/kg. Pts received a median of 6 prior systemic regimens, including >1 novel AR therapy, and 59% received prior chemotherapy for mCRPC. Median PSA at baseline was 251 ng/mL (range: 0.05 – 5000). No DLTs have been observed. The most common grade >3 TRAEs were cytokine release syndrome (CRS) (3 pts) and transient elevated liver transaminases (2 pts) that occurred concurrently with CRS. All CRS events resolved and pts were successfully re-treated. Short-term steroid premedication was effective in limiting CRS and allowing long-term weekly treatment. HPN424 demonstrated dose proportional increase in Cmax and AUC with a geometric mean T1/2 of 30.5 hours. Dose-dependent, transient increases in peripheral cytokine and chemokine levels were observed. Reduction in circulating tumor cells (CTCs) was seen in 11 of 19 pts with measurable CTC at baseline. Six pts had PSA decreases from baseline ranging from -3.8% to -76%, including 2 pts with PSA decline ≥50%. Ten of 20 pts (50%) with > 18 weeks follow-up remained on study beyond week 18 and includes 8 pts on study > 24 weeks. Conclusions: HPN424 represents a novel half-life extended PSMA-targeting T-cell engager that can be safely administered once weekly. AEs have been transient and manageable. Cytokine increases indicate T-cell activation. CTC reductions in subset of pts suggest target engagement. Early signs of clinical activity include PSA reductions and time on study, including 8 pts on study > 24 weeks. Dose escalation is ongoing, including exploration of step dosing. Clinical trial information: NCT03577028 .
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