Preclinical Characterization of ARX517, a Site-Specific Stable PSMA-Targeted Antibody-Drug Conjugate for the Treatment of Metastatic Castration-Resistant Prostate Cancer

Abstract

Metastatic castration-resistant prostate cancer (mCRPC) is an advanced disease in which patients ultimately fail standard-of-care androgen deprivation therapies and exhibit poor survival rates. The prostate-specific membrane antigen (PSMA) has been validated as an mCRPC tumor antigen with overexpression in tumors and low expression in healthy tissues. Using our proprietary technology for incorporating synthetic amino acids into proteins at selected sites, we have developed ARX517, an antibody-drug conjugate composed of a humanized anti-PSMA antibody site-specifically conjugated to a tubulin inhibitor at a drug-to-antibody ratio of 2. After binding PSMA, ARX517 is internalized and catabolized, leading to cytotoxic payload delivery and apoptosis. To minimize premature payload release and maximize delivery to tumor cells, ARX517 employs a noncleavable polyethylene glycol linker and stable oxime conjugation enabled via synthetic amino acid protein incorporation to ensure its overall stability. In vitro studies demonstrate that ARX517 selectively induces cytotoxicity of PSMA-expressing tumor cell lines. ARX517 exhibited a long terminal half-life and high serum exposure in mice and dose-dependent antitumor activity in both enzalutamide-sensitive and -resistant cell line-derived xenograft and patient-derived xenograft models of prostate cancer. Repeat-dose toxicokinetic studies in nonhuman primates demonstrated that ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 inhibited tumor growth in diverse mCRPC models, demonstrated a tolerable safety profile in monkeys, and had a wide therapeutic index based on preclinical exposure data. Based on the encouraging preclinical data, ARX517 is currently being evaluated in a phase I clinical trial (NCT04662580).

Figure 1.

ARX517 binds comparably to human and cynomolgus monkey PSMA and exhibits robust in vitro cytotoxicity in PSMA-expressing lines. A, huJ591 mAb or ARX517 was immobilized onto biosensors, and association/dissociation with serially diluted PSMA from the indicated species was measured via biolayer interferometry. Sensorgram traces for huJ591 mAb are shown. Affinities were calculated from PSMA binding curves that were globally fitted using a 1:1 stoichiometry binding model. B, Schematic representation of ARX517 in which drug-linker AS269 is conjugated via a stable oxime bond to the pAF residues incorporated at the HC ALA114 site in the huJ591 mAb. C, Dose–response curves show relative cell viability (% of untreated control samples) in cells treated for 4 days with serially diluted ARX517 or a positive control, cell-permeable auristatin, MMAE.

Figure 2.

Analytic characterization of the ARX517 confirmed site of AS269 conjugation, controlled DAR), high purity with minimal aggregation, and high thermal stability postconjugation. A, Peptide maps of unconjugated mAb and ARX517 using the reverse-phase HPLC method with mass spectrometric detection are shown. B, HIC method with UV detection at 280 nm resolved ARX517 unconjugated mAb, DAR1, and DAR2 species. C, Size exclusion chromatography HPLC with UV detection at 280 nm quantified ARX517 size variants. The percent area of high molecular weight, monomer, and low molecular weight species were calculated using Agilent ChemStation software. D, The T_m values of unconjugated mAb and ARX517 were determined using a differential scanning calorimeter. HMW, high molecular weight; LMW, low molecular weight.

Figure 3.

Linear PK and prolonged stability with minimal payload release following ARX517 dosing in mice and cynomolgus monkeys. nu/nu mice (A) without and (B) with C4-2 tumors (n = 5 mice/group) were dosed intravenously with ARX517. Blood samples were collected at the indicated time points and analyzed in qualified TA and intact ADC bioanalytic assays. Blood samples were collected from cynomolgus monkeys (n = 6/sex/group) following ARX517 dosing. C, TA, ADC, and (D) pAF-AS269 serum concentrations were quantified using validated assays (see “Materials and Methods”). Shown are mean ± SD (upper error bars) for TA, ADC, and pAF-AS269 (time points with n ≥5 quantifiable concentrations).

Figure 4.

ARX517 inhibits tumor growth in multiple prostate CDX and PDX models. All graphs show mean TVs ± SEM over time. A, In the MDA-PCa-2b murine xenograft model, MDA-PCa-2b cells were implanted subcutaneously into the flank of male nu/nu mice (n = 10/group). When tumors reached 100–200 mm³, mice were given a single intravenous dose of the indicated test articles (dotted line, day 14). % TGI was calculated based on tumor wet weights at day 45 after cell implantation. Statistical analysis: *, P < 0.05; **, P < 0.01, calculated using the nonparametric Mann–Whitney t test. In the enzalutamide-sensitive (B) TM00298 and (C) CTG-2440 PDX models, mice [(B) NOD/SCIDγ mice, n = 9/group; (C) NOG mice, n = 10/group for vehicle and 1 mg/kg ARX517, n = 9/group for other cohorts] were subcutaneously implanted with patient-derived tumor cells. When tumors reached 100–200 mm³, mice were dosed intravenously with ARX517 once weekly for four total doses (dotted lines show ARX517 doses) and/or enzalutamide orally in B daily for 28 days or in C daily for 5 days, followed by no dosing for 2 days, in a week for 4 weeks. % TGI was calculated based on TV measurements at study end. Statistical analysis: *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were calculated in B using nonparametric Mann–Whitney t test and in C using one-way ANOVA followed by the Tukey multiple comparisons test. D and E, nu/nu mice (D; n = 10/group; except n = 5 for the ARX517 10 mg/kg group) or NCG mice (E; n = 7/group for enzalutamide and combination treatment groups; n = 10/group for other cohorts) were subcutaneously implanted with enzalutamide-resistant C4-2 tumor cells in Matrigel. When tumors reached 200–500 mm³, mice were dosed intravenously with the indicated test articles (dotted lines show ARX517 doses). % TGI in E was calculated based on TV measurements at study end. Statistical analysis: **, P < 0.01; ***, P < 0.001 calculated using a two-way ANOVA with repeated measures and Tukey post hoc test.