Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers

Abstract

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

Graphical abstract

Figure 1.

STRO-002 is a homogeneous anti-FolRα ADC. A, The structure of STRO-002, which consists of a high affinity anti-FolRα antibody (SP8166) conjugated to a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239). The released cytotoxic warhead SC209 is indicated in the red box. B, The binding kinetics of SP8166 to as determined by SPR shows that the anti-FolRα antibody SP8166 binds to the human FolRα ECD with high affinity. C, SP8166 binds with high affinity to human FolRα overexpressed on 293T cells. Only week binding was observed to highly related human FolRβ expressed on 293T cells, while no binding on 293T cells. D, SP8166 binds to cynomolgus monkey FolRα overexpressed on CHO cells, while no binding observed on CHO- parental cells. E, Deconvoluted LC-MS chromatogram of intact STRO-002, indicating a single dominant peak with calculated DAR of 3.95.

Figure 2.

Characterization of the novel cytotoxin 3-amino-hemiasterlin (SC209) released by STRO-002. A, Internalization time course of fluorescently labeled SP8166 into Igrov1 and OVCAR3 cells as determined by FACS-based assay. Total internalized signal is measured by FACS after removing the antibody bound to the cell surface. Percent (%) internalization is the ratio of FACS signal after and before cell surface antibody removal. B, Representative fluorescent microscopy images of internalized fluorescently labeled SP8166 in Igrov1 cells. C,In vitro tubulin polymerization assay shows SC209 is a potent inhibitor of tubulin polymerization, similar to other known tubulin inhibitors (MMAE and Maytansine). D, Cytotoxic activity of STRO-002 in target positive Igrov1 cells compared with the free cytotoxin SC209 and the drug-linker SC239. STRO-002 is highly efficient at delivering SC209 into the target positive cells. E, SC209-induced cytotoxicity on P-gP expressing MES-SA/MX2 cells in the presence and absence of P-gP inhibitor GF120918, compared with know P-gP substrates DM4 and MMAE. Cell killing EC₅₀ fold change with and without GF120918 are indicated on the cell killing curves.

Figure 3.

In vitro and in vivo stability of STRO-002. A, STRO-002 antibody stability in human plasma and cyno plasma measured by anti-FolRα ELISA shows STRO-002 antibody is stable up to 21 days in plasma. B, Drug linker stability of STRO-002 in human and cyno plasma measured by LC/MS demonstrates STRO-002 DAR is stable up to 21 days in plasma. C, PK profile of STRO-002 total antibody in non–tumor-bearing mice. STRO-002 displayed PK profile similar to other antibodies. D, STRO-002 drug linker is stable for up to 21 days in mice indicated as DAR determined by LC/MS. E, Release of SC209 in Igrov1 tumors bearing mice dosed with a single intravenous bolus dose of 5 mg/kg STRO-002. SC209 is only detected in tumor, and not in plasma (LLOQ, 0.02 ng/mL).

Figure 4.

Specific cell killing activity of STRO-002 on FolRα-positive cells and bystander cell killing activity on FolRα-negative cells. A, STRO-002 showed potent cell killing activity in FolRα-positive Igrov1 and OVSAHO cells, while no killing was observed in FolRα-negative A549 cells. The specific killing of Igrov1 and OVSAHO cells can be competed off by adding unconjugated anti-FolRα antibody SP8166. B, Bystander killing of FolRα-negative Daudi cells observed when cocultured with target positive Igrov1 cells, but no killing observed when cocultured with FolRα-negative A549 cells. C, FolRα ECD concentration in the patient serum samples was detected by ELISA. STRO-002 cytotoxicity was not affected by FolRα ECD at concentrations higher than what have been detected in patient blood samples.

Figure 5.

In vivo efficacy of STRO-002 as monotherapy and in combined with approved SOC therapies in ovarian models. In all studies, treatment was initiated in established tumors [∼100–150 mm³ with a single dose of STRO-002 or specified test article at the indicated dose unless otherwise noted. A, Single dose of 10 mg/kg STRO-002 induced tumor regression in Igrov1 tumors. Equivalent doses of control test articles had no activity. B, Dose-dependent STRO-002 efficacy is observed in Igrov1 tumors starting at 2.5 mg/kg. C and D, OVCAR3 tumors showed similar dose responsiveness to STRO-002 in established (C) and advanced [400 mm³ (D) tumors. STRO-002 combination treatment with carboplatin (E) or anti-VEGF antibody Avastin (F) improved efficacy in Igrov1 and OV90 tumors, respectively.

Figure 6.

Robust STRO-002 monotherapy efficacy in endometrial PDX models with different levels of FolRα expression. A, Representative IHC images of FolRα expression in endometrial PDX models. B, Bar graph shows mean TGI for 20 endometrial PDX tumors in response to weekly 10 mg/kg STRO-002 treatment. Models are color coded based on FolRα expression levels and lines indicate 50% and 100% TGI. C, Table summarizes the number of models with statistically significant TGI and incidence of response for PDX models grouped by expression level. D, Representative tumor growth curves of endometrial PDX models that express negative, low, moderate, and high FolRα with significant efficacy in response to STRO-002 treatment.