Targeting Folate Receptor Alpha Research Articles

The effect of paclitaxel- and fisetin-loaded PBM nanoparticles on apoptosis and reversal of drug resistance gene ABCG2 in ovarian cancer

BackgroundHigh-grade serous ovarian cancer (OvCa) is the most common type of epithelial OvCa. It is usually diagnosed in advanced stages, leaving a woman’s chance of survival below 50%. Despite traditional chemotherapeutic therapies, there is often a high recurrence rate following initial treatments. Hence, a targeted drug delivery system is needed to attack the cancer cells and induce apoptosis, overcome acquired drug resistance, and protect normal cells from cytotoxicity. The present study shows that targeting folate receptor alpha (FRα) through planetary ball milling (PBM) nanoparticles (NPs) induces apoptosis in OvCa cells.ResultsHuman tissue microarrays (TMAs) show overexpression of FRα in Stage IV OvCa tissues compared to matched normal tissues. They provide a focus for a targeted delivery system. We formulated PBM nanoparticles encapsulated with paclitaxel (PTX) or fisetin (Fis) and conjugated with folic acid (FA). The cytotoxic effect of these PBM NPs reduced the concentration of the toxic chemotherapy drug PTX by five-fold. The combined treatment of PTX-FA NPs and Fis-FA NPs inhibited cell proliferation and induced apoptosis more extensively than the individual drugs alone. Apoptosis of OvCa cells, determined by flow cytometry, showed an increase from 14.4 to 80.4% (OVCAR3 cells) and from 2.69 to 90.0% (CAOV3 cells) in the number of apoptotic cells. Also, expressions of the pro-apoptotic markers, BAK and active caspase-3, were increased after PTX-FA + Fis-FA PBM NP treatment. In addition to looking at targeted treatment effects on apoptosis, drug resistance was investigated. Drug resistance in OvCa cells was reversed by ABCG2, an ABC-transporter marker.ConclusionsOur study shows that PTX-FA and Fis-FA PBM NPs directly target platinum-resistant OvCa cells, induce cytotoxic/apoptotic effects, and reverse multi-drug resistance (MDR). These findings allow us to create new clinical applications using PTX-FA and Fis-FA combination nanoparticles to treat drug-resistant cancers.

Signaling via a CD28/CD40 chimeric costimulatory antigen receptor (CoStAR™), targeting folate receptor alpha, enhances T cell activity and augments tumor reactivity of tumor infiltrating lymphocytes.

Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.

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

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).

Abstract P5-08-02: MORAb-202, an antibody-drug-conjugate (ADC) targeting folate receptor alpha (FRα), exhibits durable anti-tumor efficacy in PDx models of TNBC

Abstract Background MORAb-202 is an ADC consisting of a FRα-targeting antibody (farletuzumab) paired with a cathepsin B-cleavable linker and an eribulin payload. FRα is overexpressed in various solid tumors including TNBC where 30-40% are enriched for FRα expression. MORAb-202 is currently in phase 1 clinical trials in patients with advanced solid tumors expressing FRα including ovarian cancer, endometrial cancer, TNBC and NSCLC. Methods In vitro characterization of MORAb-202 was conducted against over 60 various cancer cell lines expressing various level of FRα. In vitro co-culture system was also developed to evaluate bystander effect of MORAb-202. The anti-tumor activity of MORAb-202 at single dose (5 mg/kg) by comparison with eribulin (3.2 mg/kg as 32x molarity of eribulin in MORAb-202 at 5 mg/kg) was evaluated in TNBC PDx models established from primary and metastatic tumors, with different levels of FRα expression. Target engagement and bystander effect of MORAb-202 in the tumor microenvironment (TME) were also evaluated by IHC/IF methods. Dose dependent anti-tumor activity and the PK/PD profile of the single administration of MORAb-202 were examined in a human cancer CDx model (NCI-H2110). Soluble FRα (sFRα) is one of PD biomarker candidates measured in this model. The toxicology study of MORAb-202 was conducted in cynomolgus monkey. Results MORAb-202 showed cytotoxicity to FRα-positive cells in vitro, with limited off-target killing of FRα-negative cells. There was linear correlation observed between IC50 of MORAb-202 and MFI of FRα, while no relationship between IC50 of eribulin and MFI of FRα. More importantly, MORAb-202 showed 100x potent in vitro bystander cytotoxic effect in co-culture with FRα-positive/negative cells. Next, In vivo antitumor efficacy studies of MORAb-202 in TNBC PDx models exhibited durable activity proportional to tumor FRα expression. Single treatment of MORAb-202 showed long-lasting anti-tumor activity on TNBC PDx models superior to a group treated with eribulin. No body weight loss was observed in a group of MORAb-202, while transient body weight loss (~10%) was observed in the eribulin group. IHC/IF studies in tumors collected post treatment of MORAb-202 revealed that target engagement of MORAb-202 and its bystander effect on the TME could account for the anti-tumor activity in the models. PK/PD & anti-tumor activity of single administration of MORAb-202 (5 dose levels) were examined in human cancer CDx models (NCI-H2110). PK profiles of intact and total MORAb-202 were comparable. PD profile were assessed using sFRα, a potential biomarker candidate for clinic. In this model, spike of the sFRα was observed at 1 to 2 days post MORAb-202 treatment and returned to baseline levels by Day 4 post treatment. In cynomolgus monkeys, the AUC after the 2nd treatment of MORAb-202 on Day 22 was almost similar with that after the 1st treatment on Day 1. Elimination half-life T1/2 of MORAb-202 in plasma was nearly 5 days, supporting Q3W schedule in human clinical trials. The major toxicity observed with MORAb-202 treatment was hematologic toxicity. Conclusion These findings suggest MORAb-202 may be a promising ADC for TNBC and warrants further clinical investigation in this setting. Citation Format: Keiji Furuuchi, Katherine Rybinski, James Fulmer, Tomoyuki Moriyama, Brian Drozdowski, Allis Soto, Shawn Fernando, Kerrianne Wilson, Andrew Milinichik, Mary Lou Dula, Keigo Tanaka, Xin Cheng, Earl Albone, Toshimitsu Uenaka. MORAb-202, an antibody-drug-conjugate (ADC) targeting folate receptor alpha (FRα), exhibits durable anti-tumor efficacy in PDx models of TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-02.

High Expression of Folate Receptor Alpha (FOLR1) is Associated With Aggressive Tumor Behavior, Poor Response to Chemoradiotherapy, and Worse Survival in Rectal Cancer.

Objectives: Recently, molecular medicine targeting Folate Receptor Alpha (FOLR1), which mediates intracellular folate uptake and tumor cell proliferation, has been identified in several malignancies. However, the association between FOLR1 expression and rectal cancer remains unclear. Methods: Immunostaining of FOLR1 was performed on biopsy specimens from 172 rectal cancer patients undergoing preoperative chemoradiotherapy (CRT). FOLR1 expression was measured and divided into low (0+-2+) or high (3+-4+) level. Correlations between FOLR1 status and clinicopathologic features, tumor regression grade, disease-specific survival (DSS), local recurrence-free survival, and metastasis-free survival (MeFS) were analyzed, retrospectively. Results: High FOLR1 expression was significantly associated with advanced post-treatment tumor and nodal status (T3-4; N1-2, P = .001), vascular invasion (P = .042), perineural invasion (P = .012), and poor regression change after CRT (P = .001). In uni- and multi-variable survival analysis, FOLR1 overexpression remained a significant predictor of lower DSS (hazard ratio [HR], 2.328; 95% confidence interval [CI], 1.014-5.344; P = .046) and MeFS (HR, 2.177; 95% CI, 1.000-1.1286; P = .050). Conclusion: These results indicate that high FOLR1 status is associated with aggressive tumor behavior, poor response to CRT, and worse survival. Therefore, FOLR1 expression at initial biopsy may be useful in predicting outcomes and also be a target for the exploration of FOLR1-based therapeutic agents.

Revisiting antibody-drug conjugates and their predictive biomarkers in platinum-resistant ovarian cancer.

Until to date, platinum derived drugs are still the backbone of treating ovarian cancer (OC). Most patients treated with platinum-based chemotherapy develop resistance during the course of their management. The treatment of platinum-resistant ovarian cancer (PROC) is challenging. Few therapeutic options are available for patients with this aggressive disease. Besides, there are liminal advances regarding new anticancer drugs as well as validated predictive biomarkers of clinical outcomes in this setting. The enrollment of PROC patients in interventional studies is limited as compared to newly launched clinical trials for platinum-sensitive OC. Enthusiastically, the emergence of antibody-drug conjugates (ADCs) has provided promising findings for further clinical development in PROC. ADCs have the advantage to selectively deliver cytotoxic drugs to cancer cells expressing several of antigens using specific monoclonal antibodies based on the concept of immune bioconjugation. This innovative class of therapeutics showed encouraging early signs of clinical efficacy in PROC particularly mirvetuximab soravtansine that has been successfully introduced into three randomized and controlled phase III studies. In this review, the evidence from clinical trials supporting the development of ADCs targeting folate receptor alpha, sodium-dependent phosphate transporter 2B, dipeptidase 3, mesothelin, mucin 16, and tissue factor using various cytotoxic payloads in PROC is reviewed.

Fourth-generation chimeric antigen receptor T cells targeting folate receptor alpha antigen expressed on breast cancer cells for adoptive T cell therapy.

Treatment of breast cancer (BC) by standard methods is effective in the early stage, but ineffective in the advanced stage of disease. To develop an adoptive T cell therapy for advanced and severe BC, we generated fourth-generation chimeric antigen receptor (CAR) T cells targeting folate receptor alpha antigen (FRα) expressed on BC cells, and preclinically evaluated their anti-BC activities. The fourth-generation FRα-CAR T cells containing extracellular FRα-specific single-chain variable fragment (scFv) and three intracellular costimulatory domains (CD28, 4-1BB, and CD27) linked to CD3ζ were generated using a lentiviral system, and then were evaluated for their anti-BC activities in two-dimensional and three-dimensional (spheroid) cultures. When our fourth-generation FRα-CAR T cells were cocultured with FRα-expressing MDA-MB-231 BC cell line at an effector to target ratio of 20:1, these CAR T cells specifically lysed 88.7 ± 10.6% of the target cells. Interestingly, the cytotoxic lysis of FRα-CAR T cells was more pronounced in target cells with higher surface FRα expression. This specific cytotoxicity of the CAR T cells was not observed when cocultured with FRα-negative MCF10A normal breast-like cell line at the same ratio (34.3 ± 4.7%). When they were cocultured with MDA-MD-231 spheroid, the FRα-CAR T cells exhibited antitumor activity marked with spheroid size reduction and breakage. This proof-of-concept study thus shows the feasibility of using these fourth-generation FRα-CAR T cells for adoptive T cell therapy in BC.

Abstract NT-090: PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER

Abstract OBJECTIVES: Folate receptor alpha (FolRα) is a cell-surface glycoprotein, highly expressed in ovarian and endometrial adenocarcinoma, and thus a promising target for cancer therapy using antibody drug conjugates (ADCs). Most ADCs currently in development are generated by random attachment of the cytotoxic payload to the antibody and result in a heterogeneous mixture, comprised of many different forms that are likely to vary in stability and activity, and therefore may be suboptimal therapeutic agents. We have employed an E. coli cell-free expression system (XpressCFTM) and site-specific conjugation technology, to generate STRO-002, a novel homogenous FolRα-targeting ADC. STRO-002 was optimized by selection of the antibody, drug-linker, conjugation site and drug-antibody ratio (DAR) that conferred the best pharmacological properties. We have conducted preclinical studies to evaluate the stability of STRO-002 and characterize the pharmacological properties of the cytotoxic metabolite SC209. In vitro cytotoxicity assays and in vivo efficacy studies were conducted to evaluate the activity of STRO-002 in multiple ovarian cancer cell lines and xenografts. IND enabling toxicology studies were conducted to determine the safety profiles for STRO-002 and its metabolite SC209 in cynomolgous monkeys and rats, respectively. RESULTS: Based on optimization studies, the anti-FolRα human IgG1 antibody (H01/SP8166) conjugated to a proprietary cleavable drug-linker (SC239) was selected for the lead ADC STRO-002. SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity. Based on most favorable anti-tumor activity, positions 180 and 404 on each heavy chain were selected for conjugation of SC239 to SP8166 to yield an ADC with DAR of ~ 4. The drug-linkage in STRO-002 is highly stable and the released warhead, SC209, is a very weak substrate for cellular drug-resistance efflux pumps and is cleared rapidly from plasma. STRO-002 has potent but highly specific cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and anti-tumor efficacy in ovarian xenograft models. STRO-002 exhibits dose-dependent tumor growth inhibition in Igrov-1 tumor xenografts at a single dose and complete regression is achieved in Igrov-1 and OVCAR-3 tumors with a single dose at 10 and 5 mg/kg, respectively. In addition, administration of STRO-002 in combination with carboplatin confers added benefit in efficacy in Igrov-1 tumors. Toxicology studies show favorable safety profiles for STRO-002 and SC209. The main toxicity finding in monkeys dosed up to 9 mg/kg consists of reversible hematopoietic/lymphoid tissue toxicity, which is considered antigen-independent and is consistent with the anti-proliferative effects of SC209 observed in single-dose toxicology studies in rats. No evidence of ocular toxicity due to SC209 were observed in either species. CONCLUSIONS: STRO-002 is a highly specific FolRα targeting ADC with minimal drug moiety release in circulation and the potential for an improved safety and activity profile, and a reduced risk of tumor drug resistance. Our data supports the advancement of STRO-002 to the clinic as a potential treatment of FolRα expressing malignancies such as ovarian cancer. Citation Format: Cristina Abrahams, Stellanie Krimm, Xiaofan Li, Sihong Zhou, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Dharti Kothari, Abigail Yu, Robert Henningsen, Cuong Tran, Gang Yin, James Zawada, Julie Hang, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Jr., Trevor Hallam. PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-090.

Abstract 1782: Discovery and activity of STRO-002, a novel ADC targeting folate receptor alpha for ovarian and endometrial cancer

Abstract Folate receptor alpha (FolRα) is a glycosylphosphatidylinositol linked cell-surface glycoprotein that is widely expressed in serous and epithelial ovarian cancer, endometrial adenocarcinoma, non-small cell lung cancer and triple negative breast cancer. In contrast, FolRα expression is highly restricted on normal tissues, making it a highly promising target for cancer therapy using antibody drug conjugates (ADCs). We have designed a novel, FolRα-targeting ADC, STRO-002, with potent cytotoxic activity on FolRα expressing tumors in vitro and in vivo, including in cells with low expression levels (~0.2 million copies/cell) of FolRα. STRO-002 contains the anti-FolRa human IgG1 antibody (SP8166) conjugated to a proprietary cleavable drug-linker (SC239). SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity and is a weak substrate for efflux pumps. SP8166 was discovered and optimized using a Fab ribosome display selection and screening platform based on Sutro's Xpress CF+TM system. Four non-natural amino acid p-azidomethyl phenylalanine (pAMF) residues are incorporated into SP8166 at two defined sites on each heavy chain. These sites were selected based on optimal stability and activity in vitro and in vivo. The SC239 drug-linker is conjugated via a cleavable valine citrulline p-aminobenzyl carbamate linker functionalized with dibenzocyclooctyne (DBCO). The rapid and selective reaction of DBCO and pAMF results in a well-defined, homogeneous ADC with a drug-antibody ratio (DAR) of ~4. STRO-002 has potent cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and demonstrates strong anti-tumor response in KB, Igrov1 and OvCAR3 xenograft models in vivo. On Igrov1 xenografts, STRO-002 exhibits dose-dependent tumor growth inhibition starting at a single dose as low as 2.5 mg/kg. Evaluation of in vivo activity of STRO-002 in additional xenograft and PDX models, as well as in combination studies with chemotherapeutic agents is ongoing. Data from exploratory safety studies of STRO-002 in cynomolgus monkey and SC209 (active catabolite) in rats show a favorable safety profile. Our data suggests that STRO-002 is a promising clinical candidate for ovarian cancer, including tumors with low expression levels of FolRα, and IND enabling studies are currently being conducted. Citation Format: Xiaofan Li, Cristina Abrahams, Sihong Zhou, Stellanie Krimm, Robert Henningsen, Heather Stephenson, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Cuong Tran, Gang Yin, James Zawada, Ganapathy Sarma, Joy Chen, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Trevor Hallam. Discovery and activity of STRO-002, a novel ADC targeting folate receptor alpha for ovarian and endometrial cancer [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 1782.

Remarkable in vitro and in vivo activity of IMGN853, an antibody-drug conjugate targeting folate receptor alpha linked to the tubulin-disrupting maytansinoid DM4, in biologically aggressive (type II) endometrial cancers

Remarkable in vitro and in vivo activity of IMGN853, an antibody-drug conjugate targeting folate receptor alpha linked to the tubulin-disrupting maytansinoid DM4, in biologically aggressive (type II) endometrial cancers

Preclinical activity and safety of STRO-002, a novel ADC targeting folate receptor alpha for ovarian cancer

Objective: Folate receptor alpha (FolRa) is a cell-surface glycoprotein, highly expressed in ovarian and endometrial adenocarcinoma, and is thus a promising target for cancer therapy using antibody drug conjugates (ADCs). We have employed an E. coli cell-free expression system (XpressCFTM) and site-specific conjugation technology to generate STRO-002, a novel, site-specific, and homogenous FolRa-targeting ADC.

In Vitro and In Vivo Activity of IMGN853, an Antibody-Drug Conjugate Targeting Folate Receptor Alpha Linked to DM4, in Biologically Aggressive Endometrial Cancers.

Grade 3 endometrioid and uterine serous carcinomas (USC) account for the vast majority of endometrial cancer deaths. The purpose of this study was to determine folic acid receptor alpha (FRα) expression in these biologically aggressive (type II) endometrial cancers and evaluate FRα as a targetable receptor for IMGN853 (mirvetuximab soravtansine). The expression of FRα was evaluated by immunohistochemistry (IHC) and flow cytometry in 90 endometrioid and USC samples. The in vitro cytotoxic activity and bystander effect were studied in primary uterine cancer cell lines expressing differential levels of FRα. In vivo antitumor efficacy of IMGN853 was evaluated in xenograft/patient-derived xenograft (PDX) models. Semiquantitative IHC analysis indicated that 41% of the USC patients overexpress FRα. Further, overexpression of FRα (i.e., 2+) was detected via flow cytometry in 22% (2/9) of primary endometrioid and in 27% (3/11) of primary USC cell lines. Increased cytotoxicity was seen with IMGN853 treatment compared with control in 2+ expressing uterine tumor cell lines. In contrast, tumor cell lines with low FRα showed no difference when exposed to IMGN853 versus control. IMGN853 induced bystander killing of FRα = 0 tumor cells. In an endometrioid xenograft model (END(K)265), harboring 2+ FRα, IMGN853 treatment showed complete resolution of tumors (P < 0.001). Treatment with IMGN853 in the USC PDX model (BIO(K)1), expressing 2+ FRα, induced twofold increase in median survival (P < 0.001). IMGN853 shows impressive antitumor activity in biologically aggressive FRα 2+ uterine cancers. These preclinical data suggest that patients with chemotherapy resistant/recurrent endometrial cancer overexpressing FRα may benefit from this treatment. Mol Cancer Ther; 17(5); 1003-11. ©2018 AACR.

Highlights of This Issue

Targeting cyclinD:CDK4/6:Rb signaling pathway through CDK4/6 inhibition has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, O'Brien and colleagues investigated the preclinical activity of the CDK4/6 inhibitor, abemaciclib, in each of the known molecular subtypes of breast cancer. Measurement of specific biomarkers can be used to identify sensitive sub-populations beyond the ER+ group and within the HER2-amplified and TNBC. In addition, they show that abemaciclib combinations improve upon responses observed with standard of care therapeutics in these sensitive sub-populations, including cytotoxic chemotherapies. These data support further clinical development of abemaciclib as mono-therapy or as a combination partner in biomarker selected ER+, HER2-amplified and TNBCs.Osimertinib, a third-generation EGFR TKI with activity against the activating mutations (Ex19del and L858R) and the T790M EGFR mutation, is approved for the treatment of T790M-positive NSCLC. EGFR exon 20 insertions (Ex20Ins), the 3rd most prevalent EGFR activating mutations, are unresponsive to 1st and 2nd generation EGFR inhibitors. Here, Floc'h and colleagues report that osimertinib and its metabolite inhibit proliferation in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-Ex20Ins mutant tumor xenograft and patient derived xenografts in vivo. Together, these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC.Advanced stage neuroblastoma has limited therapeutic options once conventional therapy fails. Moreover, chemotherapy is not targeted, leading to dose-limiting toxicity and for neuroblastoma survivors, potential life-long side effects. Here, Sagnella and colleagues report the efficacy of bispecific antibody-targeted delivery of a bacterially-derived nanocell loaded with doxorubicin (EGFREDVTMDox). Neuroblastoma cells grown as spheroids displayed increased sensitivity to cell death induced by EGFREDVTMDox compared to doxorubicin alone. In vivo, EGFREDVTMDox significantly reduced tumor growth in orthotopic models of neuroblastoma compared to free doxorubicin. The use of targeted and drug-loaded EDVTM provides a potential new approach for the treatment of aggressive pediatric cancers.Type II endometrial cancers, including uterine serous carcinoma, clear cell and poorly differentiated endometrioid adenocarcinoma account for a disproportionate number of endometrial cancer deaths. IMGN853 is an antibody-drug conjugate linked to DM4 targeting Folate Receptor Alpha (FRα), a receptor overexpressed in biologically aggressive endometrial tumors. Here, Altwerger and colleagues demonstrate that IMGN853 may have impressive preclinical activity against Type II endometrial cancers with high (2+) FRα expression. Due to its cleavable linker, IMGN853 may be active against heterogeneous FRα expressing tumors. Clinical studies with IMGN853 in patients harboring biologically aggressive endometrial cancers overexpressing FRα are warranted.

Abstract B115: MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts

Abstract Triple-negative breast cancer (TNBC) is a highly metastatic disease with very poor long-term prognosis. Because of the lack of well-defined molecular targets, traditional chemotherapy is currently the only therapeutic regimen for TNBC. Within this agent class, eribulin has been approved for metastatic breast cancer and the prespecified subgroup analysis in phase 3 clinical trial (study301) revealed that eribulin demonstrated the greatest therapeutic benefit on overall survival of TNBC patients among all other breast cancer subtypes. Folate receptor alpha (FRA) is a GPI-linked membrane glycoprotein that has limited expression in normal tissue, but is highly overexpressed on a large number of cancers of epithelial origin, including non-small cell lung cancer (NSCLC), ovarian cancer, and TNBC. These features make FRA an attractive target for the treatment of TNBC using targeted therapeutic approaches. Farletuzumab, a humanized monoclonal antibody targeting FRA, has been investigated in multiple clinical trials enrolling patients overexpressing FRA. MORAb-202, a novel antibody-drug conjugate (ADC) consisting of farletuzumab conjugated to eribulin, targets FRA overexpressed by a variety of tumor types including TNBC. Upon binding and internalization, it is hypothesized that the liberated eribulin elicits both a direct cytotoxic effect on FRA-positive tumor cells and a bystander effect on FRA-negative tumor cells and, importantly, tumor-associated stromal cells within the tumor microenvironment. To address this hypothesis, we have evaluated the in vivo efficacy of MORAb-202 against a FRA-positive TNBC patient-derived xenograft (PDX) model. Single administration of 5 mg/kg MORAb-202 mediated tumor regression statistically compared to vehicle control (P&amp;lt;0.0001). Additionally, one animal treated with MORAb-202 showed complete and durable tumor regression. In order to examine the effects of MORAb-202 treatment on the tumor microenvironment, we harvested tumors both prior to treatment and five days post-treatment. Immunofluorescent staining was used to assess the target-specific engagement of MORAb-202 to FRA-positive regions of the tumor. Strong membrane staining of MORAb-202 was observed, while no staining was seen in the adjacent stromal regions. MORAb-202 also had demonstrable effects on the tumor microenvironment via reduction of the number of FRA-negative cancer-associated fibroblasts (CAF) present. To further evaluate whether these effects on CAF are part of the mode of action (MOA) of MORAb-202, we tested the antitumor efficacy and effects on the tumor microenvironment in a surrogate FRA-positive in vivo model. Tumors were isolated on days 3, 5, 7, and 9 post-treatment. The reduction in CAF, visualized by alpha-smooth muscle actin staining, was directly proportional to overall tumor reduction. In both studies, single-dose MORAb-202 showed dramatically greater efficacy than molar equivalent dosing of eribulin alone. Histologic analyses revealed unique modes of action of MORAb-202 over antibody or payload alone. These results suggest a potential benefit of MORAb-202 in the treatment of TNBC. Citation Format: Katherine A. Rybinski, Christopher Maddage, Xin Cheng, Earl Albone, Luigi Grasso, Keiji Furuuchi, Toshimitsu Uenaka. MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B115.

Radiosensitization of triple negative breast cancer with gold nanosphere conjugates targeting the folate receptor

Despite geometric localization capabilities of current radiation therapy (RT) techniques to the tumor, the need for specificity at the cellular level remains. Gold nanoparticles (AuNPs) decorated with tumor-targeting peptides and antibodies can hone in on tumors, get internalized, and sensitize tumors to RT. Extending this paradigm to triple-negative breast cancer (TNBC) has been challenging due to its lack of classical tumor markers. Targeting folate receptor alpha (FRα), overexpressed by MDA-MB-231 TNBC cells, with AuNP-folate conjugates showed substantial AuNP internalization, but mitogenic effects of folate offset anti-tumor effects of AuNP radiosensitization in xenograft mouse studies. To overcome this problem, we switched the targeting moiety on AuNPs from an agonist of FRα, to anti-FRα Ig, an antibody (Ab) that binds to FRα without inducing proliferative or mitogenic effects. Pegylated 20 nm AuNPs (pAuNPs) were synthesized and conjugated to nonspecific rabbit IgG Ab (rAuNPs) or targeted anti-FRα Ab (aAuNPs). AuNPs were characterized by zeta potential, hydrodynamic diameters and absorption spectra. Uptake by MDA-MB-231 cells was analyzed qualitatively using dark field microscopy. In-vitro radiosensitization potential by aAuNPs was assessed via clonogenic assay using clinically relevant, 6MV RT. Surface plasmon resonance peaks of pAuNPs, rAuNPs, and aAuNPs were observed at 521±2 nm. AuNP zeta potentials were: -34±6 mV (bare AuNPs), -8±4 mV (pAuNPs), -1±6 mV (rAuNPs), and -5±5 mV (aAuNPs). Hydrodynamic diameters determined by dynamic light scattering were: 33±12 nm (bare AuNPs), 56±18 nm (pAuNPs), 64±22 nm (rAuNPs), and 62±17 nm (aAuNPs). aAuNPs demonstrated significant radiosensitization with a dose enhancement factor of 1.15 calculated at 10% cell survival relative to RT alone. AuNPs conjugated to anti-FRα Ab sensitize triple negative breast cancer cells to radiation and present a viable strategy to target cancer cells at the molecular level.

Harnessing IMGN853-mediated cell cytoxicity response by modulating FRα expression in ovarian cancer.

e17061 Background: Ovarian cancer (OC) is the fifth leading cause of cancer-related death among women in the United States. Standard treatment includes debulking surgery followed by platinum based chemotherapy. While most tumors respond to this treatment, 70% of the tumors recur and develop into a chemo-resistant disease. There is a need for new therapies targeting recurrent chemoresistant OC. To address this need, Mirvetuximab Soravtansine (ImmunoGen, Waltham, MA) has developed an antigen-drug conjugate (ADC) containing DM4, a highly potent maytansinoid derivative that induces the cell cycle arrest, conjugated to an antibody targeting folate receptor alpha (FRα). Mirvetuximab Soravtansine (IMGN853), was tested in Phase I clinical trials on women with recurrent OC and showed low grade toxicity profile and activity in platinum-resistant disease. The goal of this study is to characterize the mechanism(s) leading to IMGN853 resistance in OC and test whether anticancer drugs targeting these mechanisms could be used in combination with IMGN853 for an additive/synergistic therapeutic effect. Methods: In vitro experiments were performed to detect and modulate FRα expression. Results: We show that sensitivity of OC cell lines to IMGN853 correlates with the expression levels of FRα (R = 0.82). Long-term exposure of sensitive cells to sublethal doses of IMGN853 induces drug resistance which correlates with decreased FRα expression. Anti-cancer drugs such as dexamethasone (Dex), which induces glucocorticoid receptors, or epigenetic modulators (Trichostatin A and 5-Azacytidine) induce FRα expression in SKOV3 and OVCAR8 OC cells, suggesting the possibility to use these drugs to maintain sensitivity of OC cells to IMGN853 and increase its therapeutic window. The therapeutic benefits of combining each of these drugs with IMGN853 will be further tested in mice using ovarian cancer patients’ derived xenografts with different FRα expression levels. Conclusions: These findings have clinical implications as they indicate that OC sensitivity to IMGN853 is modulated in part by changes in FRα levels. Resistance to this drug may be overcome by simultaneous treatment with Dex or other anti-cancer drugs such as TSA or AZA.

Abstract 4531: Effects of drug load on therapeutic index for antibody-maytansinoid conjugates

Abstract Antibody-drug conjugates (ADCs) are being actively pursued as a new modality to treat cancer following the regulatory approval of Adcetris@ and Kadcyla@. ADCs consist of a cytotoxic agent, or drug, conjugated to a targeting antibody (Ab) through a linker. The two approved ADCs (and most ADCs now in the clinic) are heterogeneous conjugates with an average molar drug to Ab ratio (DAR) of 3-4 (potentially ranging from 0-8 for individual molecules). To understand the effects of different DAR ranges on the preclinical properties of ADCs using a maytansinoid cytotoxic agent, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor alpha) or an uncleavable linker (J2898A-SMCC-DM1 targeting epidermal growth factor receptor) with varying DAR, and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. Both M9346A-sulfo-SPDB-DM4 and J2898A-SMCC-DM1 conjugates with low (average ∼2, range 0-4) to very high DAR (average 10, range 7-14) were prepared in good yield, high monomer content and low free drug levels. At constant Ab concentration, the in vitro potency consistently increased with increasing DAR. We then characterized the in vivo disposition of these ADCs. First, pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but at an average DAR around 9-10 rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR conjugates rapidly distribute to the liver, with the maximum%ID/g for this organ at 24-28% compared with 7-10% for lower DAR conjugates (all at 2-6 h post-injection). We further studied the efficacy of these ADCs in mouse xenograft models at both constant antibody and constant maytansinoid doses. At constant Ab dose, conjugates with average DAR ∼6 are more efficacious than conjugates with DAR ∼3. However, DAR 9-10 conjugates are either less or similarly active compared to DAR 6 conjugates, consistent with the observed rapid clearance. At constant maytansinoid dose, conjugates with 2 - 6 DAR show similar efficacy, while those with 9-10 DAR were again less active. Tolerability studies monitoring body weight loss show that conjugates with different DAR, even as high as 9-10, have comparable tolerability based on total administered maytansinoid dose. In summary, our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR in the range between 2 - 6 have better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs, but suggest the exploration of lower or higher DAR depending on the biology of the target antigen. Citation Format: Xiuxia Sun, Jose F. Ponte, Nicholas C. Yoder, Jennifer Coccia, Leanne Lanieri, Rassol Laleau, Qifeng Qiu, Rui Wu, Erica Hong, Megan Bogalhas, Lintao Wang, Erin K. Maloney, Olga Ab, Hans K. Erickson, Thomas A. Keating, Ravi Chari, John M. Lambert. Effects of drug load on therapeutic index for antibody-maytansinoid conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2015-4531

Intraoperative Molecular Imaging of Lung Adenocarcinoma Can Identify Residual Tumor Cells at the Surgical Margins.

During lung surgery, identification of surgical margins is challenging. We hypothesized that molecular imaging with a fluorescent probe to pulmonary adenocarcinomas could enhance residual tumor during resection. Mice with flank tumors received a contrast agent targeting folate receptor alpha. Optimal dose and time of injection was established. Margin detection was compared using traditional methods versus molecular imaging. A pilot study was then performed in three humans with lung adenocarcinoma. The peak tumor-to-background ratio (TBR) of murine tumors was 3.9. Fluorescence peaked at 2 h and was not improved beyond 0.1 mg/kg. Traditional inspection identified 30% of mice with positive margins. Molecular imaging identified an additional 50% of residual tumor deposits (p < 0.05). The fluorescent probe visually enhanced all human tumors with a mean TBR of 3.5. Molecular imaging is an important adjunct to traditional inspection to identify surgical margins after tumor resection.