FOLR1 Expression Research Articles

Characterizing the genomic landscape through the lens of FOLR1 status in low and high grade serous ovarian carcinoma

ObjectiveTargeted therapy in folate receptor alpha (FOLR1)-positive high grade serous ovarian carcinoma (HGSOC) is now a mainstay for platinum-resistant disease. However, the rate of FOLR1-positivity in low grade serous ovarian carcinoma (LGSOC) is not well documented. Less common than HGSOC, LGSOC tends to respond poorly to traditional platinum-based chemotherapeutic regimens, particularly in recurrence. Thus, there is an urgent need to identify molecular targets that may assist in identifying more efficacious treatments for LGSOC. In this work, we assessed the genomic and transcriptomic landscapes in FOLR1-positive/negative LGSOC compared to its high-grade counterpart. MethodsUsing a large precision oncology database, next-generation sequencing and immunohistochemistry was performed on a cohort of 281 LGSOC and 5086 HGSOC. Associated MAPK activation was calculated based on NGS results and patient survival analysis was completed stratified by molecular alteration. ResultsCompared with LGSOC (24.6 %), HGSOC tumors have significantly higher prevalence of FOLR1+ status (43.5 %) and significantly higher PD-L1+ status. Conversely, LGSOC had higher prevalence of KRAS and NRAS mutations, with a near exclusivity for BRAF mutation compared to HGSOC. FOLR1- LGSOC and HGSOC had similar prevalences of T cell-inflamed tumors, though FOLR1+ LGSOC had a significantly lower prevalence of T-Cell inflamed tumors than FOLR1+ HGSOC. MAPK activation, quantified via MAPK activation score (MPAS), was significantly higher in low-grade tumors compared to HGSOC, yet no difference between FOLR1+ vs FOLR1- LGSOC was observed. ConclusionsThough less than in high-grade disease, a notable portion of low-grade tumors were FOLR1+, suggesting FOLR1 expression in LGSOC could be a viable target for this rare histology, particularly in the recurrent setting.

Alteration in folate carrier expression via histone deacetylase inhibition in BeWo human placental choriocarcinoma cells

Folates are essential nutrients for fetal development during pregnancy. Valproic acid (VPA), an inhibitor of histone deacetylases (HDACs), alters the expression of folate carriers in placental cells; however, the underlying mechanisms remain unclear. Here, we aimed to determine the profiles of folate carriers (folate receptor alpha [FOLR1], solute carrier [SLC]-19A1, and SLC46A1) after inhibition of HDACs, especially class I and IIa HDACs, using different inhibitors and gene knockdown tests. Quantitative polymerase chain reaction revealed that BeWo cells (a trophoblast model) expressed HDACs and folate carriers, similar to human placental villi. FOLR1 expression was upregulated by VPA, apicidin, and trichostatin A, but downregulated by MS-275 after 24 h treatment. VPA and apicidin upregulated the expression of SLC46A1. These inhibitors downregulated SLC19A1 expression. TMP269 (a class IIa inhibitor) did not affect folate carrier levels. HDAC1/2 knockdown upregulated FOLR1 and SLC46A1 levels, whereas HDAC1/3 knockdown downregulated FOLR1 levels. Our findings suggest that the pharmacological inhibition of class I HDACs alters the expression of folate carriers in BeWo cells. By contrast, HDAC inhibitors exert different regulatory effects on folate carriers. Moreover, HDAC1/2 inhibition may be a potential mechanism involved in altering FOLR1 and SLC46A1 levels.

Characterizing FOLR1 expression in low-grade serous ovarian carcinoma.

Characterizing FOLR1 expression in low-grade serous ovarian carcinoma.

Dolutegravir induces FOLR1 expression during brain organoid development.

During the first month of pregnancy, the brain and spinal cord are formed through a process called neurulation. However, this process can be altered by low serum levels of folic acid, environmental factors, or genetic predispositions. In 2018, a surveillance study in Botswana, a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs, found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result, the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs, we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing, Optical Coherence Tomography (OCT), Optical Coherence Elastography (OCE), and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast, reverberant OCE measurements indicate decreased organoid volumes and internal stiffness.

Abstract LB127: A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model

Abstract Folate receptor alpha (FOLR1) is specifically overexpressed in malignant tumors of epithelial origin, including ovarian, lung and breast cancers. While antibody-drug conjugate (ADC) therapies targeting FOLR1 have shown promise in clinical trials, there is still a large proportion of patients, particularly those with low FOLR1 expression, who do not respond, indicating new therapeutic strategies are needed. MUC1 is another tumor-associated antigen that is likewise overexpressed in epithelial ovarian cancer, breast cancer, and NSCLC, among other cancers; analysis of ovarian PDX tumors by immunohistochemistry (IHC) suggests that FOLR1 and MUC1 are both highly expressed in this cancer type. We therefore hypothesized that dual targeting of FOLR1 and MUC1 could provide therapeutic benefit in ovarian cancer as well as a broad range of tumors. We generated a FOLR1 x MUC1 bispecific antibody (bsAb) from anti-FOLR1 and anti-MUC1 monoclonal antibodies that were produced using RenLite® fully human common light chain mice. These parental monoclonal antibodies recognize both human and cynomolgus monkey antigens. Anti-FOLR1 antibodies were highly specific for FOLR1, as they did not recognize other FOLR family members. The anti-MUC1 antibody was previously confirmed to target MUC1-C, the juxtamembrane domain of MUC1, ensuring tumor cell recognition regardless of MUC1 cleavage. Assessment of the novel FORL1 x MUC1 bsAb indicates binding to several ovarian, breast and NSCLC cell lines, and shows enhanced internalization in MDA-MB-468 and T47D cell lines when compared to parental antibodies. Subsequently, the FORL1 x MUC1 bsAb was conjugated to vcMMAE with a DAR of 4, to generate a bispecific ADC, BCG023. BCG023 exhibited superior tumor growth inhibition compared to benchmark ADCs conjugated with the same payload and DAR in a low FOLR1-expressing NSCLC PDX model. These data suggest that targeting FOLR1 and MUC1 with a bispecific ADC is a promising therapeutic strategy for the treatment of ovarian and other tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Chaoshe Guo, Yi Yang. A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB127.

Abstract 2913: Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model

Abstract Introduction: The tumor extracellular matrix (ECM) is a physical barrier which obstructs trafficking of CAR T cells to tumors. We hypothesize that co-expressing ECM remodeling/degrading enzymes heparanase (HPSE) or PH-20 with a CAR directed against folate receptor 1 (FolR1) will overcome the physical tumor microenvironment barrier and improve anti-tumor efficacy of anti-FolR1 CAR in a human ovarian cancer model. Methods: ECM CARs were constructed by combining anti-FolR1 ScFv with a CD8 hinge and transmembrane domain, 4-1BB, and CD3ζ endodomains, followed by a P2A element and full-length HPSE or GPI-anchored PH-20 sequence. CAR T cells were prepared from CD4+ and CD8+ T cells of healthy donors by lentiviral transduction. CAR expression was evaluated by flow cytometry; HPSE and PH-20 expression were measured via ELISA and Western blotting, respectively. CAR-mediated killing and cytokine release were observed in overnight co-culture assays with FolR1-expressing tumor cells. ECM CARs’ migration through CultrexTM and sodium hyaluronate matrices were assessed for HPSE and PH-20 function, respectively. ECM CARs’ efficacy was evaluated in vivo using a FolR1-expressing subcutaneous OV90 ovarian cancer xenograft model in NOD SCID Gamma (NSG) mice, and representative OV90 tumors were harvested at day 7 post-CAR T treatment for immunohistochemical staining. Results: FolR1 expression was confirmed in ovarian cancer cell lines and substrates targeted by HPSE (perlecan, glypican 3, CD138) and PH-20 (hyaluronan) were detected by immunofluorescence in human ovarian tumors’ sections. Lentiviral transduced primary T cells yielded robust expression of FolR1 CAR and ECM enzymes. Anti-FolR1 CAR and ECM CARs secreted pro-inflammatory cytokines IL-2, TNFα, IFNƳ and lysed OVCAR3 tumor cells with similar efficacy. ECM CARs with HPSE or PH-20 exhibited enhanced migration through ECM-rich matrices as compared to anti-FolR1 CAR alone. In vivo, all anti-FolR1 CAR T cells rejected OV90 tumors and extended survival compared to tumor alone or untransduced T cells (UTD). However, ECM CARs co-expressing HPSE or PH-20 accelerated tumor regression as compared to anti-FolR1 CAR alone. Tumor histology revealed CAR T cell infiltration (CD3+, CD8+) in mice treated with anti-FolR1 CARs but not UTD, indicating that anti-FolR1 CARs can effectively infiltrate OV90 tumors. Imaging studies are underway to evaluate the function of ECM CARs in the tumor microenvironment. Conclusions: The anti-FolR1 CARs boosted with ECM enzymes HPSE or PH-20 demonstrated specific lysis of FolR1-expressing cell lines, improved penetration through ECM-rich matrices in vitro, and accelerated tumor regression in an ovarian cancer model in vivo. Therefore, ECM CARs may help improve clinical outcomes in patients with ovarian tumors. Citation Format: Brittany L. Steimle, Peirong Hu, Ngoc Tran, Jia-Jye Lee, Kathleen Baysac, Kun Luo, Xu Wang, Genqing Liang, Saule Nurmukhambetova, Tri Tran, Pradyot Dash, Dina Schneider. Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2913.

Developing Folate-Conjugated miR-34a Therapeutic for Prostate Cancer: Challenges and Promises.

Prostate cancer (PCa) remains a common cancer with high mortality in men due to its heterogeneity and the emergence of drug resistance. A critical factor contributing to its lethality is the presence of prostate cancer stem cells (PCSCs), which can self-renew, long-term propagate tumors, and mediate treatment resistance. MicroRNA-34a (miR-34a) has shown promise as an anti-PCSC therapeutic by targeting critical molecules involved in cancer stem cell (CSC) survival and functions. Despite extensive efforts, the development of miR-34a therapeutics still faces challenges, including non-specific delivery and delivery-associated toxicity. One emerging delivery approach is ligand-mediated conjugation, aiming to achieve specific delivery of miR-34a to cancer cells, thereby enhancing efficacy while minimizing toxicity. Folate-conjugated miR-34a (folate-miR-34a) has demonstrated promising anti-tumor efficacy in breast and lung cancers by targeting folate receptor α (FOLR1). Here, we first show that miR-34a, a TP53 transcriptional target, is reduced in PCa that harbors TP53 loss or mutations and that miR-34a mimic, when transfected into PCa cells, downregulated multiple miR-34a targets and inhibited cell growth. When exploring the therapeutic potential of folate-miR-34a, we found that folate-miR-34a exhibited impressive inhibitory effects on breast, ovarian, and cervical cancer cells but showed minimal effects on and targeted delivery to PCa cells due to a lack of appreciable expression of FOLR1 in PCa cells. Folate-miR-34a also did not display any apparent effect on PCa cells expressing prostate-specific membrane antigen (PMSA) despite the reported folate's binding capability to PSMA. These results highlight challenges in the specific delivery of folate-miR-34a to PCa due to a lack of target (receptor) expression. Our study offers novel insights into the challenges and promises within the field and casts light on the development of ligand-conjugated miR-34a therapeutics for PCa.

Folate receptor overexpression induces toxicity in a diet-dependent manner in C. elegans

Folate receptor (FR) alpha (FOLR1) and beta (FOLR2) are membrane-anchored folate transporters that are expressed at low levels in normal tissues, while their expression is strongly increased in several cancers. Intriguingly, although the function of these receptors in, for example, development and cancer has been studied intensively, their role in aging is still unknown. To address this, we utilized Caenorhabditis elegans, in which FOLR-1 is the sole ortholog of folate receptors. We found that the loss of FOLR-1 does not affect reproduction, physical condition, proteostasis or lifespan, indicating that it is not required for folate transport to maintain health. Interestingly, we found that FOLR-1 is detectably expressed only in uterine-vulval cells, and that the histone-binding protein LIN-53 inhibits its expression in other tissues. Furthermore, whereas knockdown of lin-53 is known to shorten lifespan, we found that the loss of FOLR-1 partially rescues this phenotype, suggesting that elevated folr-1 expression is detrimental for health. Indeed, our data demonstrate that overexpression of folr-1 is toxic, and that this phenotype is dependent on diet. Altogether, this work could serve as a basis for further studies to elucidate the organismal effects of abnormal FR expression in diseases such as cancer.

Abstract 3189: Pre-clinical evaluation of novel folate receptor 1 directed CAR T cells for high grade serous ovarian cancer

Abstract Purpose: Treatment of ovarian cancer remains challenging. A high unmet clinical need for targeted, efficient, and persisting drugs still is a major issue for clinicians and patients diagnosed with ovarian cancer, particularly platinum-resistant and-refractory, recurrent disease. Genetically modified T cells expressing chimeric antigen receptors (CARs) have shown efficient, as well as persisting anti-tumor functionality in hematological malignancies. However, solid cancers have not yet been as efficiently treated with CAR T cells, potentially due to the lack of appropriate targets. Therefore, we thoroughly characterized FOLR1 as CAR T cell target for ovarian cancer, designed novel CAR T cells, and assessed these drug candidates preclinically in vitro and in vivo. Experimental Design: We performed ultra-high content imaging on high-grade serous epithelial ovarian cancer as well as healthy tissue samples to profile the expression of the tumor-associated antigen FOLR1. Subsequently, we designed a series of CAR T cell candidates based on different monoclonal antibodies targeting FOLR1, in order to leverage on clinical experience with these binders, particularly safety aspects. The CAR designs differed in the VL-VH orientation and the spacer domain of the CAR construct. The preclinical assessment of various FOLR1-directed CAR T cell constructs was evaluated against various ovarian cancer cell lines in vitro and in vivo. Results: In this study we show high and consistent expression of the tumor-associated antigen FOLR1 on primary ovarian cancer samples, namely high-grade serous ovarian cancer. Moreover, FOLR1 expression is low, restricted, and polarized in healthy tissues. We generated diverse FOLR1-specific CAR T cells and characterized their efficacy and specificity in vitro as well as in an ovarian cancer xenograft model. CAR T cells rapidly and efficiently eradicate xenograft tumors. This anti-tumor response is accompanied by CAR T cell expansion, FOLR1-dependent cytokine secretion, and infiltration of CAR T cells into the tumor. Conclusions: These findings demonstrate the feasibility of a preclinical CAR T cell approach targeting FOLR1 as treatment option for ovarian cancer and potentially other FOLR1-expressing tumors. Citation Format: Christoph Herbel, Julie Daigre, Manuel Martinez-Osuna, Maria Bethke, Larissa Steiner, Janina Brauner, Jens Kopatz, Paurush Praveen, Dominik Eckardt, Andreas Bosio. Pre-clinical evaluation of novel folate receptor 1 directed CAR T cells for high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3189.

Abstract 1544: MBK-103, a potent novel conjugation platform-based antibody-drug conjugate, changing therapeutic options in folate receptor alpha positive cancer patients

Abstract Folate receptor alpha (FOLR1) is a clinically validated target, that is overexpressed extracellularly in numerous solid malignancies with a high unmet medical need. Ovarian, non-small cell lung and breast adenocarcinomas are among indications with the highest frequency of FOLR1-positive patients with one out of two patients showing upregulated topoisomerase I expression. MBK-103, an antibody-drug conjugate targeting FOLR1, is based on Mablink’s novel proprietary hydrophilic drug-linker platform. It is comprised of: (i) an Fc-attenuated humanized IgG1 monoclonal antibody, that selectively binds to FOLR1; (ii) a polysarcosine hydrophobicity masking entity allowing for a high drug-to-antibody ratio (DAR) of 8, while improving the pharmacokinetics and tolerability of the drug; (iii) a proprietary dipeptide cleavable unit and iv) exatecan, a potent topoisomerase I inhibitor. MBK-103 showed excellent ex vivo stability in human plasma enabling a prolonged half-life, similar to that of the unconjugated monoclonal antibody. This led to an increased drug exposure, resulting in a potent antitumor efficacy observed in a collection of more than ten in vivo mouse tumor models with a diverse FOLR1 expression, already at doses as low as 1-3 mg/kg. In addition, the distinct mode of action of exatecan along with the unique characteristics of the proprietary drug-linker platform resulted in the in vivo antitumor potency obtained also in colorectal cancer models, in which tubulin-inhibiting agents exhibit only limited therapeutic effects. The compound was also very well tolerated in cynomolgus monkeys with HNSTD at 50 mg/kg and even when administered repeatedly. High conjugation yields along with 100% homogeneity are major drivers of the successful ongoing manufacturing process with planned IND submission in Q4/2023. Citation Format: Warren Viricel, Louise Conilh, Edouard Leroy, Jean-Guillaume Lafay, Frederique Brune, Lenka Kyrych Sadilkova, Charles Dumontet. MBK-103, a potent novel conjugation platform-based antibody-drug conjugate, changing therapeutic options in folate receptor alpha positive cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1544.

KDM6B Variants May Contribute to the Pathophysiology of Human Cerebral Folate Deficiency.

(1) Background: The genetic etiology of most patients with cerebral folate deficiency (CFD) remains poorly understood. KDM6B variants were reported to cause neurodevelopmental diseases; however, the association between KDM6B and CFD is unknown; (2) Methods: Exome sequencing (ES) was performed on 48 isolated CFD cases. The effect of KDM6B variants on KDM6B protein expression, Histone H3 lysine 27 epigenetic modification and FOLR1 expression were examined in vitro. For each patient, serum FOLR1 autoantibodies were measured; (3) Results: Six KDM6B variants were identified in five CFD patients, which accounts for 10% of our CFD cohort cases. Functional experiments indicated that these KDM6B variants decreased the amount of KDM6B protein, which resulted in elevated H3K27me2, lower H3K27Ac and decreased FOLR1 protein concentrations. In addition, FOLR1 autoantibodies have been identified in serum; (4) Conclusion: Our study raises the possibility that KDM6B may be a novel CFD candidate gene in humans. Variants in KDM6B could downregulate FOLR1 gene expression, and might also predispose carriers to the development of FOLR1 autoantibodies.

Pediatric Acute Myeloid Leukemia with Co-Occurring BCR::ABL and CBFA2T3::GLIS2 Dual Fusion with Deep Response to FOLR1-Targeting Antibody Drug Conjugate Stro-002 and Tyrosine Kinase Inhibitor

Introduction: Pediatric acute myeloid leukemia (AML) is characterized by significant cytogenetic and molecular heterogeneity. AML with CBFA2T3::GLIS2 fusion (CBF::GLIS) involving infants and very young children is a rare but uniformly refractory disease with high mortality. Laboratory data demonstrates that this fusion is sufficient for leukemic transformation without recurrent secondary variants. These studies have also linked CBF::GLIS fusion to induction of folate receptor alpha (FOLR1) expression in transformed cells (Meshinchi et al, 2020). Incidence of Ph+ de novo (BCR::ABL minor breakpoint, p190 hybrid) disease ranges from 0.5-3% of all AML cases across the age spectrum (Soupir et al, 2017). We, for the first-time, report dual existence of both fusions in a child with AML and discuss novel interventions including combination of a BCR::ABL tyrosine kinase inhibitor (TKI) and antifolate therapy consisting of methotrexate (MTX) and FLOR1-specific antibody-drug conjugate (ADC) STRO-002 (Sutro Biopharma) based on available data regarding its in-vitro preclinical activity in CBF::GLIS disease (Meshinchi et al, ASH abstract, 2021). Case: A 2-year-old girl of Vietnamese descent received a bone marrow examination for bicytopenia and macrocytosis. Flow cytometry confirmed the poor prognostic RAM immunophenotype AML (CD45 dim, HLA-DR negative, CD38 dim, CD56 bright) and FOLR1 expression. Marrow aspirate smears showed numerous hypercellular spicules and blasts with scant cytoplasm and some blebbing but no granules or Auer rods. Marrow exhibited 100% cellularity. CNS was not involved. Review of diagnostic genomic profile mid induction showed a BCR::ABL minor breakpoint fusion as well as CBF::GLIS fusion. The child began AML induction on COG AAML1831 trial randomized to the experimental arm with CPX-351 and gemtuzumab ozogamicin (GO). Due to lack of response to AML therapy at end of Induction (EOI) I, she was taken off protocol and started on modified ALL regimen for Induction II consisting of cytarabine, low dose weekly methotrexate and bimonthly peg-asparaginase with addition of an oral TKI, dasatinib. EOI II marrow examination showed improvement with reduction in blasts (11%) and detectable BCR::ABL but undetectable CBF::GLIS by Q-RT-PCR. Induction III included low dose weekly methotrexate, cytarabine, GO, and dasatinib. EOI III marrow examination showed complete morphologic remission (CR) with no evidence of minimal residual disease (MRD) or fusion transcripts of either clone. Due to availability of a matched sibling donor, the child underwent hematopoietic stem cell transplantation (HSCT); however, was noted to have 3% blasts on day +83 marrow examination despite previously commencing dasatinib post-transplant prophylaxis. BCR::ABL and CBF::GLIS transcripts were again detectable at low levels. Dasatinib dosage was maximized and weekly MTX and bimonthly peg-asparaginase were reintroduced, but the marrow disease burden increased significantly to 78% blasts positive only for CBF::GLIS fusion. Given availability of FOLR1 directed ADC on single-patient compassionate use basis, patient received single agent STRO-001 on a bi-monthly basis. With rising ANC, evaluation of marrow after 2nd STRO-002 dose remarkably showed CR with MRD negative remission by flow and molecular studies. This time remission was consolidated with monthly STRO-002 and donor lymphocyte infusions (DLI). Currently, patient remains in MRD-negative remission after the 3 of 4 planned STRO-002/DLI maintenance. Patient has maintained a 100% Lansky play score and tolerated the regimen with only prolonged initial cytopenia requiring supportive care as a toxicity. Plan is to continue TKI and monthly STRO-002 as maintenance therapy. Conclusion: Complete genomic sequencing has revolutionized pediatric AML categorization. It is quite conceivable that (BCR::ABL, CBF::GLIS) dual fusion disease will become a more common finding in future. Although CBF::GLIS AML is uniformly refractory to conventional therapy, targeted therapy with FOLR1-directed STRO-002 was highly effective in eliminating CBF::GLIS clone in this patient, and combination with DLI appears to be feasible. Long-term follow-up is required to assess durability of remission. Additional testing of this approach in a larger patient population is needed to determine the role of STRO-002 in this high-risk pediatric AML population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Folate receptor genes were up-regulated in epithelial ovarian cancer and partly associated with patients’ prognosis

Abstract Objective The present work aimed to investigate folate receptor (FOLR1, FOLR2, FOLR3) expression, functional enrichment, signaling pathway and prognosis in ovarian cancer patients by integrated bioinformatics analysis. Methods Folate receptor (FOLR1, FOLR2, and FOLR3) mRNA expression level between epithelial ovarian cancer and corresponding normal ovarian tissue of cancer patients was compared through the TCGA database by GEPIA online analysis tool. The protein–protein interaction (PPI) network of FOLR1, FOLR2, FOLR3, and related genes were constructed through the STRING database. GO and KEGG enrichment of FOLR1, FOLR2, FOLR3, and relevant genes were analyzed. Overall survival (OS) and progression-free survival (PFS) between FOLR1, FOLR2, and FOLR3 mRNA high and low expression epithelial ovarian cancer patients were compared by log-rank test. Results FOLR and FOLR3 mRNA expression in epithelial ovarian cancer tissue were significantly higher than that of corresponding normal ovarian tissue of cancer patients (P < 0.05) The PPI network showed 53 nodes and 298 edges with the average node degree of 11.2. The local clustering coefficient was 0.744, which indicated that the protein–protein enrichment was statistically significant (P < 1.0 × 10−16). Folate receptor (FOLR1, FOLR2, and FOLR3) and relevant genes were mainly enriched in folic acid transport, methotrexate transmembrane transporter activity, antifolate resistance for biological process, molecular function, and KEGG pathway, respectively. The PFS of FOLR1 and FOLR3 high expression epithelial ovarian cancer patients was significantly lower compared to low-expression subjects with statistical significance [hazard ratio (HRFOLR1) = 1.26, 95% confidence interval (CI): 1.09–1.45, P < 0.05, HRFOLR3 = 1.22, 95% CI: 1.06–1.40, P < 0.05]. However, the OS was not statistically different between FOLR1, FOLR2, and FOLR3 low and high expression groups. Conclusion Folate receptor (FOLR1, FOLR2, and FOLR3) genes were up-regulated in epithelial ovarian cancer and partly associated with patient’s poor prognosis.

Abstract 1075: Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate

Abstract Acute myeloid leukemia (AML) in infants is a unique subtype of AML that is driven by oncogenic fusions, resulting in a highly proliferative disease. The CBFA2T3-GLIS2 oncogenic fusion that is seen exclusively in infants and young children <3 years is the most refractory AML with near uniform mortality with conventional therapies. As part of our Target Pediatric AML (TpAML) discovery effort to define novel therapeutic targets, we discovered folate receptor alpha (FRα, FOLR1) transcript to be expressed in CBF-GLIS positive AML and verified cell surface expression of FOLR1 on leukemic cells. We present pre-clinical efficacy of a novel FOLR1 directed therapy (ELU001) in CBF-GLIS AML.ELU001 is a novel ~6 nm-diameter FOLR1-targeted C’Dot-Drug-Conjugate (CDC) incorporating ~21 exatecan molecules as a payload that is designed to treat cancers with improved safety and efficacy as compared to antibody-drug conjugates. A Phase 1 clinical trial of ELU001 in adult patients with tumors that overexpress FOLR1 is currently underway (NCT05001282). In this study, we determined the potential utility of ELU001 for CBFA2T3-GLIS2 AML by evaluating its preclinical efficacy in AML models. We confirmed the binding of ELU001 to AML cells expressing FOLR1 by flow cytometry using MV4;11 AML cell line transduced with an FOLR1 expression construct (MV4;11 FOLR1+). We show that MV4;11 FOLR1+ cells readily bind to ELU001 but not MV4;11 parental cells. Consistent with target-dependent binding, in vitro cytotoxicity studies demonstrated that MV4;11 FOLR1+ cells were highly susceptible to ELU001 (IC50=30 pM) while MV4;11 parental cells were only minimally affected by treatment. To evaluate in vivo efficacy, we transplanted MV4;11 FOLR1+ cells transduced with Luciferase-expression construct into immune deficient NSG mice at 1M cells per mouse and treated the mice the following week with different doses of ELU001 (vehicle, 0.3mg/kg Q3Dx6, 0.5 mg/kg Q3Dx3, and 0.65mg/kg Q3Dx3). Leukemia burden was assessed by whole-body bioluminescence (IVIS) imaging, performed weekly. IVIS imaging showed that ELU001 effectively eradicated the leukemia in vivoat all three doses tested resulting in enhanced survival while mice that received the vehicle treatment exhibited disease progression and eventually succumb to the disease. We confirmed that the leukemia was absent in ELU001-treated mice at the study endpoint of 120 days post treatment by flow cytometric analysis of the bone marrow, spleen and liver tissues harvested at necropsy. ELU001 was well tolerated with an apparent maximum tolerance dose of 1.50 mg/kg/mouse (0.5 mg/kg exatecan, Q3DX3). In summary, we demonstrated that ELU001 is highly effective at eliminating FOLR1 positive AML cells in vitro and in vivo, providing the preclinical data to support further assessment of ELU001 in clinical trials for pediatric patients with CBFA2T3-GLIS2 fusions overexpressing FOLR1. Citation Format: Quy Le, Feng Chen, Thao Tang, Cynthia Nourigat McKay, Fei Wu, Melik Turker, Thomas Gardinier, Vaibhav Patel, Aranapakam Venkatesan, Tin Khor, Kai Ma, Gregory P. Adams, Soheil Meshinchi. Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1075.

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos

Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. Although the oocytes contain endogenous pool of folates for development, the present study investigated the effect of external folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 μM), were in vitro fertilized and cultured. Cumulus expansion markers (PTX3 and PTGS2) in cumulus cells were highly upregulated after 50 μM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 μM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 μM folic acid in maturation medium improves oocyte maturation, the blastocyst production rate, reduces ROS production as well as upregulate the expression of FOLR1 and folate metabolism enzyme, MTR.

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.

Development of (−)-epigallocatechin-3-gallate-loaded folate receptor-targeted nanoparticles for prostate cancer treatment

Abstract In continuation of our previous studies, we developed polymeric epigallocatechin 3-gallate (EGCG)-loaded nanoparticles (NPs) coupled with folic acid (FA), able to dually bind the human folate receptor alpha (FOLR1), and prostate-specific membrane antigen (PSMA+) in prostate cancer (PCa) model. After a preliminary computational molecular recognition of NP′ ligand binding on the FOLR1 active site, we synthesized the biocompatible block-copolymer PLGA–PEG–FA to prepare EGCG-targeted NPs (EGCG-T-NPs). The obtained NPs were characterized by various analytical techniques, and anticancer efficacy was determined by different sets of experiments in a 3D culture of PCa using PC3 and 22Rv1 cell lines. Results showed a significant reduction in spheroid size by EGCG-T-NPs, especially in PSMA+ (22Rv1) cells. The targeted NPs significantly enhanced the antiproliferative activity of EGCG against PCa cell lines, especially toward the PSMA+ cells, known to have higher FOLR1 expression. We did not observe any changes in the reactive oxygen species formation in both studied cell lines. However, significant changes in mitochondrial depolarization (15%) and polarization (18%) were recorded in response to EGCG-T-NP compared to control in 22Rv1. Similarly, EGCG-T-NP treatment also showed an increase in the number of dead apoptotic cells in 22Rv1 spheroids. Collectively, the obtained results support our hypothesis about the role of these targeted nanoprototypes in the increasing cellular uptake of EGCG payload into PCa cells, thus enhancing its antitumor efficacy.

Targeting FOLR1 in High-Risk CBF2AT3-GLIS2 AML with Stro-002 FOLR1-Directed Antibody-Drug Conjugate

Cryptic inv(16)(p13.3q24.3) leading to the CBFA2T3-GLIS2 (CBF/GLIS) oncogenic fusion is exclusively seen in infants with AML and is associated with adverse outcome. Infants with this fusion are uniformly refractory to conventional therapies and despite intensive and myeloablative therapies, virtually all patients relapse with survival less than 15% (Smith et. al. 2020). In the effort to discover actionable targets for this highly refractory leukemia, we interrogated the genome and transcriptome of nearly 3,000 AML cases, including 45 cases of CBF/GLIS AML, and contrasted this to the transcriptome in normal hematopoietic tissues.Initial computational effort to identify CBF/GLIS-specific targets focused on genes that are overexpressed in CBF/GLIS AML and silent in normal hematopoietic tissues providing us with 194 candidate genes. Filtering this library of genes based on cell surface expression and prevalence in target population narrowed the target gene list to six cell surface encoding genes (FOLR1, FRAS1, HPSE2, KLRF2, MEGF10 and PCDH19). FOLR1 encodes folate receptor alpha and given available therapeutic options with FOLR1-targeting agents, this gene was selected for further studies and therapeutic assessment of available targeted therapies. Based on RNA-seq data, FOLR1 is uniquely expressed in CBF/GLIS AML as it is absent in other AML and in normal hematopoietic cells (Figure 1A) providing an opportunity to specifically target leukemia cells while sparing normal hematopoiesis. Cell surface expression of FOLR1 on AML blasts was confirmed by flow cytometry, which further shows AML-restricted expression of FOLR1 on AML cells but not in normal hematopoietic subsets in individual CBF/GLIS patient samples (Figure 1B, C). We previously showed that forced expression of CBF/GLIS fusion transcript in cord blood hematopoietic stem/ progenitor cells (CB HSPCs) induces malignant transformation that fully recapitulates primary CBF/GLIS AML (Hylkema et. al. 2019 ASH). We also demonstrated that CBF/GLIS-transduced CB HSPCs upregulate FOLR1 expression indicating that FOLR1 expression is causally associated with CBF/GLIS expression (Figure 1D).Given cell surface expression of FOLR1 on CBF/GLIS AML cells, we next investigated the preclinical efficacy of targeting FOLR1 using FOLR1-directed site-specific antibody-drug conjugate (STRO-002, Sutro Biopharma). In vitro efficacy of STRO-002 was tested against MV4;11 AML cell line engineered to express FOLR1 (MV4;11 FOLR1+, Figure 1D) and CBF/GLIS-transduced CB HPSCs. STRO-002 exhibited high cytotoxicity against MV4;11 FOLR1+ and CBF/GLIS-transduced CB HPSCs with IC50s of 0.1nM and 4.2nM, respectively (Figure 1E). In vivo efficacy of STRO-002 was evaluated in MV4;11 FOLR1+ NSG xenograft models, which showed potent activity that led to complete leukemia clearance after 3 weekly doses of STRO-002 at 2.5 and 5 mg/kg (Figure 1F). The anti-leukemia effects of STRO-002 resulted in significant increase in survival (p=0.002, Figure 1G). STRO-002 showed similar in vivo efficacy in the xenograft mice bearing CBF/GLIS-transduced CB HSPCs (Figure 1H) suggesting highly potent activity of STRO-002 against FOLR1+ AML cells.In this study, we utilize a computational approach to discover AML-restricted targets (high expression in AML, silent in normal) that are previously elusive to identify actionable targets for high-risk CBF/GLIS AML. From this discovery pipeline, we demonstrate FOLR1 to be highly and uniquely expressed in CBF/GLIS AML but not in normal counterparts, providing a strategy to target leukemia cells without impacting normal hematopoiesis. Finally, we demonstrate that targeting FOLR1 with STRO-002 antibody drug conjugate effectively eliminates CBF/GLIS-positive AML in vitro and in vivo, providing a promising approach to eradicate leukemia and improve outcomes in this high-risk AML subtype. [Display omitted] DisclosuresHylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Project Stella: Development and Preclinical Assessment of FOLR1-Directed Chimeric Antigen Receptor T Cells in CBF2AT3-GLIS2/RAM AML

Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML.Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A).Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D).To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G).Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. [Display omitted] DisclosuresHylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Phase I study of mirvetuximab soravtansine (MIRV) and rucaparib for recurrent endometrial, platinum-resistant ovarian, fallopian tube or primary peritoneal cancer

Phase I study of mirvetuximab soravtansine (MIRV) and rucaparib for recurrent endometrial, platinum-resistant ovarian, fallopian tube or primary peritoneal cancer