Follicle-Stimulating Hormone Receptor as a Target in the Redirected T-cell Therapy for Cancer

The follicle-stimulating hormone receptor (FSHR) is a G-protein–coupled receptor that is primarily expressed in the granulosa cells of the ovary and the Sertoli cells of the testis of adults (1, 2). Although the role of FSHR expression in the development and progression of ovarian cancer is not fully understood, one hypothesis currently being tested is that gonadotrophin signals, including those transmitted through FSHR, may be involved in the transformation and progression of normal ovarian surface epithelium (OSE) to neoplastic OSE. Indeed, ovarian cancer is more common in conditions in which gonadotropin levels are elevated, such as in postmenopausal women (3). FSHR mRNA can be detected in ovarian cancer (4), although protein detection by IHC has been challenging and requires further investigation. In vitro results from overexpressing cell lines suggest that the protein may play a role as a stimulator of cancer cell proliferation (5), and treatment of some ovarian cancer cells with FSH, the ligand for FSHR, can result in growth stimulation in a dose- and time-dependent manner in vitro (6), which suggests that FSHR-targeted therapy could be effective in ovarian cancer. Furthermore, recent biodistribution studies from Radu and colleagues show ubiquitous ectopic expression of FSHR in the tumor-associated blood vessels of various tumor types, including prostate adenocarcinoma, urothelial carcinoma, renal cell carcinoma, and ovarian cancer, without detectable expression in surrounding healthy vasculature (7, 8), although this finding has yet to be confirmed by others. The biologic significance of its expression in tumor blood vessels remains unknown, but the FSHR expression level correlates with the response to antiangiogenic therapies and suggests that anti-FSHR therapy could be used broadly as a tumor vasculature disruption agent across solid malignancies. Thus, FSHR expression by both ovarian cancer cells and its tumor-associated vasculature (4, 9) may provide a unique opportunity to deliver synergistic effects from a single, anti-FSHR therapy, thus making FSHR targeting particularly attractive in ovarian cancer.

T-cell targeting of antigens on the cancer cell surface can now be achieved through the genetic modification of primary T cells to express a chimeric antigen receptor (CAR). Adoptive immunotherapy using CAR T cells specific for CD19 has been shown to induce durable remissions in hematologic malignancies (10); however, targeting of solid tumors has not yet achieved parallel success. Multiple factors may limit the efficacy and safety of this form of therapy in solid tumors, one of which is the risk associated with on-target, off-tumor toxicity. For example, CD19 CAR therapy induces profound B-cell aplasia through coordinate elimination of healthy and malignant CD19⁺ B lymphocytes (10), and transfer of ERBB2 CAR T cells following lymphodepleting chemotherapy was associated with rapid-onset respiratory arrest and death, presumably via cytokine release by CAR T cells triggered by the recognition of low levels of ERBB2 on lung epithelial cells (11). The potential for on-target, off-tumor toxicity from CAR T-cell therapy has limited more widespread translation of this promising therapy to other malignancies. Still, the targeting of bona fide cancer/testis antigens, such as NY-ESO-1, with expression restricted to the ovary, male germ cells in the testis, and malignant cells but not somatic tissue, has paved the path for safe and effective gene-modified T-cell therapy (12). In the absence of tumor-restricted antigens, other antigens that are expressed by tumor cells and cells of healthy organs with nonessential function for patient survival, such as reproductive organs, may be attractive targets for CAR-based immunotherapy. FSHR is a member of this class of antigens. The restricted expression pattern of abundant FSHR in ovarian cancer (2, 4, 13), tumor vessels (7, 8), and gonadal tissues (1), relative to that found in adult somatic tissues (14), lowers the risk for on-target, off-tumor toxicity and makes FSHR an appealing target for T-cell–based immunotherapy.

In this study, we developed a strategy for treatment of human ovarian cancer via the redirection of primary human T cells against FSHR and validated for the first time the suitability of FSHR as a target of T-cell–based immunotherapy. Anti-FSHR immunoreceptors(FSHR-IR), specific for human and mouse FSHR, were created using peptides derived from the natural FSHR ligand, FSH, for redirected T-cell activity. Herein, we report the results of in vitro testing of FSHR-redirected T cells for recognition and reactivity against human FSHR-expressing ovarian cancer cells, and the in vivo efficacy of anti–FSHR-IR T-cell therapy in a xenograft model of human ovarian cancer. This study serves as a foundation for the future development of FSHR-targeted immunotherapies.

Amino acid sequences encoding FSH peptides, including flanked 3′-BamHI and 5′-NheI restriction sites, were synthesized. DNA products were digested with BamHI and NheI enzymes and ligated into pELNS, a third-generation self-inactivating lentiviral expression vector, containing human CD8alpha-hinge and transmembrane region fused to CD3z or CD28–CD3z signaling endodomains, under an EF-1α promoter. Resulting constructs were designated anti-FSHR IR-z and anti–FSHR-28z, respectively.

High-titer replication-defective lentiviral vectors were produced and concentrated as described previously (15). Briefly, 293T cells were transfected with pVSV-G (VSV glycoprotein expression plasmid), pRSV.REV (Rev expression plasmid), pMDLg/p.RRE (Gag/Pol expression plasmid), and pELNS transfer plasmid using Lipofectamine 2000 (Invitrogen). The viral supernatant was harvested at 24 and 48 hours after transfection.

Primary human CD4⁺ and CD8⁺ T cells isolated from healthy volunteer donors were purchased from the Human Immunology Core at University of Pennsylvania (Philadelphia, PA), activated, and transduced with lentiviral vectors as described previously (16).

The immortalized normal fetal renal 293T cell line and human cell lines used in immune-based assays, including CaOV3, CaOV434, SKOV3, and OVCAR3, were purchased from and maintained according to the ATCC. The murine mesothelioma cell line AE17 and the murine ovarian cancer cell line ID8 were provided by Steven M. Albelda and George Coukos at the University of Pennsylvania, respectively. Cells were transfected with firefly luciferase as described previously (16).

APC-Cy7 mouse anti-human-CD3, FITC–anti-human-CD4, APC–anti-human-CD8, PE–human-CD45, and APC–human-CD69 antibodies were purchased from Biolegend. FSHR expression was detected using clone6266717 (R&D Systems). T-cell transduction was measured by GFP transgene expression. 7-Aminoactinomycin D (AAD; Biolegend) was used to assess viability. For in vivo T-cell quantification, 50 μL blood was obtained from mice via retro-orbital bleeding and labeled for human CD45, CD3, and CD8. Cell numbers were quantified using BD TruCount tubes according to the manufacturer's instructions. Flow cytometry data were analyzed using FlowJo software.

Total RNA was extracted from 5 × 10⁶ viable tumor cells using an RNeasy Mini kit (Qiagen). RNA quantity and quality were verified using a NanoDrop 2000 spectrophotometer (Thermo). cDNA was generated from 1 μg total RNA using the High-Capacity-RNA-to-cDNA kit (Applied Biosystems). The human FSHR was PCR amplified using the following primers: 5′-CTCACCAAGCTTCGAGTCATCCAA-3′ and 5′-GCTCATCTAGTTGGGTTCCATT-3′ (Gene ID: 2492), mouse FSHR 5′-GGGATCTGGATGTCATCACT-3′ and 5′-GGAGAACACATCTGCCTCTA-3′ (Gene ID: 14309).

Cytokine release assays were performed by coculture of 1 × 10⁵ FSHR-IR T cells with FSHR-expressing CaOV3 and FSHR⁻ 293T cells, or mouse FSHR-expressing ID8, as described previously (16). After 16 hours, coculture supernatants were assayed for the presence of cytokines using an ELISA kit (Biolegend) and Cytokine Bead Array (BD Biosciences) according to the manufacturer's instructions.

fLuc-transduced targets were plated at 1 × 10⁴ per well in triplicate. T cells were added at the indicated effector:target (E:T) ratios. Cocultures were incubated overnight in phenol-free conditioned medium. The Extended-Glow Bioluminescent Reporter Gene Assay (Applied Biosystems) was used to measure residual luciferase activity from remaining targets, and lysis was calculated as follows: Percent lysis = 100 − [(average signal from T-cell–treated wells)/(average signal from untreated target wells) × 100].

NOD/SCID/γ-chain^−/− (NSG) mice were bred, treated, and maintained under pathogen-free conditions in-house under the University of Pennsylvania IACUC–approved protocols. Six- to 12-week-old female mice were purchased from the University of Pennsylvania Stem Cell and Xenograft Core, and 5 × 10⁶ CaOV3-fLuc tumor cells were inoculated s.c. (5 mice per group). Twenty and 25 days later, mice were injected i.v. with 6 × 10⁶ T cells. Tumor growth was assessed by weekly caliper measurements. Tumor volume was calculated using the following formula: V = 1/2 (length × width²), where length is the greatest longitudinal diameter and width is the greatest transverse diameter.

The Student t test was used to evaluate differences in T-cell–specific cytolysis and cytokine secretion. GraphPad Prism 4.0 (GraphPad Software) was used for the statistical calculations. P < 0.05 was considered statistically significant.

The chimeric immunoreceptor was constructed for the redirection of primary human T cells against FSHR-expressing targets. FSH, the natural ligand for FSHR, was selected as the binding moiety for the immunoreceptor. FSH is a heterodimeric glycoprotein gonadotropin, consisting of α and β subunits, and several peptides derived from either the FSH beta subunit alone or mixed β/α subunits are reported to specifically bind to FSHR, as high affinity FSHR agonists or antagonists (16). On the basis of their predicted binding affinity for FSHR, we selected multiple anti-FSHR peptides for the construction of a panel of chimeric immunoreceptors shown in Table 1. Some FSHR-binding sites have been reported, including amino acid fragments 33–53, 51–65, and 81–95 of the FSHβ-chain (17–19). In particular, FSHβ 33–53 peptide appears to be functional when covalently attached to nanoparticles, providing high-selectivity nanoparticle delivery to FSHR-expressing ovarian tumors (20). FSH peptides were cloned into previously validated lentiviral constructs with the intracellular CD3z domain alone or with the CD28 costimulatory signaling domain in tandem (16) and referred to as anti–FSHR-IR-z and anti–FSHR-IR-28z (Fig. 1A). We reproducibly achieved a high transduction efficiency of primary human T cells, as measured by coexpression of GFP (65%–80%; Fig. 1B). Transduction efficiencies of all anti–FSHR-IR constructs were essentially identical.

First, established human ovarian cancer cell lines were characterized for cell-surface FSHR expression. FSHR was detected on CaOV3 and CaOV434 cancer cells, whereas OVCAR3, SKOV3, and 293T (Fig. 1C), as well as OVCAR5 and OVCAR 8 (not shown), cancer cells lacked detectable FSHR expression. FSHR expression in CaOV3 and CaOv434 was confirmed by PCR using RT-PCR primers (Fig. 1D). The 18S was used as an internal control (Supplementary Fig. S1). To evaluate the FSHR-specific response of anti–FSHR-IR T cells, the panel of anti–FSHR-IR T cells was cocultured with FSHR⁺ or FSHR⁻ cells overnight, and supernatants were subsequently assayed for the secretion of the cytokine, IFNγ. Anti–FSHR-IR-28z, comprised of extracellularly expressed peptide 33–53β, agonist A, and antagonist A, produced the highest levels of IFNγ following coculture with FSHR-expressing CaOV3, but not against FSHR⁻ 293T cells (Fig. 2A). Anti-FSHR 81–95β IR-28z and anti-FSHR α+β IR-28z (α-chain + β-chain of FSH) produced much lower levels of IFNγ against FSHR⁺ targets, associated with predicted lower affinity of those constructs to FSHR. First-generation anti–FSHR-IR-z T cells also secreted IFNγ in a similar pattern (data not shown). Similarly, when tested for immunoreactivity against the murine FSHR-expressing cell line ID8, anti-FSHR 33–53β-28z, anti-FSHR 81–95β-28z and anti-FSHR agonist A-28z IR T cells all exhibited moderate and specific immune recognition (Fig. 2B), consistent with the high (93%) homology between human and mouse FSHRs and known binding of human FSH, and FSH-derived peptides, to murine FSHR (21). This observation is of special interest, given the opportunity to address potential, but unpredicted, toxicity issues associated with targeting of FSHR in preclinical models. Notably, none of the anti–FSHR-IR constructs tested were capable of recognizing immobilized recombinant human FSHR protein commercially produced from Escherichia coli (Fig. 2C), consistent with previous reports demonstrating that multiple posttranslational modifications of both FSHR and FSH are required for proper folding and binding of the ligand to the receptor (22).

Anti-FSHR 33–53β-28z and anti-FSHR agonist A-28z IR constructs were selected for further evaluation, based on their high immunoreactivity against FSHR⁺ tumor cells. We first determined whether anti–FSHR-IR T cells that encounter FSHR antigen release T_H1-type cytokines other than IFNγ, as has been reported for conventional scFv-based CARs (16). As shown in Fig. 3A, both anti-FSHR 33–53β-28z and anti-FSHR agonist A-28z IR T cells produce multiple proinflammatory cytokines, including TNFα, MIP1α, and IL2, when stimulated with FSHR-expressing CaOV3 or ID8 cell lines. This is in line with results from previous studies using CARs with a CD28 costimulatory domain (23, 24). IFNγ and TNFα production was significantly lower in response to ID8 cells, with almost negligible levels of IL2 detected. This may be due to a lower affinity of the anti–FSHR-IRs for mouse FSHR than human FSHR protein, or antigen density, as our antihuman antibody was unable to detect murine FSHR. Anti–FSHR-IR T cells did, however, specifically upregulate cell-surface expression of CD69, an activation marker, when cocultured with FSHR⁺ CaOV3 or ID8 cells. FSHR-deficient cell lines did not affect the CD69 level, confirming antigen-specific reactivity against both mouse and human FSHR⁺ cell lines. Control GFP and anti-FSHR 51–65β-28z T cells did not respond to either cell line (Fig. 3B). Collectively, these data indicate that anti–FSHR-IRs can confer T cells with the capacity to specifically recognize human and mouse FSHR protein expressed on the tumor cell surface.

To evaluate the lytic proficiency of anti–FSHR-IR T cells, we first engineered FSHR⁺ and FSHR⁻ target cell lines to constitutively express firefly luciferase. Following overnight coculture of target cells with gene-engineered T cells, the percentage of specific lysis was calculated on the basis of a residual luciferase signal. Both anti-FSHR 33–53β-28z and anti-FSHR agonist A-28z IR T cells showed dose-dependent lysis of FSHR⁺ targets (Fig. 3C). As observed in IFNγ release assays, anti–FSHR-IR T cells also exhibited specific lytic activity against the mouse FSHR⁺ ID8 cell line.

To assess the antitumor effect of anti-FSHR T cells in vivo, we established human ovarian cancer xenografts in NOD/SCID/IL2Rγ^−/− (NSG) mice by s.c. flank injection of the CaOV3 cancer cell line and then administered two i.v. injections of gene-engineered T cells at days 20 and 25, similar to our previous studies (15, 24). Consistent with their in vitro function, both anti-FSHR 33–53β-28z and anti-FSHR agonist A-28z IR T cells mediated the suppression of established tumor outgrowth that was statistically superior to the control, GFP-engineered T-cell–treated group, which provided no benefit (Fig. 4A). Because objective clinical response to T-cell transfer therapy is often associated with the persistence of T cells after infusion (25), we measured peripheral blood for the continued persistence of engineered human T cells in vivo via TruCount bead-based counting (Fig. 4B). Consistent with the antitumor response, mice treated with anti-FSHR 33–53β-28z or anti-FSHR agonist A-28z IR T cells had increased peripheral blood CD3⁺ T-cell counts compared with controls at 3 and 5 weeks after T-cell infusion, although only at the level of statistical significance in the anti-FSHR agonist A-28z IR T-cell group. No overt immune-related toxicity or immunopathology was observed.

In summary, we have successfully exploited a novel therapeutic antigen, the FSHR protein, expressed on the tumor cell surface, to deliver T-cell–based immune therapy to patients with confirmed expression of FSHR in their tumor. Given its highly restricted, abundant expression in cancer and gonadal tissues, the rationale for FSHR targeting in solid human malignancy is strong. FSHR may also serve as a target for cancer-associated vessel disruption (7, 8), although we were unable to detect FSHR protein in the blood vessels of human or mouse cancers by immunohistochemical analysis using commercially available antibodies. Possible risks associated with on-target/off-tissue toxicities appear limited. FSHR-deficient mice are vital but infertile with decreased size of ovaries and uterus (26), suggesting possible toxicities may be restricted to reproductive organs. However, in the scenario of a patient diagnosed with ovarian cancer, these organs are virtually nonessential and often surgically removed. In contrast, male FSHRKO mice exhibit reduced numbers of spermatocytes, which would suggest possible targeted toxicity against testis, an immunoprivileged organ (27). Our study shows that anti-FSHR IR T cells are capable of targeting and killing FSHR-expressing cancer cells, resulting in significant suppression of FSHR-expressing tumor outgrowth in vivo. Our findings warrant the examination of other molecules for FSHR targeting to create higher-affinity anti-FSHR chimeric immunoreceptors. Furthermore, the activity of peptide-based anti-FSHR-IRs may also be improved by optimization of other components of the immunoreceptor including modification of the hinge length (28), enhancement of affinity of peptide, or the use of scFv-based immunoreceptors, although such peptides and scFvs have not been described. Moving forward, it will be important to test this FSHR-targeted therapy in tumor models where FSHR is exclusively expressed by tumor blood vessels, compared with simultaneous expression on both tumor cells and its associated vasculature. This is of special interest given its potential for application not only in ovarian cancer but also across many solid cancer types.

No potential conflicts of interest were disclosed.

Conception and design: K. Urbanska, C. Stashwick, D.J. Powell Jr

Development of methodology: K. Urbanska, C. Stashwick, D.J. Powell Jr

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Urbanska, C. Stashwick, M. Poussin, D.J. Powell Jr

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Urbanska, M. Poussin, D.J. Powell Jr

Writing, review, and/or revision of the manuscript: K. Urbanska, C. Stashwick, D.J. Powell Jr

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Urbanska, M. Poussin, D.J. Powell Jr

Study supervision: K. Urbanska, D.J. Powell Jr

Support for imaging and animal studies was provided by the Small Animal Imaging Facility and Stem Cell/Xenograft Core at the University of Pennsylvania, respectively. K. Urbanska is now employed by Janssen Research & Development, LLC, Oncology Discovery Research, Spring House, PA 19477.

This work was supported by grants from the NIH (RO1-CA168900; D.J. Powell Jr) and a kind gift from the Bethesda Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.