IgA Fc-folate conjugate activates and recruits neutrophils to directly target triple-negative breast cancer cells

Abstract

Purpose: According to the American Cancer Society, 1 in 8 women in the U.S. will develop breast cancer, with triple-negative breast cancer (TNBC) comprising 15-20% of all breast cancer cases. TNBC is an aggressive subtype due to its high metastatic potential and lack of targeted therapy. Recently, folate receptor alpha (FRA) is found to be expressed on 80% of TNBC with high expression correlating with poor prognosis. In this study, we examined whether binding IgA Fc-folate molecules to FRA receptors on TNBC cells can elicit and induce neutrophils (PMNs), by binding their FcαR1 receptors, to destroy TNBC cells.

Methods: FRA was analyzed on TNBC cells and binding assays were performed using ³H-folate. Fc-folate was synthesized by linking Fc fragments of IgA via amine groups to folate. Binding specificity and antibody-dependent cellular cytotoxicity (ADCC) potential of Fc-folate to FcαR1 were confirmed by measuring PMN adhesion and myeloperoxidase (MPO) release in a cell-based ELISA. Fc-folate binding to FRA-expressing TNBC cells inducing PMNs to destroy these cells was determined using ⁵¹Cr-release and calcein-labeling assays.

Results: Our results demonstrate expression of FRA on TNBC cells at levels consistent with folate binding. Fc-folate binds with high affinity to FRA compared to whole IgA-folate and induces MPO release from PMN when bound to FcαR1. Fc-folate inhibited binding of ³H-folate to TNBC cells and induced significant cell lysis of TNBC cells when incubated in the presence of PMNs.

Conclusion: These findings support the hypothesis that an IgA Fc-folate conjugate can destroy TNBC cells by eliciting PMN-mediated ADCC.

Keywords: Antibody-dependent cellular cytotoxicity; FcαR1; Folate receptor alpha; IgA Fc-folate; Triple-negative breast cancer.

Figure 1.. Fc-folate conjugate triggers neutrophil (PMN) –mediated tumor cell lysis by antibody-dependent cellular-cytotoxicity (ADCC).

Binding of the folate component of the conjugate to its folate receptor (FRA) present on tumor cells allows binding of the Fc fragment to its receptor (FcαR1) on the PMN. This binding will cross-link FcαR1 receptors, triggering ADCC through the release of cytotoxic enzymes resulting in tumor cell death.

Figure 2.. FRA expression and ³H-folate binding in TNBC cells.

(A) Cancer cells were grown to confluence in folate-free RPMI and 10% FBS containing ~0.02 mM (physiological level) of folate (plating medium). FRA message was measured by RT-qPCR. (B) Cancer cells were plated at 2.5 × 10⁵ cells/well in 24-well plates in plating medium and incubated overnight. Binding experiments were performed on ice in cells stripped of folate by washing with cold low pH buffer (pH 3) twice prior to incubation in 61 μCi/ml ³H-folate (100 nM) in PBS for 1 hr. After one hour incubation, cells were washed with PBS to remove unbound folate and remaining ³H-folate eluted using cold low pH buffer and counted using a Perkin Elmer liquid scintillation analyzer. For each cell line, background cpm values from control wells (containing both ³H-folate and 1mM cold folate) were subtracted from wells containing only ³H-folate and resulting cpm values were used to determine nmoles of ³H-folate bound. Results are plotted as average nmoles of ³H-folate bound per one million cells (counted in parallel). Human ovarian cancer cells infected with non-silenced control vector (iGROV1) or shFRA were used to confirm ³H-folate binding specificity. Results represent the mean ± SD of n = 3 independent experiments performed in triplicate.

Figure 3.. Confirmation of Fc-fragmentation by gel electrophoresis.

SDS-PAGE (10% Bis-Tris) results of reduced samples from the IgA digest purified using the human IgA affinity matrix. Fc fragmentation was confirmed showing a band around 30 kd in the elution fraction (Fc fragment) and lack of bands at 25 and 45 kd corresponding to Fab fragments which are present at high levels in the wash fraction (Fab fragment). The presence of heavy and light chains in the elution fraction indicates incomplete digestion of IgA.

Figure 4.. IgA- and Fc-folate conjugates inhibit binding of ³H-folate.

(A) iGROV1 cells were plated at 2.5 × 10⁵ cells/well in plating medium. Binding assays were performed as described in Figure 2 but in the presence of increasing amounts of folate conjugates which compete with ³H-folate for FRA binding. Results represent the mean ± SD of n = 3 independent experiments each performed in duplicate. IC50 values were determined using GraphPad Prism version 6.0. (B) Specificity of ³H-folate and Fc-folate for FRA was demonstrated using FRA-expressing and shFRA-infected iGROV1s as well as normal human epithelial cells in the same binding assay as described in (A) but using 3 μM Fc-folate conjugate and 1 mg/ml whole IgA. Results represent the mean ± SD of n = 3 independent experiments each performed in duplicate where * P<0.01 compared to ³H-folate alone.

Figure 5.. IgA and Fc-folates bind PMNs and induce ADCC.

(A) ELISA plates were triplicate coated overnight with varying concentrations of IgA, Fc fragment, their corresponding folate conjugates or BSA (negative control) prior to addition of BCECF-AM labeled PMNs at 4 × 10⁵ cells/well in plating medium. After incubation for 30 minutes, unattached PMNs were washed away and remaining cells lysed in 2% SDS, transferred to black plates and fluorescence measured (ex: 485/20 and em: 528/20). Results represent the mean ± SD of n = 3 independent experiments performed in triplicate. (B) PMN-induced MPO release was measured in the clarified supernatant collected 6 hours after plating of unlabeled PMNs in the ELISA experiment described in Part A. Results represent the mean ± SD of n = 3 independent experiments performed in triplicate where *P<0.01 compared to samples containing PMN alone.

Figure 6.. TNBC cells bind Fc-folate and trigger cell lysis.

(A) Binding assays were performed in high FRA-expressing TNBC cells as described in Figure 2. Results represent the mean ± SD of n = 3 independent experiments performed in triplicate where * P<0.01 compared to ³H-folate alone. (B) TNBC cells were plated at 1 × 10⁵ cells/well in 96-well plates overnight in low folate RPMI containing 10% and then labeled with ⁵¹Cr (250 μCi/ml) for 4 hours. Excess ⁵¹Cr was washed away and cells were incubated overnight (12-15 hours) in same medium and in co-culture with PMNs in the presence and absence of Fc-folate (3 μM) at a target to effector ratio of 1:60 (T:E). Cell lysis for each cell line was determined by measuring ⁵¹Cr released into the supernatant. For each cell line, resulting cpm values in the supernatant from control wells (TNBC cells with no PMNs) were subtracted from all PMN-containing wells. Results are expressed as percent of maximum control lysis where maximum control lysis of each cell line was determined by lysing additional control wells with 2% SDS. Results represent mean ± SD of n=2 experiments performed in triplicate where * P<0.01 compared to samples containing PMN alone. (C) TNBC cells were plated at 1 × 10⁵ cells/well in 96-well black plates overnight in low folate RPMI containing 10% FBS and then labeled with calcein-AM for 45 minutes. Excess dye was washed away with PBS and cells were incubated for 8 hours in co-culture with PMN and in the presence and absence of Fc-folate (3 μM) at a target to effector ratio of 1:50 (T:E). After 8 hours, wells were washed and remaining adherent cells were lysed with 2% SDS and detected by measuring calcein fluorescence (ex: 485/20 and em: 528/20). Cell viability for each cell line is expressed as percent of control where control represents the TNBC cells without PMN treatment. Results are the mean ± SEM of n=3 independent experiments performed in triplicate where *P<0.01 compared to samples containing PMN alone.