# FcγR3A polymorphism influences natural killer cell activation and response to anti-PD-L1 (avelumab) in gestational trophoblastic neoplasia

Adrien Msika, Virginie Mathias, Marina Boudigou, Mathilde Chambon, Valérie Dubois, Touria Hajri, Jean-Pierre Lotz, Md, Jérôme Massardier, Pierre Descargues, Laurence Gladieff, et al.

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## ► To cite this version:

Adrien Msika, Virginie Mathias, Marina Boudigou, Mathilde Chambon, Valérie Dubois, et al. FcγR3A polymorphism influences natural killer cell activation and response to anti-PD-L1 (avelumab) in gestational trophoblastic neoplasia. American Journal of Obstetrics and Gynecology, 2024, 25 (1), pp.244. 10.1016/j.ajog.2024.09.115. hal-04751244

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# FcγR3A polymorphism influences natural killer cell activation and response to anti-PD-L1 (avelumab) in gestational trophoblastic neoplasia

Adrien Msika, MSc; Virginie Mathias, MS; Marina Boudigou, PhD; Mathilde Chambon, MS; Valérie Dubois, MD; Touria Hajri, MS; Jean-Pierre Lotz, MD, PhD; Jérôme Massardier, MD; Pierre Descargues, MD; Laurence Gladieff, MD; Florence Joly, MD, PhD; Coriolan Lebreton, MD; Delphine Maucort-Boulch, MD, PhD; Sylvie Bin, MD; Pascal Rousset, MD, PhD; Fabienne Allias, MD; Lucie Gaillot-Durand, MD; Mojgan Devouassoux-Shisheboran, MD, PhD; Nicolas Lemaitre, MS; Nadia Alfaidy, PhD; Carole Langlois-Jacques, MS; Marine Alves-Ferreira, MD; François Golfier, MD, PhD; Benoit You, MD, PhD; Olivier Thaunat, MD, PhD; Pierre-Adrien Bolze, MD, PhD; Alice Koenig, MD, PhD

**BACKGROUND:** Low-risk gestational trophoblastic neoplasia are currently receiving monochemotherapy as first-line therapy. In the case of a resistance, a second-line monochemotherapy or polychemotherapy is proposed. As an alternative to these toxic and historic chemotherapy agents, the efficacy of the anti-PD-L1 monoclonal antibody (avelumab) was assessed in the TROPHIMMUN phase II trial Cohort A. Avelumab yielded a 53% cure rate with an acceptable tolerance profile, including normal further pregnancy and delivery. Beyond the blockade of PD-1/PD-L1 interactions, avelumab effect could rely on the induction of antibody-dependent cell-mediated cytotoxicity mediated by FcγR3A-expressing natural killer cells.

**OBJECTIVE:** This translational study aimed at testing whether antibody-dependent cell-mediated cytotoxicity is involved in avelumab efficacy on gestational trophoblastic neoplasia and if FcγR3A affinity polymorphism could help predicting the response to avelumab in gestational trophoblastic neoplasia.

**STUDY DESIGN:** The expression of PD-L1 by the tumor and the phenotype of natural killer cells infiltrating gestational trophoblastic neoplasia were verified by performing transcriptomic and proteomic analyses. Then, JEG-3 choriocarcinoma cells were cocultured with human natural killer cells in the presence and absence of avelumab. The impact of FcγR3A functional polymorphism was assessed on the activation status of natural killer cells and the viability of JEG-3 choriocarcinoma cells. Finally, the data from TROPHIMMUN trial were re-analyzed to determine the

impact of the FcγR3A polymorphism of patients on their response to avelumab.

**RESULTS:** We confirmed that FcγR3A+ natural killer cells infiltrated PD-L1-expressing gestational trophoblastic neoplasia. In vitro, avelumab-coated JEG-3 choriocarcinoma cells induced natural killer cell activation, which promoted the destruction of JEG-3 cells. Natural killer cell activation was abolished when the Fc portion of avelumab was removed, demonstrating the importance of Fcγ receptor in this process. Using this model of antibody-dependent cell-mediated cytotoxicity, we demonstrated that high-affinity FcγR3A polymorphism on natural killer cells was associated with better in vitro response to avelumab. In line with this result, patients from the TROPHIMMUN trial homozygous for the high-affinity FcγR3A polymorphism had better clinical response to avelumab.

**CONCLUSION:** Our work demonstrates that antibody-dependent cell-mediated cytotoxicity contributes to the therapeutic effect of avelumab in gestational trophoblastic neoplasia and that the individual patient response is impacted by the FcγR3A polymorphism. The FcγR3A polymorphism could be used as a biomarker to identify patients diagnosed with monochemoresistant gestational trophoblastic neoplasia who are most likely to respond to avelumab.

**Key words:** anti-PD-L1, antibody-dependent cell-mediated cytotoxicity, avelumab, FcγR3A polymorphism, gestational trophoblastic neoplasia, immunotherapy, NK cells

## Introduction

Gestational trophoblastic neoplasia (GTN) encompass a range of invasive conditions, including choriocarcinoma,

invasive mole, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. GTN may arise after 15% of complete moles and after about 1 per 67,000 to 1 per 100,000 term deliveries.¹,² Patients with low-risk GTN, defined by an International Federation of Gynecology and Obstetrics score of ≤6, typically undergo monochemotherapy, achieving cure rates of 30% to 75%.³,⁴ Patients who do not respond to first-line monochemotherapy are treated with second-line monochemotherapy, surgery, or polychemotherapy, such as the Etoposide, Methotrexate, Actinomycin-

D, Cyclophosphamide, Oncovin (Vincristine; EMA-CO) regimen (etoposide, methotrexate, and actinomycin D alternating with cyclophosphamide and vincristine).³,⁵ Surgery often implies hysterectomy, and EMA-CO is associated with potential severe hematological treatment-related adverse events and potential long-term early menopause and second malignancies.⁶ Therefore, developing new therapeutic approaches with similar efficacy but fewer adverse events is crucial.

Our team and others have reported a strong and ubiquitous PD-L1 expression

**Cite this article as:** Msika A, Mathias V, Boudigou M, et al. FcγR3A polymorphism influences natural killer cell activation and response to anti-PD-L1 (avelumab) in gestational trophoblastic neoplasia. Am J Obstet Gynecol 2025;232:381.e1-11.
## AJOG at a Glance

### Why was this study conducted?

Avelumab is an effective second-line option for mono-chemotherapy-resistant gestational trophoblastic neoplasia (GTN), curing 53% of patients. There is a need to better understand the mechanisms of action of avelumab in GTN in order to identify patients with the best chance of response.

### What are the key findings?

This study shows that avelumab can induce antibody-dependent cell-mediated cytotoxicity mediated by FcγR3A-expressing natural killer (NK) cells and that this contributes to the therapeutic effect of avelumab in GTN. We also demonstrate that high-affinity FcγR3A polymorphism is associated with improved NK cell response in vitro and better clinical response to avelumab in patients with GTN.

### What does this study add to what is already known?

The FcγR3A polymorphism could be used as a biomarker to identify patients diagnosed with monochemoresistant GTN who are most likely to respond to avelumab.

by malignant gestational trophoblast cells.⁷,⁸ In line with this, the phase II TROPHIMMUN trial tested the anti-PD-L1 antibody (avelumab) in 15 patients with monochemoresistant low-risk GTN, demonstrating a 53% cure rate.⁹ These results indicate that some patients may respond better to avelumab than others.

The efficacy of avelumab might be influenced by its mechanisms of action. Avelumab blocks the PD-L1/PD-1 interaction, restoring anti-tumor immunity primarily through T cells. To kill tumor cells, CD8+ T cells must recognize antigenic peptides presented by cognate human leukocyte antigen (HLA) class I molecules on tumor cells. In GTN, the extent of this is questionable as trophoblasts are thought to express few classical HLA class I molecules.¹⁰ Avelumab may also facilitate the recruitment of NK cells, which represent a predominant population at the tumor border in gestational trophoblastic diseases,¹¹ to target tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) via FcγR3A.¹² Interestingly, a single-nucleotide polymorphism (SNP) in FcγR3A (rs396991) which enhances FcγR3A's binding capacity to IgG is described.¹³

This study aims to determine if ADCC is a mechanism of action of avelumab in patients with GTN and if identifying FcγR3A polymorphism at diagnosis can

predict which patients will respond better to avelumab.

## Materials and methods
### Patient selection and study design

The study included 15 patients with monochemoresistant GTN from the TROPHIMMUN phase II clinical trial (NCT03135769) (cohort A)⁹ and one additional patient treated with avelumab under compassionate use. They received avelumab 10 mg/kg intravenously every 2 weeks until human chorionic gonadotropin (hCG) normalization according to each laboratory serum threshold, followed by 3 consolidation cycles. The study was conducted according to French biomedical research and the Declaration of Helsinki. All patients provided informed consent for the use of their clinical data and biological samples in research.

### Cell preparation and cultures

The JEG-3 cell line was obtained from DSMZ (#ACC463, Braunschweig, Germany) and cultured in Ham's F12K medium (ThermoFisher, Courtaboeuf, France) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µM streptomycin. They were used between passages 1 and 15.

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient centrifugation (Eurobio,

Courtaboeuf, France). When stipulated, PBMCs were frozen in human albumin 80% and dimethyl sulfoxide 20%. NK cells were then purified (>80%) using magnetic enrichment kits (EasySep Human NK Cell Enrichment Kit, Stemcell, Grenoble, France) and cultured overnight at 37°C in complete RPMI medium (RPMI Glutamax with 10% FBS, 100 U/mL penicillin, 100 µM streptomycin) supplemented with 50 ng/mL of recombinant human IL-15.¹⁴

### Flow cytometry

JEG-3 were stained with anti-PD-L1 AF488 (clone MIH1, 5 µL; Thermofischer), anti-PD-1 APC (clone EH12.2H7, 5 µL, Biolegend), anti-HLA-G PE (clone 87 G, 5 µL, Thermofischer), or corresponding isotype controls. In some cases, cells were pre-incubated with avelumab (Merck KGaA, Darmstadt, Germany, 300 µg/mL) or IgG1 isotype control or F(ab')₂ of avelumab (90 µg/mL) for 30 minutes at 4°C.

NK cells were stained with anti-CD56 PE-Texas-Red (clone NCAM16.2, 5 µL; BD Biosciences, Le Pont de Claix, France), anti-CD3 BV510 (clone SK7, 5 µL; BD Biosciences), and additional markers such as anti-CD16 APC (clone 3G8, 5 µL; BD Biosciences), anti-ILT2 AF700 (clone HP-F1, 5 µL; Thermofischer) or anti-KIR2DL4 PE-Cy5 (clone mab#33, 5 µL; Thermofischer), anti-PD-1 BV421 (clone MIH4, 5 µL; BD Biosciences), or corresponding isotype controls. For PD-1, CD8 T cells incubated with soluble anti-CD3 (0.5 µL/1 million cells) overnight were used as positive control.

Data were acquired using an LSR FORTESSA flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.7.2 (Tree Star Inc, Ashland, OR, USA).

### NK cell activation in vitro

JEG-3 were treated with avelumab, IgG1 isotype control, or avelumab-F(ab')₂, then co-cultured with NK cells at a ratio of 1:1. Anti-CD107a FITC (clone eBioH4A3, 5 µL; Invitrogen; ThermoFisher, Courtaboeuf, France) was added at the start of the co-culture. After 1 hour, GolgiStop (BD Biosciences) was
added. After 4 hours of co-culture, the cells were harvested and stained with anti-CD3 BV 510 (clone SK7, 5 µL), anti-CD56 PE-Texas-Red (clone NCAM16.2, 5 µL), anti-CD107a FITC, and a viability dye (Fixable Viability Dye, 1/1000; Invitrogen). The cells were then fixed, permeabilized (Cytofix/Cytoperm fixation/permeabilization kit; BD Biosciences), and stained with anti-interferon-γ PE (clone 4S.B3, 5 µL; eBiosciences; ThermoFisher).

## Analysis of JEG-3 viability

JEG-3 were treated with avelumab or IgG1 isotype control, seeded at 1.10⁴ per well, and incubated for 24 hours. Purified NK cells primed with IL-15 were then added at different effector-to-target ratios (1:1, 5:1, 10:1, 20:1). JEG-3 viability was monitored over 5 hours using the xCEL-Ligence RTCA SP instrument (ACEA Biosciences, San Diego, CA, USA). JEG-3 viability in the avelumab condition was normalized to the IgG1 isotype control at the same ratio for each donor.

## FcγR3A genotyping

DNA was isolated from PBMCs using the chemagic DNA blood kit (Perkin Elmer, Villebon-sur-Yvette, France) according to manufacturer's instructions. Genotyping of FcγR3A-V/F158 (rs396991) was carried out using TaqMan SNP genotyping Assay and TaqMan Universal PCR Master Mix.

## Transcriptomic data analysis

To assess PD-1, PD-L1, and HLA-G mRNA expression in GTN, transcriptomic data from 53 choriocarcinoma samples were analyzed as described previously (study NCT03488 901).¹⁵ In brief, macrodissection was employed to exclude peritumoral and necrotic tissues from the samples. RNA was extracted from the formalin-fixed paraffin-embedded (FFPE) samples. A total of two to six 5 µm slides were utilized. The slides were initially dewaxed with two baths of D-limonene (2 minutes) and a bath of absolute ethanol (2 minutes), and RNA extraction was subsequently conducted using a High Pure FFPET RNA Isolation Kit (Roche, Switzerland, #06483852001).

For immune infiltrate assessment in GTN, the CIBERSORT algorithm was used to analyze the public data set GSE135727.¹⁶ A custom signature matrix was generated using dataset GSE97217¹⁷ to estimate the proportion of decidual and peripheral NK cell subsets.

## Pathology

HLA-G, PD-1, and PD-L1 expressions were detected by immunohistochemistry (IHC) using FFPE specimens. Primary antibodies used were against HLA-G (1/50, clone 4H84; Abcam, Cambridge, United Kingdom), PD1 (1/100, clone NAT105; Abcam), or PD-L1 (0.25 µg/mL, clone MKP1A07310; Merck KGaA). The positive control for HLA-G and PD-L1 staining was a normal placenta, while healthy lymph node sections were used as a control for PD-1 staining. Slides were counterstained with hematoxylin and visualized using the BenchMark XT (Ventana Roche).

For immunofluorescence, FFPE tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Tissue sections were blocked and then incubated with anti-CD56 (1/75, clone E7X9M; Cell Signaling Technology) and anti-CD16 (1/100, clone 2H7; Leica Biosystems, Wetzlar, Germany) antibodies. Secondary antibodies (AF488 goat anti-mouse IgG and AF546 goat anti-rabbit IgG [1/1000, ThermoFisher]) were incubated for 1 h. Nuclei were stained with NucBlue reagent (ThermoFisher). Validation of the specificity of anti-CD16 and CD56 antibodies was carried out on healthy lymph node sections. Imaging was performed using the Axio Imager Apotome fluorescence microscope with Zen system 2012 software (Carl Zeiss Microscopy, Oberkochen, Germany).

Negative controls were performed with an isotype control for all experiments.

## Statistical analysis

Data were expressed as mean ± SD. Differences between groups were evaluated using the Mann-Whitney unpaired rank test, Wilcoxon matched-pairs signed rank test, or Kruskal-Wallis comparison test. Statistical analyses

were performed using Prism software (Version 8.1.1; GraphPad Software Inc., La Jolla, CA).

## Results

### FcγR3A+ NK cells are infiltrating PD-L1-expressing gestational trophoblastic neoplasia

First, the expression of PD-L1 on GTN was investigated. In 53 macrodissected choriocarcinoma samples, a significant expression of PD-L1 mRNA was found (Figure 1, A). PD-L1 protein expression was then confirmed in each trophoblast subtype of 21 choriocarcinoma cases and scored according to the Allred Total Score, which combines intensity and proportion of expression on a scale of 0 to 8.¹⁹ The results showed high levels of PD-L1 expression in both syncytiotrophoblast and cytotrophoblast contingents of all the tumors (Figure 1, B–D). Notably, choriocarcinoma samples also highly expressed the immunoregulatory molecule HLA-G (Figure 1, A, E, and F). Of note, PD-1 expression was not detected in the tumor lesions (Figure 1, A, G, and H).

Then, we aimed to characterize the nature of immune cells infiltrating the tumor lesions since the presence of NK cells expressing FcγR3A in close proximity to tumor cells is a prerequisite for ADCC-induced NK cell activation. For this, we used a publicly available database (GSE135727) containing the transcriptomic data obtained from the total RNA sequencing of 2 choriocarcinomas, 4 epithelioid trophoblastic tumors, and 4 placental site trophoblastic tumors.¹⁶ To estimate the relative leukocyte distribution, the CIBERSORT algorithm was applied to the transcriptomic data.¹⁸ For this, we used the LM22 gene signature matrix that accurately distinguishes 11 major types of leukocytes. The most represented populations were monocytes/macrophages, T cells, and NK cells (Figure 1, I). Of note, among T cells, CD8⁺ T cells were in the minority, representing only 0% to 11% of the total immune cells (Figure 1, I). NK cells represented between 2% and 49% of the immune cells. Interestingly, they were particularly abundant in the choriocarcinoma cases (around 32%) (Figure 1, I).
<caption>FIGURE 1</caption>

FcγR3A+ NK cells are present in the micro environment of PD-L1 + expressing GTN

<figure><img src="image_4.png" /></figure>

A, PD-1, PD-L1, and HLA-G mRNA expression was estimated in 53 choriocarcinoma. B and C, PD-L1 expression was determined after immunohistochemical staining of 21 choriocarcinoma. Representative images are depicted. * Represents choriocarcinoma, + represents normal myometrium, and § represents peri-tumoral infiltrate. D, Quantification of PD-L1 protein expression in diverse trophoblast subtypes, including syncytiotrophoblast (SCT) and villous cytotrophoblast (CT), using the Allred total score (ATS) in 21 choriocarcinomas. E—H, HLA-G and PD-1 expressions were determined after immunohistochemical staining. Representative images are depicted. * Represents choriocarcinoma, + represents normal myometrium, and § represents peri tumoral infiltrate. I, The CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) algorithm¹⁸ was applied to the public bulk transcriptomic data set GSE135727,¹⁶ which corresponds to 4 placental site trophoblastic tumors (PSTT), 4 epithelioid trophoblastic tumors (ETT), and 2 choriocarcinomas (CC). The averaged relative distribution of 11 leukocytes types was compared by the type of Gestational Trophoblastic Neoplasia (GTN). The data obtained for each sample as well as the means for each GTN type are represented. J, CIBERSORT algorithm with
To further characterize the NK cells present in the tumors, we re-analyzed the dataset using CIBERSORT and a custom signature to distinguish decidual from peripheral NK cells (GSE97217).¹⁷ Regardless of the tumor type, the NK cells present were predominantly of peripheral origin (Figure 1, J). The presence of peripheral NK cells expressing FcγR3A in choriocarcinoma infiltrate was confirmed at the proteomic level (Figure 1, K).

These data suggest that FcγR3A+ NK cells are present in peritumor infiltrates of PD-L1 expressing GTN and could thus be involved in avelumab-mediated anti-tumor effects.

## Choriocarcinoma cells trigger NK cell activation in the presence of avelumab

We hypothesized that ADCC-induced NK cell activation could be one of the mechanisms responsible for the therapeutic potential of avelumab in GTN patients. To verify this hypothesis, we developed an in vitro model in which purified NK cells were co-cultured with JEG-3 human choriocarcinoma cells, in the presence of either avelumab or an IgG1 isotype control.

To ascertain that the JEG-3 cell line was an appropriate surrogate for PD-L1+ GTN, the membranous expression of PD-L1 was confirmed by flow cytometry (Figure 2, A). Then, JEG-3 choriocarcinoma cells were incubated with avelumab or isotype control at a concentration similar to patients' blood concentration of avelumab (300 µg/ mL) (Supplemental Figure 1, A).²⁰ In the presence of avelumab, no residual PD-L1 staining was detected (Supplemental Figure 1, A) suggesting that, at this concentration, all PD-L1 molecules on JEG-3 were bound by avelumab. Similar to primary GTN (see Figure 1, A, E-H), JEG-3 cells also

expressed HLA-G but not PD-1 (Figure 2, A).

Then, the expression of FcγR3A+ was evaluated on NK cells, and as expected, nearly all NK cells exhibited high levels of FcγR3A+ (Figure 2, B). In addition, we found that a variable proportion of NK cells expressed ILT2 but not KIR2DL4, which are inhibitory receptors that can interact with HLA-G on GTN (Figure 2, B). Finally, in order not to overlook a potential improvement of NK cell activation through the blocking of the PD-1/PD-L1 axis,²¹ the absence of PD-1 expression on NK cells was confirmed (Supplemental Figure 1, B).

NK cells from 10 healthy volunteers were mixed with JEG-3 that have been pre-incubated with avelumab or its isotype control (Figure 2, C). After 4 hours of co-culture, NK cell activation status (ie, extracellular membranous expression of CD107a and intra-cellular expression of interferon-γ) was assessed at the single-cell level by flow cytometry. In presence of avelumab, NK cells expressed significantly higher levels of both CD107a and interferon-γ (Figure 2, D and E). Taken together, these data confirm that JEG-3 coated with avelumab can trigger NK cell activation, and that this process does not seem to be impeded by the expression of HLA-G on JEG-3.

To demonstrate that NK cell activation in our model was dependent on the binding of their Fcγ receptor (FcγR3A) to the Fc region of avelumab bound to choriocarcinoma cells, we treated avelumab with pepsin. After verifying the ability of avelumab-F(ab')₂ to bind PD-L1 (Supplemental Figure 1, A), NK cell activation status was assessed by flow cytometry after a 4-hour co-culture with JEG-3 in the presence of avelumab-F(ab')₂ (Figure 2, C-E). Similar to what happened in the presence of isotype control, no NK cell activation was found

in the presence of avelumab-F(ab')₂ (Figure 2, D and E).

These data demonstrate that NK cells can be activated by avelumab bound on choriocarcinoma cells through an Fc binding.

## Avelumab-induced NK cell activation damages choriocarcinoma cells

Having shown that avelumab can induce NK cell activation, we wanted to demonstrate its effect on JEG-3. To this end, the death of adherent JEG-3 was monitored by real-time impedance measurement in the co-culture model described above (Figure 2, C). We compared the survival of JEG-3 exposed to NK cells at different effector:target ratios in the presence of avelumab or its isotype control. This in vitro experiment, replicated with 10 different donors, showed that in the presence of avelumab, JEG-3 survival was progressively reduced as the effector to target ratio was increased (Figure 3, A and B).

Thus, these in vitro data support the notion that NK cell activation by avelumab induces choriocarcinoma cell death through the ADCC mechanism.

## FcγR3A polymorphism influences avelumab response in patients with gestational trophoblastic neoplasia

Then, we sought to investigate the validity of our hypothesis in vivo. To this end, the data from the TROPHIMMUN phase II trial (cohort A) were re-analyzed.⁹ As it has been described that FcγR3A polymorphism could modulate response to monoclonal antibodies, in the context of cancers, and we assessed whether it could modulate the response to avelumab of patients with GTN. Of the 16 patients, 31.3% (n=5), 43.7% (n=7), and 25% (n=4) carried no high-affinity alleles (F/F), 1 high-affinity allele (F/V), and 2 high-affinity alleles (V/V),

the "impute cell fractions" module with a custom signature matrix was applied the public bulk transcriptomic data set GSE135727 to estimate the proportions of CD49a+ (decidual NK cells, dNK) and CD49b+ (peripheral NK cells, pNK) NK cells. K, The presence of FcγR3A+ NK cells were determined by double immunofluorescence staining against CD56 and CD16 in choriocarcinoma samples. A representative image is depicted. Arrows indicate double positive cells.

*γδ T cell*, gamma-delta T cell; *CD4*, CD4-positive T cell; *CD8*, CD8-positive T cell; *DC*, dendritic cell; *Eo*, eosinophil; *MC*, mast cell; *Mono/Mφs*, monocyte/macrophage; *PMN*, polymorphonuclear neutrophil.
<caption>FIGURE 2</caption>

JEG-3 choriocarcinoma cells trigger ADCC-induced activation of FcγR3A + NK cells in presence of avelumab

<figure><img src="image_5.png" /></figure>

A, The expression of PD-L1, HLA-G, and PD-1 was assessed on the surface of JEG-3 choriocarcinoma cells. Representative flow cytometry profiles are shown. B, The expression of CD16, ILT-2, and KIR2DL4 was evaluated on NK cells after an overnight stimulation with IL-15 (50 ng/ml). Representative flow cytometry profiles are shown. C-E, Purified NK cells from 10 healthy volunteers were co-cultured with JEG-3 choriocarcinoma cells which have been pre-incubated with either avelumab isotype control (300 µg/mL) or avelumab (300 µg/mL) or F(ab')₂ of avelumab (90 µg/mL). After 4 hours of co-culture, the activation status of NK cells was assessed at the single cell level by flow cytometry. C, Schematic representation of the experimental model. D, Expression of CD107a (LAMP1) on NK cell surface after a 4-hour co-culture. Left panels: representative flow cytometry profiles. Right panel: individual values according to the 3 conditions. ***P<.001; Kruskal-Wallis comparison test. E, Intracellular staining for Interferon γ in NK cells after a 4-hour co-culture. Left panels: representative flow cytometry profiles. Right panel: individual values according to the 3 conditions. ***P<.001; Kruskal-Wallis comparison test.

respectively. In line with our hypothesis, the rate of complete response to avelumab was 40%, 57%, and 75% in the patients with respectively no high-

affinity alleles (F/F), 1 (F/V), or 2 high-affinity alleles (V/V) of FcγR3A (Figure 4, A). However, the number of patients available for analysis was too

low to perform statistical comparison. Therefore, we went on demonstrating that FcγR3A polymorphism impacts patients' NK cell level of activation in the
<figure><img src="image_6.png" /><figcaption>**FIGURE 3**
ADCC-induced NK cell activation is harmful for JEG-3 choriocarcinoma cells</figcaption></figure>

A and B, Purified NK cells from 10 healthy volunteers were co-cultured with adherent JEG-3 choriocarcinoma cells, which have been pre-incubated with either avelumab isotype control (300 µg/mL) or avelumab (300 µg/mL) at different effector:target (E:T) ratios (1:1, 5:1, 10:1, and 20:1). The viability of JEG-3 was assessed by real-time impedance measurement. Real-time impedance data of the experimental co-culture under the avelumab condition were normalized over the isotype control condition for each E:T ratio. A, Representative individual impedance profiles (mean ± standard error) at each E:T ratio for one donor are shown. B, Area under the curves from 10 independent experiments are shown (mean ± standard error). \*\*P<.01; Wilcoxon signed rank test.

presence of avelumab using the in vitro ADCC model described above (Figure 2, C). NK cell activation status was assessed by flow cytometry for patients with available PBMCs (n=13). The NK cells of patients with 2 FcγR3A high-affinity alleles (V/V) (n=4) expressed higher levels of CD107a and interferon-γ than the patients with no high-affinity allele (F/F) (n=5) (Figure 4, B and C).

To strengthen the data obtained from the patients, we conducted additional experiments with 10 healthy volunteers carrying either 2 FcγR3A low-affinity alleles (F/F) or 2 high-affinity alleles (V/V). The results showed that a higher percentage of NK cells with 2 FcγR3A high-affinity alleles (V/V) expressed CD107a and interferon-γ, in the presence of avelumab (Figure 4, D). This increased activation resulted in greater JEG-3 cell death at the various effector:target ratios tested (Figure 4, E–G).

All together, these data suggest that FcγR3A polymorphism may influence ADCC activity mediated by NK cells in patients who received avelumab as treatment for monochemoresistant GTN.

## Comment

### Principal findings

We found that avelumab induces NK cell-mediated ADCC, leading to the killing of PD-L1+ choriocarcinoma cells. Notably, this effect appears unaffected by the expression of the immunomodulatory molecule HLA-G by the tumor. In addition, our work shows that patients with monochemoresistant GTN who possess two high-affinity FcγR3A alleles are more likely to respond to avelumab. This is due to the enhanced ability of their NK cells to be activated and kill tumor cells, suggesting that FcγR3A polymorphism plays a crucial role in mediating the therapeutic efficacy of avelumab.

### Results in the context of what is known

Our findings that avelumab can induce NK cell activation by ADCC in GTN align with previous studies that have reported similar mechanisms of action for avelumab in other PD-L1+ cancers, such as meningiomas and triple-negative breast cancer.²²⁻²⁴ Furthermore, the modulation of avelumab efficacy by

FcγR3A polymorphism in GTN is consistent with numerous studies that have shown a relationship between NK cell ADCC, FcγR3A polymorphism, and clinical responses in various cancers treated with antibodies like rituximab, trastuzumab, and cetuximab.²⁵⁻²⁷

### Clinical implications

Our data suggest that anti-PD-L1 agents such as avelumab, which can induce NK cell activation by ADCC, may be more effective in patients with monochemoresistant GTN. Determining the FcγR3A polymorphism at the time of diagnosis could serve as a biomarker to predict treatment response. This approach could personalize and optimize the therapeutic strategy for patients with GTN, potentially improving clinical outcomes.

### Research implications

For patients with low-affinity FcγR3A, complementary NK cell therapy could be proposed to enhance NK cell activation by ADCC in the presence of avelumab. This approach was recently tested in patients with B-cell non-Hodgkin
<caption>FIGURE 4</caption>

Impact of FcγR3A polymorphism in patients with monochemoresistant GTN treated with avelumab

<figure><img src="image_7.png" /></figure>
lymphoma who received rituximab, high-dose chemotherapy, and autologous stem cell transplantation in combination with ex vivo expanded cord blood NK cells.²⁸ Interestingly, FcγR3A expression on cord blood NK cells was positively associated with the outcome, and the presence of 2 high-affinity alleles for FcγR3A on cord blood NK cells was positively associated with a better outcome.²⁸ Our study, combined with these findings, provides a strong rationale for testing the association of ex vivo expanded NK cells in combination with anti-PD-L1 antibodies for monochemoresistant GTN in patients with low-affinity FcγR3A alleles.

## Strengths and limitations

A major strength of this study is its translational design, incorporating prospective clinical data and confirming findings through in vitro experiments using samples from patients and healthy volunteers. Although the number of patients may seem limited, this study was conducted in one of the largest cohorts of monochemoresistant GTN. By focusing on patients with monochemoresistant GTN, we ensured a homogeneous patient population, excluding those who had received various chemotherapies, which could bias the interpretation of anti-PD-L1 activity. However, this focus resulted in a small sample size, which is a limitation.

## Conclusion

NK cell ADCC and FcγR3A polymorphism are associated with clinical

response in monochemoresistant GTN. Assessing FcγR3A polymorphism at the time of GTN diagnosis may help clinicians predict the clinical efficacy of avelumab in treating monochemoresistant GTN. This conclusion is supported by observations that patients with two high-affinity FcγR3A alleles respond better to avelumab, with their NK cells showing greater activation and killing of choriocarcinoma cells in vitro. Future research should focus on validating these findings in vivo and exploring complementary NK cellular therapies for patients with low-affinity FcγR3A.

## Acknowledgments

We thank Christelle Forcet and Violaine Tribollet from the Institut de génomique fonctionnelle, Lyon, France for their help with xCELLigence experiments. We also want to express our deep gratitude to Nathalie Dufay, Gaelle Cavillon, and Joffrey Ghirardi from NeuroBioTec, Lyon, France thanks of whom the collection of samples was made possible. We also thank Dr Marie Cohen from Geneva University Hospitals for the provision of cell lines. Finally, our grateful thanks are also extended to Aurélie Gaultier and Garance Tondeur from the Pool Biotech/PRTO, Lyon, France. The French Reference Center for Trophoblastic Diseases was accredited by the Institut National du Cancer (INCa) and the European Reference Network (EuraCan G2 sub domain).

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## Author and article information

From the Centre Français de Référence des Maladies Trophoblastiques, Hospices Civils de Lyon, Hôpital Lyon Sud, Pierre Bénite, France (Msika, Hajri, Lotz, Massardier, Descargues, Allias and Gaillot-Durand); Centre international de recherche en infectiologie (CIRI), INSERM U1111, Université Claude Bernard Lyon I, CNRS UMR5308, Ecole Normale Supérieure de Lyon, Lyon, France (Msika, Mathias, Boudigou, Chambon, Thaunat and Koenig); Laboratoire HLA, Etablissement Français du Sang Auvergne-RhoneAlpes, Laboratoire HLA, 111, Decines Charpieu, France (Mathias, Dubois); Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Pôle Onco-Hématologie Hôpitaux Universitaires de l'Est Parisien, Paris, France (Lotz); Service de Gynécologie

Obstétrique, Unité de Diagnostic Anténatal - Hôpital Fémme Mère Enfant, Bron, France (Massardier); Service de Chirurgie Gynécologique et Oncologique, Obstétrique - Hôpital Lyon Sud, Pierre Bénite, France (Descargues, Golfier and Bolze); Département d'Oncologie Médicale, IUCT Oncopole, Toulouse, France (Gladieff); Clinical Research Department, Centre François Baclesse, Caen, France (Joly); Département d'oncologie médicale, Institut Bergonié, Bordeaux, France (Lebreton); ARTiSt Lab, Inserm U1312, Toulouse, France (Lebreton); Université de Lyon, Lyon, France (Maucort-Boulch and Langlois-Jacques); Université Lyon 1, Villeurbanne, France (Maucort-Boulch and Langlois-Jacques); Pôle Santé Publique, Hospices Civils de Lyon (Maucort-Boulch); Service de Biostatistique et Bioinformatique, Lyon, France (Maucort-Boulch); CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Évolutive, Équipe Biostatistique-Santé, Villeurbanne, France (Maucort-Boulch); Hospices Civils de Lyon, Unité Recherche et Epidémiologie Cliniques - Pôle de Santé Publique, Hôpital Lyon Sud, Pierre Bénite, France (Bin, Alves-Ferreira); Université Lyon 1 Faculté de Médecine et de Maïeutique Lyon-Sud Charles Mérieux - Centre Français de Référence des Maladies Trophoblastiques - Hospices Civils de Lyon, Hôpital Lyon Sud - Centre pour l'Innovation en Cancérologie de Lyon (CICLY), EA3738, Pierre Bénite, France (Rousset, Devouassoux-Shisheboran, Golfier, You and Bolze); Service de radiologie. Hôpital Lyon Sud, Pierre Bénite, France (Rousset); Hospices Civils de Lyon, Service de Pathologie - Hôpital Lyon Sud, Pierre Bénite, France (Allias, Gaillot-Durand and Devouassoux-Shisheboran); Interdisciplinary Research Institute of Grenoble, IRIGBiosanté, University Grenoble Alpes, INSERM, CEA, UMR 1292, Grenoble, France (Lemaitre and Alfaidy); Université de Lyon, Lyon, France (Langlois-Jacques); Université Lyon 1, Villeurbanne, France (Langlois-Jacques); Hospices Civils de Lyon, Pôle Santé Publique, Service de Biostatistique et Bioinformatique, Lyon, France (Langlois-Jacques); CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Évolutive, Équipe Biostatistique-Santé, Villeurbanne, France (Langlois-Jacques); Medical Oncology, Institut de Cancérologie des Hospices Civils de Lyon, Centre d'Investigation de Thérapeutiques en Oncologie et Hématologie de Lyon, Hôpital Lyon Sud, Pierre Bénite, France (You); and Service de Transplantation, Néphrologie et Immunologie Clinique, Hôpital Edouard Herriot, Lyon, France (Thaunat and Koenig).

Received July 21, 2024; revised Sept. 23, 2024; accepted Sept. 24, 2024.

P.-A.B. and A.K. contributed equally to this work.

The authors report no conflict of interest.

This study was financially supported by Merck KGaA (CrossRef Funder ID: 10.13039/100009945) and was previously conducted under an alliance between Merck and Pfizer. Merck was not involved in the study design. Merck only reviewed the manuscript for medical accuracy before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors.

Corresponding author: Pierre-Adrien Bolze, MD, PhD. pierre-adrien.bolze@chu-lyon.fr
SUPPLEMENTAL FIGURE

PD-L1 expression in JEG-3 choriocarcinoma cells, PD-1 expression in immune cells and clinical characteristics of patients

<figure><img src="image_8.png" /></figure>

C

FIGO score at enrollment in TROPHIMMUN trial

0-2 0

3-4 5 (31)

5-6 7 (43)

≥ 7 4 (25)

Antecedent pregnancy and pathology

Complete hydatiform mole on curettage specimens 16 (100)

Prior treatment before avelumab

Methotrexate 16 (100)

EMA-CO 1 (6)

Actinomycin D 1 (6)

Hysterectomy 1 (6)

hCG normalization 9 (56)

A, The expression of PD-L1 was assessed on the surface of JEG-3 choriocarcinoma cells pre-incubated with avelumab isotype control (iso ctrl) (300 µg/mL), with avelumab (300 µg/mL), or with F(ab')2 of avelumab (90 µg/mL). Representative flow cytometry profiles are shown. B, The expression of PD-1 was evaluated on CD8 T cells and NK cells after an overnight stimulation with soluble anti-CD3 or IL-15 (50 ng/ml), respectively. Representative flow cytometry profiles are shown. C, Demographics and clinical characteristics of patients from the TROPHIMMUN trial (n=15) and one additional patient treated with avelumab under compassionate use (n=1).

FIGO, International Federation of Gynecology and Obstetrics; hCG, human chorionic gonadotropin; EMA-CO, Etoposide, Methotrexate, Actinomycin-D, Cyclophosphamide, Oncovin (Vincristine).