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Abstract
Background While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized.
Methods BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition.
Results BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice.
Conclusion Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.
- Antibody Specificity
- Drug Evaluation, Preclinical
- Immune Checkpoint Inhibitors
- Immune Evation
- Immunotherapy
Data availability statement
Data are available in a public, open access repository.
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- Antibody Specificity
- Drug Evaluation, Preclinical
- Immune Checkpoint Inhibitors
- Immune Evation
- Immunotherapy
WHAT IS ALREADY KNOWN ON THIS TOPIC
Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has only benefited a limited subset of patients with cancer to date. While butyrophilin 1A1 (BTN1A1) has previously been reported for its potential to suppress T-cell activity, its clinical relevance as an immune checkpoint molecule has yet to be assessed.
WHAT THIS STUDY ADDS
High levels of basal BTN1A1 expression were observed in several solid human tumor types. BTN1A1 expression by tumor cells was associated with the suppression of T cell-mediated effector activity and tumor clearance, and the knockout or antibody-mediated blockade of BTN1A1 was sufficient to enhance antitumor immunity in vitro and in vivo in mice. Strikingly, the expression of BTN1A1 was found to be mutually exclusive with that of PD-L1 owing to its ability to regulate Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY
BTN1A1 holds promise as a novel immune checkpoint that may be amenable to antibody-mediated blockade in human patients with a range of cancer types. The mutual exclusivity of BTN1A1 and PD-L1 expression further suggests that such immunotherapeutic treatment will benefit patients refractory to anti-PD-1/anti-PD-L1 treatment.
Introduction
Immune checkpoints control the delicate balance between autoimmunity and unrestrained pathology. Tumor antigens are ideal targets for host immune recognition,1 2 and tumors hijack these immune checkpoints to evade such detection. The development of antibodies specific for the best-studied immune checkpoints, including programmed cell death-ligand 1 (PD-L1), its cognate receptor programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),3 has revolutionized the treatment of many cancers and spurred efforts to therapeutically target other immune checkpoint proteins.4 5 However, the efficacy of anti-PD-1/anti-PD-L1 monotherapy in solid tumors is limited, with few patients achieving durable long-term responses.6 Moreover, only a subset of patients respond to PD-1/PD-L1 blockade, and both primary and acquired resistance hamper durable efficacy.7 The identification of additional immune checkpoints and the ability to reliably target these molecules thus remains a major area of unmet medical need with significant potential to improve the prognosis of patients with a range of cancers.
Butyrophilin (BTN) and butyrophilin-like (BTNL) proteins are members of the B7-related immunoglobulin superfamily that exhibit an array of immunoregulatory activities.8 9 A series of detailed studies have revealed the intricate mechanisms whereby BTN3A1 and BTNL3 directly bind to the T-cell receptor (TCR) of the Vγ9Vδ2 subset of γδ T cells, which are important for peripheral immunosurveillance,10 11 thereby enabling robust TCR activation in response to cognate antigen detection.12–14 Murine BTNL1 and BTNL6 coordinate the TCR-dependent activation of intestinal Vγ7+γδ T cells, while BTNL3 and BTNL8 expression on colonic epithelial cells similarly facilitate TCR-dependent Vγ4+γδ T-cell responses.15 Butyrophilin 1A1 (BTN1A1) is a BTN family protein that serves as an essential regulator of milk-lipid droplet formation and milk production in mammals.16–18 While there is limited evidence that BTN1A1 may additionally be expressed in secondary lymphoid organs and can suppress T-cell activation together with BTN2A2,19 its regulatory role and clinical relevance in this context remain to be fully explored. BTN1A1−/− mice have also been developed, and recent systematic analyses of these animals have suggested that the loss of this BTN family member is associated with enhanced inflammatory activity and activation of STAT3 signaling, further suggesting a potential regulatory role for BTN1A1 as a suppressor or immune-related and inflammatory response induction.20 Given that BTN proteins serve as B7-related context-dependent immunosuppressive or immunostimulatory mediators, they represent promising candidate immune checkpoint proteins.
Based on these prior reports, we hypothesized that BTN1A1 may be a novel immune checkpoint molecule with the potential to suppress T cell-mediated tumor clearance. Consistent with this hypothesis, in the present study, we found that BTN1A1 was upregulated across a broad range of solid tumor types. We further determined that it was capable of suppressing T-cell activation and tumor clearance both in vitro and in vivo. Strikingly, BTN1A1 expression was mutually exclusive to that of PD-L1, and the intracellular domain of BTN1A1 was found to bind to Janus-activated kinase (JAK)1, subsequently suppressing PD-L1 expression induced by the JAK/signal transducer and activator of transcription (STAT) pathway. BTN1A1 was also found to inhibit T-cell activation, and the knockout or antibody-mediated blockade of BTN1A1 was sufficient to suppress tumor growth in vivo in immunocompetent but not immunodeficient contexts. These findings highlight BTN1A1 as a novel, PD-1/PD-L1-independent immune checkpoint protein that holds great promise as a clinical target in solid tumors.
Methods
Antibodies, plasmids, and cell lines
Antibodies used in this study were purchased from Cell Signaling, Sigma, and BioLegend or were developed internally. BTN1A1 cloning for stable cell line generation was performed using the pCDH lentiviral vector with a FLAG tag. The fusion of the BTN1A1 extracellular domain (ECD) with hIgG1-Fc2 was performed using the pFuse-hIgG1-Fc2 vector (InvivoGen, San Diego, California, USA). All cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified eagle medium (DMEM)/F-12 or Roswell park memorial institute (RPMI)-1640 media supplemented with 10% fetal bovine serum (FBS). Cells stably expressing BTN1A1 were selected using puromycin (InvivoGen). Cells were transiently transfected with DNA sequences encoding BTN1A1 or other appropriate targets using polyethyleneimine (PEI; Polysciences, Warrington, Pennsylvania, USA) and Lipofectamine 2000 (Life Technologies, Carlsbad, California, USA).
Mice
Female BALB/cJ and C57BL/6J mice (Jackson Laboratory, Maine, USA) used for the present study were 8 weeks old with body weight (BW) values ranging from 17.0 to 23.0 g. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and an NIH 31 Modified and Irradiated Lab Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’cobs Laboratory Animal Bedding in static microisolators under controlled conditions (40–60% humidity, 20–22°C, 12 hours light/dark cycle). The STCube Animal Facility specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals concerning restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at STCube is accredited by the American Association for Laboratory Animal Science and approved by the internal Institutional Animal Care and Use Committee. C57BL6/J and Jax 001303 NOD CB17-Prkds (NOD SCID) mice (6−8-week-old females) were purchased from The Jackson Laboratory. BTN1A1KO mice were generated in the Genetically Engineered Murine Model Core of the University of Virginia (Charlottesville, Virginia, USA).
Stable cell line preparation
The BTN1A1 gene was purchased from Origene (Rockville, Maryland, USA), and cloned into the pCDH lentiviral expression vector to establish BTN1A1-FLAG expressing cell lines using standard molecular biological techniques. The pCDH-puro-BTN1A1-FLAG expression vector was used as a template to generate the BTN1A1-FLAG N55Q, N215Q, and 2NQ (N55Q/N215Q) mutants via site-directed mutagenesis. All constructs were validated via enzymatic digestion and DNA sequencing. To generate cells stably expressing BTN1A1, plasmids were transfected into HEK293T cells with the Lipofectamine LTX transfection reagent. Media was changed 24 hours later, and lentivirus-containing media was collected at 24 hours intervals, centrifuged to eliminate cell debris, and passed through 0.45 µm filters. Cells were seeded at 50% confluence 12 hours before infection, and the media was replaced with lentivirus-containing media. At 24 hours post-infection, media was replaced with fresh media, and infected cells were then selected with 1 µg/mL puromycin (InvivoGen). B16-F0-Ova cells stably expressing murine BTN1A1 (mBTN1A1) were similarly prepared via lentiviral transduction. Briefly, 293T cells (5×105 cells/well) were seeded overnight in 6-well plates. Then, 1 µg of lentiviral mBTN1A1 plasmid (pCDH-puro-BTN1A1-Flag expression vector subcloned with mBTN1A1 ORF), 1 µg of packaging vector (pDelta8.2), envelope vector (VSV-G) 0.375 µg in Opti-MEM (100 mL), and 1 µg of PEI in Opti-MEM (100 µL) were combined for 30 min at room temperature, after which 200 mL of this DNA/PEI mix was added to prepared cells. Cells were incubated for 72 hours, after which supernatants were collected, centrifuged (600×g, 5 min), and 0.5 mL aliquots of these supernatants were added to B16-F0-Ova cells with 1.0 mL of complete media and polybrene (8 mg/mL). On day 4 post-transduction, puromycin (10 mg/mL) was used to select transduced cells for 48 hours. Cells were then sorted by fluorescence-activated cell sorting (FACS), and clones that expressed low, medium, or high levels of mBTN1A1 were selected.
Immunohistochemistry
For the immunohistochemistry (IHC) assays, the ImmPRESS Excel Amplified HRP (peroxidase) Polymer Staining Kit (Vector Laboratories MP-7601) was used. Slides were deparaffinized in xylene and rehydrated with a graded ethanol: water series. Antigen retrieval was performed in citrate buffer (citrate pH 6.0, Dako K8005 Envision Flex Target Retrieval Solution Low pH (50×) DM829) using Dako PT link (95°C for 20 min). The slides were incubated in BLOXALL (Vector Laboratories SP-6000–100) for 10 min to block endogenous peroxidase and alkaline phosphatase activity. To block non-specific binding, the slides were incubated in 2.5% normal horse serum (Vector Laboratories, S-2012). Primary antibodies (3.0, 1.0, or 0.4 µg/mL) specific for BTN1A1 (STC-R4-17 or STC-R126, Atlas Antibodies HPA011126), PD-L1 (1:200) (Cell Signaling E1L3N XP Rabbit mAb #13684), or rabbit control IgG (3.0, 1.0, or 0.4 µg/mL) (Cell Signaling Rabbit (DA1E) mAb IgG XP Isotype Control #3900) were diluted in 2.5% horse serum and then incubated with samples for 20–60 min in a humidified chamber at room temperature. After incubating with Amplifier for 15 min, slides were incubated for 30 min with the ImmPRESS Polymer Reagent. Each target was detected using ImmPACT DAB EqV working solution (development time: ~2–10 min). All slides were then mounted using Vectashield HardSet Antifade Mounting Medium (Vector Labs) and scanned with a Vectra Quantitative Pathology Imaging System (Akoya Biosciences). Tumor tissue microarrays (TMA) were purchased from TissueArray LLC and AMSBIO. BTN1A1 and PD-L1 protein expression were determined by using Tumor Proportion Score (TPS) values, which was the percentage of viable tumor cells showing partial or complete membrane staining. A specimen was considered positive with a TPS≥5% for BTN1A1 and a TPS≥1% for PD-L1.
Western blotting and immunocytochemistry
Western blotting was performed as described previously.21 22 Image acquisition and quantitation of band intensity were performed using a Chemdoc Imager (Bio-Rad, Hercules, California, USA). For immunoprecipitation assays, cells were lysed in buffer (50 mM Tris·HCl, pH 8.0, 150 P and 0.5% Nonidet P-40) and centrifuged at 16,000×g for 30 min to remove cellular debris. Cleared lysates were subjected to immunoprecipitation with appropriate antibodies. For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized in 5% Triton X-100 for 5 min, and stained using appropriate primary antibodies. The secondary antibodies used for these experiments were anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 488 or Alexa Fluor 594, and nuclei were stained with 4’,6-diamidino-2-phenylindole (Life Technologies).
T-cell killing assay
T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using a magnetic αCD3-isolation antibody kit and were then activated by incubation with anti-CD3 (100 ng/mL) and interleukin (IL)-2 (10 ng/mL) for 72 hours. Cells were then added to pre-seeded PC-3 cancer cells or 4T1 cells in 96-well plates in RPMI-1640 containing 10% FBS, a caspase 3/7-sensitive fluorescent reagent (5 mM; Essen BioScience, 4440), and STM810 or hIgG1 isotype control (5 µg/mL). T cell-mediated killing of cancer cells was detected over time using an Incucyte ZOOM instrument (Essen BioScience) based on phase confluence and caspase reagent detection (green objects) as calculated with the Incucyte ZOOM 2016 software.
Cytokine ELISAs
Interferon (IFN)-γ and IL-2 levels in supernatants from T-cell proliferation assays were quantified using the Human IFN-γ ELISA Kit (BioLegend 430107) and IL-2 ELISA kit (BioLegend 431815) based on provided directions. Samples were diluted to an appropriate concentration such that they fell within the linear range of the corresponding standard curves, which were used to calculate cytokine levels in these samples.
Syngeneic mouse tumor models and treatments
For in vivo assays, tumor cells were cultured in RPMI medium with Earle’s Balanced Salt Solution containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin G sodium, 100 µg/mL streptomycin, and 25 µg/mL gentamicin in a humidified 37°C 5% CO2 incubator. On the day of tumor implantation, tumor cells were harvested in the exponential phase of growth and resuspended in cold phosphate buffered saline (PBS) at a concentration of 1×107 cells/mL. Tumor models were established by subcutaneously implanting 1×106 CT26 tumor cells (100 µL suspension) into the right flank of each test animal, and tumors were monitored as their volumes approached the target range of 50–80 mm3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
where w=width and l=length, in mm, of the tumor. Tumor weight was estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
Six days after tumor implantation, animals were assigned to four groups (n=13/group) with individual tumor volumes ranging from 30 to 70 mm3 and group mean tumor volumes of 50 mm3. STC109 was developed internally and used at a concentration of 14.47 mg/mL. Mouse IgG1 isotype control was purchased from Bio X Cell (MOPC-21, 6.28 mg/mL, Lot No. 701618J2). Rat IgG2a isotype control was purchased from Bio X Cell (LTF-2, 7.69 mg/mL, Lot No. 629 817O1). Anti-PD-L1 (10F.9G2) was purchased from Bio X Cell (6.31 mg/mL, Lot No.665718J1B). All reagents were stored at 4°C, and anti-mBTN1A1 (STC109) was diluted in the vehicle (PBS) to appropriate concentrations, providing a dosage of 200, 300, or 400 µg/animal in a fixed volume of 100 µL/animal. On day 0 of the study, mice were sorted into four groups (n=13/group), and dosing (intraperitoneal injection, i.p.) was initiated according to the established treatment plan. Dosing was performed three times per week for 2 weeks.
Statistical analysis
Unless noted otherwise, data in bar graphs represent the mean with SD from experiments independently repeated a minimum of three times. Survival outcomes were compared using Kaplan-Meier curves and log-rank tests. All statistical tests were two-tailed, and data were compared via Student’s t-tests unless otherwise indicated using GraphPad Prism V.9.0 (GraphPad) or Excel (Microsoft, V.16.50). The number of replicate samples is indicated in the provided figure legends. A p value<0.05 was considered statistically significant.
Supplemental methods
For further details regarding the experiments performed for the present study, see the online supplemental methodssupplemental methods text.
Supplemental material
Results
BTN1A1 is an immune checkpoint protein that suppresses CD8+ T-cell activation
An earlier study indicated that BTN1A1 can bind to the surface of activated T cells and inhibit CD4+ T-cell proliferation,19 suggesting the existence of a BTN1A1 receptor on T cells and raising the possibility that it may function in trans as an immune checkpoint protein through a mechanism akin to PD-L1. When T cells were stimulated with beads conjugated with anti-CD3, anti-CD28, and either the ECD of BTN1A1, PD-L1, or control IgG, the ECDs of BTN1A1 and PD-L1 suppressed T-cell proliferation (figure 1A,B). While pronounced apoptotic induction was observed in an empty vector (EV) control-expressing PC3 tumor cells cultured with activated T cells, PC3 cells overexpressing BTN1A1 exhibited reduced apoptotic death and a concomitant drop in IFN-γ production (figure 1C–E). Similarly, murine 4T1 tumor cells stably overexpressing mBTN1A1 constrained anti-CD3/CD28-induced T-cell proliferation relative to 4T1 cells expressing an EV (figure 1F). BTN1A1-Fc exposure suppressed anti-CD3/IL-2-induced T-cell IFN-γ secretion and clustering in a dose-dependent and time-dependent manner (figure 1G,H). Both BTN1A1 and PD-L1 were also able to suppress antigen-specific OT-I CD8+ T-cell survival to a similar extent in the context of ovalbumin peptide presentation on major histocompatibility complex-I (online supplemental figure S1A). BTN1A1-Fc was additionally confirmed to suppress the anti-CD3 (OKT3)-mediated activation of the TCR signaling pathway to a similar extent to that observed for PD-L1-Fc (figure 1I), further affirming the immunosuppressive role of this protein.
Supplemental material
BTN1A1 suppresses T-cell activation. (A, B) A bead-based T-cell proliferation assay was performed to test the ability of BTN1A1 to regulate T-cell activation, as outlined in (A). Beads were coated with anti-CD3, anti-CD28, and either human IgG (black), human PD-L1 extracellular domain (blue), or human BTN1A1 extracellular domain (red) prior to use for the stimulation of CFSE-stained T cells at the indicated T cell to bead ratios, with T-cell proliferation then being measured by flow cytometry based on CFSE dilution (B). (C, D) T cells were combined with parental or hBTN1A1-expressing PC3 cancer cells, and apoptotic cell death was monitored with a fluorescent caspase 3/7 substrate as a means of assessing the BTNA1A1-mediated inhibition of T cell-induced PC3 cell apoptosis. (E) Quantification of IFN-γ levels in PC3 cell culture supernatants was performed by ELISA. ***p<0.001 (Student’s t-test) (F) To assess the effect of mBTN1A1 on T-cell proliferation, the indicated numbers of 4T1 cells expressing mBTN1A1 were co-cultured with CFSE-labeled murine T cells, and T-cell proliferation was measured by flow cytometry based on CFSE dilution. (G) T cells were activated with anti-CD3 (1, 5, or 10 µg/mL) and IL-2 (10 ng/mL), and IFN-γ levels in culture supernatants were measured by ELISA. (H) T-cell clustering over time was measured using the real-time Incucyte ZOOM imaging system, with clustered T cells being counted with the Incucyte Image analysis software at the indicated time points. (I) Western blotting of primary T cells following T-cell receptor crosslinking using plate-bound OKT3 in the presence of BTN1A1-Fc, PD-L1, or control IgG-Fc (10 µg/mL) for 5 min. All data are representative of experiments that were repeated a minimum of three times. BTN1A1, butyrophilin 1A1; CFSE, carboxyfluorescein succinimidyl ester; EV, empty vector; hBTN1A1, human butyrophilin 1A1; IFN, interferon; IL, interleukin; mBTN1A1, murine BTN1A1; PD-L1, programmed cell death-ligand 1.
When B16-F0-OVA cells expressing varying levels of mBTN1A1 (online supplemental figure S1B–D) were subcutaneously implanted in immunocompetent C57BL/6 mice, tumor volumes increased in an mBTN1A1 dose-dependent manner without impacting murine BW (figure 2A; online supplemental figure S1D). However, tumors grew at a similar rate irrespective of mBTN1A1 dose in immunodeficient SCID mice (figure 2B; online supplemental figure S1E). This suggests that the effects of BTN1A1 expression on tumor growth are not evident under immunodeficient conditions, consistent with the potential influence of tumor-derived BTN1A1 on the ability of immune cells to inhibit tumor growth. To determine whether BTN1A1 is required for tumor cell immune evasion, we generated BTN1A1-knockout MC38 colorectal cancer cells (online supplemental figure S1F–H). Both BTN1A1-wild-type (WT) and BTN1A1-knockout (KO) cells grew comparably in vitro (online supplemental figure S1H). BTN1A1-KO mice were generated that were confirmed to lack the expression of Btn1a1 (online supplemental figure S2A–E), whereas the closely related and localized Btn2a2 gene expression was unaffected in these animals (online supplemental figure S2F–G). While we observed no developmental abnormalities in the generated mice, at 12 months of age, BTN1A1 homozygous null animals presented with lymphocytic aggregates and other autoimmune phenotypes similar to those reported in PD-1/PD-L1-KO animals,23 further supporting the functional role of BTN1A1 as an immune checkpoint protein (online supplemental figure S3).
Supplemental material
Supplemental material
BTN1A1-mediated enhancement of tumor growth is CD8+ T cell-dependent. (A) B16-OVA cells (1×106) overexpressing intermediate or high levels of murine BTN1A1 (mBTN1A1) or empty vector (EV) control constructs were engrafted into C57BL/6J mice (n=6/group). Tumor volumes were measured at the indicated time points. *p<0.05 (Student’s t-test). (B) B16-OVA cells (1×106) overexpressing BTN1A1 were engrafted into immunodeficient SCID mice (n=7/group), and tumor volumes were monitored over time. (C, D)Wild-type or BTN1A1−/− C57BL/6 mice (n=8/group) were inoculated (s.c.) with 1×105 MC38 (right flank) or MC38-BTN1A1KO (left flank) cells. Tumor growth was measured for 30 days post-implantation. (E) CD8+ T-cell depletion was achieved by anti-CD8 injection, and CD8+ T cells in the spleen following depletion were evaluated by flow cytometry. (F) Tumor growth was monitored over time in the indicated treatment groups, revealing that CD8+ T-cell depletion partially reversed the suppression of MC38 BTN1A1 KO tumor growth relative to the control (red) group. *p<0.05, **p<0.01, ***p<0.001, P****<0.0001 (Student’s t-test, n=6). All data are representative of experiments that were repeated a minimum of two times. KO, knockout; mBTN1A1, mouse butyrophilin 1A1; s.c., subcutaneous injections.
Next, we evaluated the dependency of BTN1A1-mediated immunosuppression on the host immune compartment. MC38-BTN1A1-KO tumors did not grow in immunocompetent WT C57BL/6 mice, while MC38-BTN1A1-WT tumors grew well in BTN1A1-KO animals and limited MC38-BTN1A1-KO tumor growth was observed in these KO mice, with greater variability than that evident in WT animals. This suggested that BTN1A1 expression on host cells may play at least a limited role in the regulation of MC38 tumor growth (figure 2C,D). However, MC38-BTN1A1-KO tumor growth was partially but significantly (p=0.023) restored following CD8+ T-cell depletion (figure 2E,F). BTN1A1 expression is thus required in order for MC38 tumor cells to effectively evade immune-mediated detection and elimination through CD8+ T cell-dependent mechanisms.
BTN1A1 is highly expressed in human cancers and mutually exclusive with PD-L1
The above results demonstrated that BTN1A1 can, when expressed on tumor cells, function to suppress T-cell responses in vitro and in vivo. We thus sought to conduct a more in-depth analysis of the role of tumor-derived BTN1A1 as a novel immune checkpoint. To gage the potential feasibility of BTN1A1 blockade, we developed a rabbit monoclonal antibody (STC43H11-1) for IHC staining (figure 3A,B) and used it to visualize BTN1A1 expression in a TMA. Pronounced and robust BTN1A1 expression was evident in head and neck, bladder, squamous non-small cell lung cancer (NSCLC), ovarian, cervical, prostate, and other tumor tissues, while PD-L1 expression was relatively low in both frequency and intensity (figure 3C,D, table 1). Intriguingly, minimal overlap between BTN1A1 and PD-L1 expression was observed in any analyzed tumors (figure 4C,D). Simultaneous PD-L1 and BTN1A1 detection using an OPAL multiplex system revealed that while these proteins were sometimes present on adjacent cells, they were exclusively expressed on a given cell in head and neck, colon, and ovarian tumors (figure 3E-G). We additionally quantified the number of BTN1A1+PD-L1−, BTN1A1− PD-L1+, BTN1A1− PD-L1−, or BTN1A1+PD-L1+ cells within each patient specimen as a percentage of cancer cells expressing cytokeratin (figure 3H), supporting this observation. Consistently, BTN1A1 and PD-L1 messenger RNA (mRNA) levels were negatively correlated in cell lines (online supplemental figure S4A–C), and BTN1A1 and PD-L1 protein levels were negatively correlated in both the tumor and non-tumor compartments in mice bearing B16-F0-Ova tumors expressing varying levels of mBTN1A1 (online supplemental figure S4D). Tumors expressing the highest levels of BTN1A1 exhibited negligible PD-L1 expression and appeared to induce BTN1A1 expression in the non-tumor compartment, further reducing stromal PD-L1 expression (online supplemental figure S4E,F).
Supplemental material
BTN1A1 and PD-L1 exhibit mutually exclusive expression in tumor cells. (A) Western blotting was performed using a candidate anti-mBTN1A1 antibody (STC43H11-1) to detect the overexpression of BTN1A1 in lysates from 293T cells that were or were not subject to reducing and denaturing conditions (DTT and boiling). (B) The immunohistochemical (IHC) staining of 293T-hBTN1A1 (human) and 4T1-mBTN1A1 (mouse) cells was performed using the STC43H11-1 antibody, with rabbit control IgG serving as a negative control for staining. (C) A tissue microarray (TMA) was used for IHC-based analyses of tumor tissue BTN1A1 and PD-L1 expression using monoclonal anti-BTN1A1 and monoclonal rabbit anti-PD-L1. Representative images exhibiting regions with positive BTN1A1 and PD-L1 staining are shown. (D) Mutually exclusive expression of BTN1A1 and PD-L1 in NSCLC was detected in IHC analyses of TMA cores of various tissue types. (E–G) Mutually exclusive expression of BTN1A1 and PD-L1 was detected in squamous cell carcinoma tumors of the hypopharynx (E) colon carcinoma tumors (F) and ovarian carcinoma tumors (G) through multiplexed fluorescent IHC staining for BTN1A1 (green), and PD-L1 (red). (H) Percentages of BTN1A1+PD-L1−, BTN1A1− PD-L1+, BTN1A1− PD-L1−, or BTN1A1+PD-L1+ cancer cells among CK+cancer cells were analyzed with the Akoya Biosciences phenoptrReports application. CK, cytokeratin; hBTN1A1, human butyrophilin 1A1; mBTN1A1, murine BTN1A1; NSCLC, non-small cell lung cancer; PD-L1, programmed cell death-ligand 1.
BTN1A1 is an upstream regulator of PD-L1 in tumor cells. (A) Human PC3 prostate cancer cells stably expressing empty vector (wild-type; WT), overexpressing human BTN1A1 (OE), or in which BTN1A1 was knocked out (KO) were transfected with firefly luciferase constructs under the human BTN1A1 (997 bp upstream of ORF) or PD-L1 (952 bp upstream of ORF) promoter. Luciferase activity was assessed using a luciferase substrate from Promega. The promoter region was identified using the LightSwitch Promoter Reporter GoClone Collection. *p<0.05, **p<0.01, Unpaired Student’s t-test, n=3. (B) Western blotting was performed to detect the expression of PD-L1 and interferon regulatory factor 9 in whole-cell lysates from WT, hBTN1A1 OE, and hBTN1A1 KO PC3 cells, with β-actin as a loading control. (C) The approach used to prepare FLAG-tagged BTN1A1 intracellular domain deletion constructs. The putative JAK1 binding domain (BD) is highlighted with a red box. (D) HEK293T cells were transiently co-transfected with BTN1A1 deletion mutant constructs and JAK1 plasmids for 24 hours. Cell lysates were then subjected to immunoprecipitation (IP) with an anti-FLAG antibody. Western blotting (IB) of the immunoprecipitated proteins was performed with a JAK1-specific antibody. Numbers above individual lanes correspond to the BTN1A1 deletion constructs shown in (C). (E) WT and BTN1A1-KO cells were exposed to ionizing radiation (0, 2, or 8 Gy). At 48 hours post-irradiation, levels of the indicated proteins in these cells were analyzed via western blotting. (F) A schematic overview of the proposed mechanism underlying BTN1A1-mediated regulation of PD-L1 expression via the JAK/STAT pathway. (G) The relative expression of JAK/STAT-induced genes (PD-L1, CXCL9, CXCL10, CXCL11, GBP1) was compared between PC3 and BTN1A1-OE PC3 cells after stimulation with 50 ng/mL IFN-γ for 48 hours. (H) Caco-2 and BTN1A1-KO Caco-2 cells were stimulated by 50 ng/mL IFN-γ, and total RNA was isolated from the cells at 48 hours post-stimulation. A total RNA sequencing analysis was performed to compare the expression of JAK/STAT-induced genes including CXCL9, CXCL10, and CXCL11 under conditions of IFN-γ stimulation. All data are representative of experiments that were repeated a minimum of three times (A–E). BTN1A1, butyrophilin 1A1; EV, empty vector; IFN, interferon; JAK, Janus-activated kinase; PD-L1, programmed cell death-ligand 1; STAT, signal transducer and activator of transcription.
Summary of butyrophilin 1A1 staining results in tissue microarray scores for patients with different solid and hematological tumor types
Human Caco-2 colon epithelial cells displayed basal BTN1A1 expression, and these levels rose on treatment with IFN-γ in a dose-dependent manner (online supplemental figure S4G,H), whereas PD-L1 expression was unaffected. To gain additional insight into the relationship between T cells and the expression of BTN1A1 by tumor cells, Caco-2 cells were co-cultured with either non-activated or activated T cells or were treated with recombinant IFN-γ, revealing that both activated T cells and IFN-γ were sufficient to induce Caco-2 cell BTN1A1 upregulation at the mRNA level, whereas non-activated T cells had no such effect (online supplemental figure S5A). Consistently, activated T cells promoted BTN1A1 mRNA upregulation in NCI-H226 cells, and a similar although non-significant trend toward increased BTN1A1 expression was evident in A549 cells co-cultured with activated T cells (online supplemental figure S5B). Flow cytometry analyses yielded comparable protein level findings in these three cell lines (online supplemental figure S5C), in line with the ability of activated immune cells to function as a stressor that promotes BTN1A1 upregulation within the tumor microenvironment (TME). Subsequent analyses of BTN1A1 and PD-L1 expression in the tumor and immune compartments of mice bearing LLC and 4T1 tumors further highlighted the inverse correlative relationship between these two immune checkpoints within the TME, together with the time-dependent upregulation of BTN1A1 in both tumor cells and immune cell populations (online supplemental figure S6).
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BTN1A1 regulates PD-L1 expression through the JAK/STAT pathway
Given the negative correlation between BTN1A1 and PD-L1 expression, we next explored whether BTN1A1 could repress PD-L1 expression. PC3 cells in which BTN1A1 was overexpressed or knocked out were transfected with a luciferase reporter construct driven by the BTN1A1 or PD-L1 promoter, using the empty pGL4 vector as a control. Exogenous BTN1A1 expression did not alter intrinsic BTN1A1 promoter activity but did reduce PD-L1 promoter activity, whereas BTN1A1 KO restored this reduced activity (figure 4A). Consistently, PD-L1 protein levels were, respectively, decreased and increased following BTN1A1 exogenous expression and KO (figure 4B). The PD-L1-inducing transcription factor interferon regulatory factor 9 (IRF9)24 was also upregulated in BTN1A1-KO cells (figure 4B).
IRF9 is a downstream JAK/STAT signaling mediator.25 Recent research has shown that PD-L1 expression level and PD-L1 stabilization via glycosylation are increased by JAK/STAT signaling pathway in various tumors.26 27 To assess whether BTN1A1 modulates JAK/STAT signaling, cells were transfected with JAK1 and BTN1A1-Flag constructs with increasingly large C-terminal deletions. Co-immunoprecipitation revealed that JAK1 bound to BTN1A1 when amino acids (aa) 380–526 but not aa331-526 were deleted (figure 4C,D). A JAK binding motif (PXXL) is present at aa345 (345PCVL), further supporting the ability of JAK1 to bind to aa330-380 of BTN1A1.
Under radiation-induced stress conditions, WT PC3 cells exhibited slightly increased phosphorylated signal transducer and activator of transcription 2 (pSTAT2) levels, unchanged phosphorylated signal transducer and activator of transcription 1 (pSTAT1) levels, and decreased PD-L1 expression, while BTN1A1-KO increased STAT1 and STAT2 phosphorylation and PD-L1 expression in these cells (figure 4E), suggesting that BTN1A1 can bind to JAK1 and thereby suppress downstream STAT/IRF9-mediated upregulation of target genes including PD-L1 (figure 4F).
To gain greater insight into the BTN1A1-JAK-STAT-PD-L1 pathway, we performed a NanoString gene expression analysis of both native PC3 (WT) and those overexpressing BTN1A1 (BTN1A1-OE), post IFN-γ stimulation. Analyses performed using the nCounter PanCancer IO 360 Panel in PC3 cells revealed that BTN1A1 overexpression led to the downregulation of numerous signaling pathways, including the JAK/STAT signaling pathway, which serves as a primary upstream regulator of PD-L1 (online supplemental table 1). We subsequently assessed the expression profiles of hallmark JAK/STAT signaling markers, including PD-L1, CXCL9, CXCL10, CXCL11, and GBP1.28 Remarkably, following IFN-γ stimulation, we observed a marked reduction in the expression of these genes in BTN1A1-OE cells compared with WT cells (figure 4G). To further understand the role that BTN1A1 plays as a modulator of JAK/STAT signaling in tumor cells, BTN1A1-KO Caco-2 cells were stimulated with IFN-γ, and their transcriptomic responses were analyzed via RNA sequencing (online supplemental table 2). In contrast to the NanoString data derived from BTN1A1-overexpressing cells, these BTN1A1-KO Caco-2 cells exhibited significant CXCL9, CXCL10, and CXCL11 upregulation as compared with WT cells (figure 4H). Thus, BTN1A1 indirectly reduces the transcription of PD-L1 by inhibiting the JAK/STAT pathway via hijacking JAK.
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Anti-BTN1A1 mAb treatment inhibits syngeneic tumor growth
To investigate the therapeutic potential of BTN1A1 blockade in vivo, we developed a mBTN1A1-specific monoclonal antibody (anti-mBTN1A1) that successfully disrupted the mBTN1A1-mediated suppression of induced murine T-cell activation and proliferation (online supplemental figure S7). When administered intraperitoneally to mice harboring different syngeneic model tumors (MC38, CT26, 4T1, LLC), anti-mBTN1A1 significantly suppressed tumor growth (figure 5A,B). When we assessed the therapeutic efficacy of combined anti-mBTN1A1/anti-PD-L1 treatment in these tumor models, superior suppression of MC38 and CT26 tumor growth was observed in mice that underwent combination treatment relative to animals treated with anti-mBTN1A1 alone (figure 5C,D). While anti-mBTN1A1 or radiation alone conferred only limited antitumor efficacy, together these treatments significantly slowed tumor growth (figure 5E). Single-agent anti-PD-L1 or anti-mBTN1A1 treatment was associated with improved survival in BALB/c mice bearing CT26 tumors, with superior survival outcomes in mice that underwent combination treatment with these two immune checkpoint inhibitors (figure 5F–H).
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BTN1A1 is a target for cancer immunotherapy. (A, B) MC38 (1×106), CT26 (1×106), 4T1 (1×105), or LLC (1×105) cells were implanted subcutaneously in mice (C57BL/6 or BALB/c, as appropriate) to establish syngeneic tumor models. Animals were then treated with anti-mBTN1A1 or vehicle control at the indicated time points (A). Tumor growth in individual mice at the endpoint of these studies was measured (n=6–16 mice/group; **p<0.01; unpaired two-tailed Student’s t-test) (B). (C, D) Average tumor growth was monitored in mice implanted with MC38 (C) and CT26 (D) cells and treated with anti-mBTN1A1 and/or anti-PD-L1. Differences in tumor growth kinetics between treatments were compared via ordinary one-way analysis of variances. **p<0.01. (E) Average tumor growth was monitored for mice implanted with LLC tumor cells and treated with anti-BTN1A1 (STC1029; 200 µg/dose) and/or ionizing radiation (8 Gy). (F) Average tumor growth was monitored in mice implanted CT26 cells and subjected to the indicated treatments (400 µg, STC1029 for anti-mBTN1A1, 10F.9G2 for anti-mPD-L1, all raised in rat) (n=10 mice/group) are shown. (G) Tumor volumes for mice in (F) were compared on study completion. Data are means±SD. **p<0.01, ***p<0.001; Unpaired Student’s t-test. (H) Log-rank (Mantel-Cox) tests comparing survival curves for mice inoculated with CT26 tumors as in (F). All data are representative of experiments that were repeated a minimum of three times. BTN1A1, butyrophilin 1A1; mBTN1A1, murine BTN1A1; PD-L1, programmed cell death-ligand 1.
The anti-human BTN1A1 mAb STC810 disrupts BTN1A1-dependent immunosuppression
Finally, to evaluate the therapeutic potential of BTN1A1 inhibition, we generated an anti-human BTN1A1 antibody (STC810). STC810 exhibited conformational specificity for the dimeric form of BTN1A1, whereas it bound only weakly to the monomeric BTN1A1 ECD in vitro under native conditions (online supplemental figure S8A–C). Native human BTN1A1 is glycosylated at two sites (N55 and N215), and the mutation of either of these sites markedly reduced the stability of this protein in cycloheximide-treated PC3 cells (online supplemental figure S8D). As compared with its binding to WT dimerized or tetramerized human BTN1A1, STC810 bound less robustly to BTN1A1 harboring N55Q or N215Q mutations under native conditions, and such binding was undetectable when both glycosylation sites were mutated (2NQ), while all such binding was absent under denaturing conditions (online supplemental figure S8E). STC810 additionally exhibited excellent specificity for human BTN1A1 when used to probe a panel of 293T cells expressing FLAG-tagged versions of various immune checkpoint proteins (online supplemental figure S8F). Biacore binding assays further confirmed the ability of STC810 to bind to dimeric BTN1A1-Fc with greater affinity as compared with monomeric BTN1A1-His (online supplemental figure S8G). Taken together these data show that STC810 exhibits excellent specificity for dimeric, glycosylated human BTN1A1 without any off-target immune checkpoint protein cross-reactivity, making it a promising checkpoint inhibitor candidate.
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Consistent with its antagonistic blocking antibody activity, STC810 increased STAT1/2 phosphorylation, IRF9 expression, and PD-L1 expression in treated tumor cells (figure 6A,B), in line with prior findings in BTN1A1-KO PC3 cells. Our goal was to clarify the epistatic relationship between BTN1A1 and the JAK/STAT pathway by administering STC810, given the ability of this antibody to effectively regulate BTN1A1. CXCL9 and CXCL10 were upregulated in response to IFN-γ stimulation and this upregulation was further enhanced by STC810 treatment (figure 6C). However, applying the JAK/STAT pathway inhibitor ruxolitinib was sufficient to abrogate the effects of IFN-γ and STC810 on CXCL9 and CXCL10 expression (figure 6C). These epistatic data suggest that the effects of BTN1A1 are mediated through JAK/STAT signaling pathways. STC810 also enhanced the ability of anti-CD3/CD28-activated T cells to induce target PC3 cell apoptosis (figure 6D–F). Moreover, STC810 treatment enhanced the proliferation and IFN-γ-producing activity of carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells on anti-CD3/CD28 bead stimulation to a similar degree to anti-PD-1 treatment while also greatly enhancing IL-2 production at higher T cell-to-bead ratios more readily than other tested immune checkpoint inhibitors (figure 6G–I). Overall, our studies strongly suggest that targeting the immune checkpoint protein BTN1A1 represents a viable immunotherapeutic interventional strategy.
Treatment with the anti-BTN1A1 mAb STC810 mimics the effects of knocking out BTN1A1 and suppresses tumor growth. (A) A schematic diagram of the proposed mechanism through which STC810 inhibits signaling via the JAK-STAT pathway. (B) Western blotting analysis of whole-cell lysates derived from PC3 cells treated with 40 µg/mL STC810 or IgG isotype control. PD-L1, STAT1 (pTyr 701), STAT2 (pTyr 690), or IRF9 levels were assessed at 48 hours post-antibody treatment. (C) qRT-PCR was used to assess CXCL9 and CXCL10 messenger RNA levels in PC3 cells treated with 50 ng/mL IFN-γ, 5 µM ruxolitinib, 100 µg/mL STC810, or IgG isotype control at 48 hours post-treatment. qRT-PCR experiments were repeated three times. (D–F) Schematic overview of the live-cell imaging approach used to monitor the T cell-induced apoptosis of PC3 cells overexpressing BTN1A1 (D) with representative images of T cell-induced BTN1A1-overexpressing PC3 cell apoptosis (green) in the control IgG and hSTC810 groups (E). Dose-dependent increases in cytolysis were assessed for PC3 cells expressing hBTN1A1 that were co-cultured with activated effector T cells in the presence of hSTC810 at the indicated concentrations (F). (G–I) T-cell proliferation was examined by FACS based on CFSE dilution in CFSE-labeled primary T cells co-cultured with the indicated concentrations of anti-CD3/CD28 beads conjugated with control IgG1, hSTC810, anti-hPD-1, or anti-hCTLA4 (G). IFN-γ (H) and IL-2 release (I) from these cells were detected by ELISA using supernatant samples collected from cells in (G). All data are representative of experiments that were repeated a minimum of three times. CFSE, carboxyfluorescein succinimidyl ester; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; FACS, fluorescence-activated cell sorting; hBTN1A1, human butyrophilin 1A1; IFN, interferon; IL, interleukin; IRF9, interferon regulatory factor 9; JAK, Janus-activated kinase; mAb, monoclonal antibody; PD-1, programmed cell death protein 1; PD-L1, programmed cell death-ligand 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription.
Discussion
While the emergence of immune checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1 has revolutionized the field of cancer immunotherapy, few patients achieve durable long-term responses,4–6 and these treatments can induce immune-related adverse events that may be poorly tolerated.29 Here, we identified BTN1A1 as a novel immune checkpoint protein. Further characterization revealed pronounced BTN1A1 expression that was mutually exclusive with that of PD-L1 in a range of human solid tumors. Functionally, BTN1A1 was able to suppress T-cell activation and associated cytotoxic functionality, while the KO or antibody-mediated blockade of BTNA1A1 inhibited tumor growth and enhanced intratumoral CD8+ T-cell accumulation, validating it as a bona fide immune checkpoint expressed primarily by tumor cells.
BTN1A1 was the first reported BTN.30 At least 13 immunoglobulin domain-containing B7-related BTN and BTNL proteins have since been described in humans, exhibiting distinct functional roles and cellular expression patterns.31 While BTN1A1 plays a well-characterized role as an essential regulator of mammalian milk-lipid droplet production and lactation,16–18 its function as an immune checkpoint protein has been largely unexplored. One prior study demonstrated that BTN1A1-Fc constructs suppress T-cell activation and consequent effector cytokine production.19 However, more recent studies of the immunomodulatory effects of BTN1A1 have largely been restricted to high-level analyses of their effects on the activation of different signaling pathways in murine T cells,32 although there have been many reports documenting the ability of other BTN family members to regulate immune activity.33 Consistently, BTN1A1-conjugated beads and BTN1A1-expressing tumor cells inhibited T-cell proliferation, cytokine production, and cytotoxicity in our study. BTN2A1 has been identified as a DC-SIGN ligand,34 and interactions between DC-SIGN and the breast milk-derived DC-SIGN ligand MUC1 are important mediators of immune tolerization and homeostasis in newborns.35 Given the immunosuppressive function of BTN1A1 and its presence at high levels in breast milk, BTN1A1 may play a role analogous to MUC1 in the context of breast feeding, preventing aberrant immunoreactivity in the neonatal immune system. Alternatively, it may suppress aberrant T-cell reactivity within the mammary tissue.19 In oncogenic contexts, tumor cells can hijack this mechanism to suppress T cell-mediated antitumor immunity, as evidenced by the striking upregulation of BTN1A1 across a range of human solid tumor types, providing them with an additional immune evasion strategy.
The mechanisms whereby BTN1A1 can suppress T-cell activation remain to be fully clarified. While BTNL3 and BTN3A1 can directly bind to the γδ TCR,12–14 no comparable mechanism has been described for BTN1A1. Although BTN1A1 ECD ligands remain to be characterized, we found that the BTN1A1 intracellular domain bound directly to JAK1 in tumor cells, disrupting JAK/STAT signaling and consequent PD-L1 upregulation.24 Whether this cell-intrinsic JAK/STAT suppression is directly tied to the immune checkpoint activity of BTN1A1 remains unclear, but may be of clinical relevance given the essential role of JAK1-mediated signaling in IFN-mediated innate immunity.36 Studies of BTN1A1-KO mice generated by a separate group have also suggested a role for this BTN family member as a regulator of STAT3 signaling and inflammatory activity in the context of milk lipid droplet production,20 potentially providing some degree of support for the present findings. Additional studies of endogenous BTN1A1 ligands and the relevance of this BTN1A1/JAK1 axis in oncogenic and immunological contexts are thus warranted. However, given prior evidence that JAK1 can additionally phosphorylate and thereby stabilize PD-L1 in at least certain oncogenic settings,37 and in light of the complex transcriptional, post-transcriptional, translational, and post-translational regulation of PD-L1 expression,38 further research will be required to determine whether BTN1A1 binding disrupts this regulatory interaction and to fully elucidate the complex feedback regulatory mechanisms governing the relationship between BTN1A1 and PD-L1 expression.
The largely mutually exclusive expression of BTN1A1 and PD-L1 in tumors may have important therapeutic implications. CTLA-4 and the PD-1/PD-L1 axis are commonly employed immunosuppressive pathways under physiological conditions.3–5 Much like PD-1/PD-L1-KO animals,23 our BTN1A1-KO mice developed autoimmune phenotypes although these were generally less severe than those in PD-1/PD-L1 KO animals. This suggests that BTN1A1 may be associated with reduced toxicity when subject to immune checkpoint blockade. BTN1A1+ tumors were more common than PD-L1+ tumors in our TMA study, highlighting the potentially broad therapeutic indications of BTN1A1 blockade. As BTN1A1 blockade induced intratumoral PD-L1 upregulation and PD-1 was upregulated in BTN1A1-KO Jurkat cells (online supplemental figure S7E), simultaneous targeting of BTN1A1 and the PD-1/PD-L1 axis may be beneficial. However, as BTN1A1 blockade also achieved single-agent antitumor efficacy, the value of combination anti-BTN1A1/anti-PD-1/PD-L1 treatment is likely tumor-type-dependent. Another perspective is the role of BTN1A1 in immune cells. The role of BTN1A1 as an immune checkpoint cannot be limited to tumors. BTN1A1, like PD-L1, is present in tumor cells and immune cells (online supplemental figure S6A–D). In immune cells, PD-L1 on activated T cells promotes the transition from memory T cells to suppressor-inducible regulatory T cells.39 Similar to the effects of PD-L1 on immune cells when BTN1A1 is expressed, it is possible that BTN1A1 may depress immune cells, and further studies are needed to confirm the role of BTN1A1 on immune cells.
Consistent with the observed in vitro and in vivo efficacy of our mBTN1A1 blocking antibody, our monoclonal anti-human BTN1A1 antibody, STC810, was able to reverse in vitro BTN1A1-dependent suppression of T-cell activation and associated signaling activity. Recently, in a toxicology study with cynomolgus monkeys, humanized hSTC810 was well-tolerated and phase I clinical trials have been successfully completed.40 We will be reporting on phase I clinical trial achievement imminently. Overall, our results highlight BTN1A1 as a novel immune checkpoint protein that is primarily expressed by tumor cells in a manner mutually exclusive with PD-1/PD-L1 that can potentially be targeted to extend new immunotherapeutic treatment options to patients whose tumors do not respond to anti-PD-1/PD-L1 therapy.
Supplemental material
Data availability statement
Data are available in a public, open access repository.
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Acknowledgments
Graphical figures were created using BioRender (biorender.com). We thank the current and previous members of STCube Pharmaceuticals, Inc, and STCube in Seoul, Korea. The corresponding author is SSY, and the mailing address is 401 Professional Dr, Suite 250, Gaithersburg, MD 20879.
References
Supplementary materials
Supplementary Data
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Footnotes
Contributors SSY, SHL, and HJ conceived the project. SSY supervised the project as the guarantor. Y-SK and S-HL contributed equally. SSY, Y-SK and S-HL wrote the manuscript. Y-SK, S-HL, AHP, and CW performed the experiments. All authors reviewed and edited the manuscript. SSY, Y-SK, and S-HL led the analysis of the data. HJ and SHL helped with data interpretation.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.