Research Article

BTN3A1 governs antitumor responses by coordinating αβ and γδ T cells

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Science  21 Aug 2020:
Vol. 369, Issue 6506, pp. 942-949
DOI: 10.1126/science.aay2767

BTN3A1 governs antitumor responses

T lymphocytes are immune cells that can be activated through their gamma delta (γδ) or alpha beta (αβ) receptors. Both T cell types are found in human cancers, but current immunotherapies do not harness their coordinated antitumor activity. Payne et al. found that BTN3A1 and BTN2A1, two members of the butyrophilin family of proteins, partner to activate the most abundant subset of γδ T cells in peripheral blood. Antibodies targeting BTN3A1 redirect γδ T cells to attack cancer cells while also increasing the activity of tumor-specific αβ T cells. Thus, the killing of established tumors by different T cell subsets can be achieved through BTN3A1 targeting and may provide new strategies for cancer immunotherapy.

Science, this issue p. 942

Abstract

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex–independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αβ and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αβ T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αβ T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αβ and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

The advent of immune checkpoint inhibitors has revolutionized the management of certain cancers (13). However, most solid tumors remain poorly responsive to existing immunotherapies, and the successes of PD-1–targeting antibodies for melanoma and lung cancer are not frequently observed in other tumor types. Whereas most immunotherapeutic approaches focus on boosting αβ T cell responses, other leukocyte subsets with antitumor potential infiltrate tumor beds. In ovarian cancer, a disease resistant to single checkpoint blockade, ~6% of hematopoietic cells in solid tumors (>20% of CD3+ T cells) represent γδ T cells (4), which include Vγ9Vδ2 lymphocytes, the most abundant γδ T cells in peripheral blood (58). Although γδ T cells spontaneously exhibit regulatory activity at tumor beds through the production of galectin-1 (4), there is a strong rationale for rescuing their antitumor activity in coordination with effector αβ T cells to expand the range of immunotherapy-sensitive tumors.

The extracellular domains of butyrophilin (BTN) and butyrophilin-like (BTNL) molecules are structurally related to the B7 family of costimulatory ligands, which includes PD-L1, B7-H3, and B7-H4 (9). Polymorphisms of several BTN and BTNL molecules are associated with inflammatory diseases (911), and it has been suggested that the BTN3A family of BTNs could modulate antigen-specific αβ T cell responses, though the mechanism(s) is currently unknown (1215). More recently, BTN and BTNL molecules have been found to play critical roles in modulating γδ T cell functions (16, 17), for which concurrent BTN3A1-BTN2A1 interactions are essential for T cell receptor (TCR)–dependent activation of human Vγ9Vδ2+ T cells (5, 6, 1820). In vivo, this is dependent on the binding of phosphorylated metabolites to the B30.2 intracellular domain (21, 22). However, these effects can be mimicked by stabilizing the extracellular domain of BTN3A1 with CD277 (BTN3A1-3)–specific antibodies (23), possibly through multimerization of BTN3A1 and conformational changes from its spontaneous V-shaped conformation (19, 22, 23).

We hypothesized that the suppressive function of BTN3A1 against αβ T cells occurs only in its spontaneous conformation without BTN2A1 and that antibodies targeting BTN3A1 would overcome the suppression of αβ T cells and simultaneously induce γδ T cell antitumor cytotoxicity. We report that antibodies against CD277 (anti-CD277) transform BTN3A1 from an immunosuppressive to an immunostimulatory molecule, thus dynamically eliciting coordinated αβ and γδ T cell–driven antitumor immunity to abrogate the progression of established ovarian cancer.

RESULTS

BTN3A1 is overexpressed in aggressive human tumors to suppress αβ T cell activity

To investigate the potential role of BTN3A1 in cancer, we first performed Western blot assays using protein lysates from 42 stage III/IV human high-grade serous ovarian carcinomas (HGSOCs). As shown in Fig. 1A, BTN3A1 is heavily overexpressed in malignant tissues, compared with benign ovarian tumors and normal tissues. Slightly lower amounts of a specific (~53-kDa) BTN3A1 band were found in four triple-negative breast cancers analyzed (Fig. 1B), supporting that BTN3A1 expression is not restricted to ovarian malignancies. Fluorescence-activated cell sorting (FACS) analysis of freshly dissociated ovarian and breast carcinomas showed that CD277 expression was high among myeloid and tumor cells, with weaker expression found among lymphocytes (Fig. 1, C and D). Lower CD277 expression was retained on peripheral blood mononuclear cells from healthy donors, without differences between myeloid cells and lymphocytes (fig. S1A).

Fig. 1 BTN3A1 is abundantly expressed in ovarian cancer and is associated with poor outcome.

(A) Relative expression of BTN3A1 in normal tissue (n = 12 tissue samples), benign ovarian tumors (n = 9), and ovarian serous carcinoma (n = 42). Data represent means ± SEM. (B) Expression of BTN3A1 in triple-negative breast cancer (TNBC; n = 4). (C and D) Expression of CD277 within dissociated human HGSOCs (n = 9) (C) or breast cancer of mixed histology (n = 13) (D) of dendritic cells (DCs; CD45+CD1c+CD11c+MHC-II+Zbtb46+), macrophages (Mϕ; CD45+CD1cCD11c+MHC-II+), tumor cells (CD45EpCAM+), and lymphocytes (CD45+CD1cCD11cMHC-II) after normalization against the isotype control. Data represent means ± SEM. MFI, median fluorescence intensity. (E) Representative BTN3A1 expression in indicated tissue samples as determined by immunohistochemistry. Scale bars represent 200 μm. (F) Survival outcome associated with the intensity of BTN3A1 expression within ovarian cancer specimens as assessed by immunohistochemistry of tissue microarrays corresponding to 200 independent ovarian cancer patients with clinical annotations. (G) Frequency of HGSOC-infiltrating Vγ9Vδ2 T cells among total CD3+ cells (n = 13). (H) Multiplex immunofluorescence detailing the presence of γδ T cells (red) within HGSOCs. DAPI, 4′,6-diamidino-2-phenylindole. (I) Survival outcome associated with the frequency of γδ T cells within 65 HGSOCs with clinical annotations. Statistical analysis was performed as follows: for (A), one-way analysis of variance (ANOVA); for (F) and (I), log-rank (Mantel-Cox) test for survival. *P < 0.05; **P ≤ 0.01.

Immunohistochemical analysis of 398 additional HGSOCs and 19 breast cancers of mixed histology confirmed that BTN3A1 is universally expressed at tumor beds, where it is commonly localized to the membrane and cytoplasm within epithelial cells (Fig. 1E and fig. S1B). Consistent with its immunosuppressive role, higher average BTN3A1 expression in 200 ovarian cancers with clinical annotations was significantly associated with reduced patient survival (Fig. 1F). FACs analysis of an additional 13 HGSOCs confirmed that Vγ9Vδ2 lymphocytes constituted up to 2.5% of total T cells (Fig. 1G), and γδ T cell infiltration was associated with improved patient outcome (Fig. 1, H and I).

As predicted from its similarity to other B7 family members, BTN3A1 retrovirally expressed on the surface of major histocompatibility complex class I–negative (MHC-I)CD32+ K562 artificial antigen-presenting cells (BTN-K32 aAPCs) (24) abrogated OKT3-induced activation of both CD4+ and CD8+ αβ T cells sorted from the peripheral blood of multiple donors (Fig. 2A). Similarly, HLA-A2–transduced BTN-K32 aAPCs pulsed with the NY-ESO-1 peptide SLLMWITQC (BTN-K32A2) elicited similar blunting on specific TCR-transduced αβ T cells (25) (Fig. 2B and fig. S1C). αβ T cell suppression was independent of γδ T cells (fig. S1D) and was not due to phenotypic alterations in K562 cells, because similar inhibitory effects were observed using multiple clones of BTN3A1 mock-transduced aAPCs (fig. S1E). Therefore, prognostically relevant BTN3A1 effectively suppress αβ T cell responses.

Fig. 2 BTN3A1-specific antibodies relieve αβ T cell inhibition and activate Vγ9Vδ2 T cells.

(A) Proliferation and IFN-γ release of CD4+ and CD8+ T cells cultured with OKT3-loaded (500 ng/ml) BTN3A1-K32 or mock-K32 cells at a 10:1 αβ T cell:K32 ratio after 6 days. (B) Proliferation and IFN-γ release of NY-ESO-1 TCR+ T cells cultured with HLA-A2+ BTN3A1-K32 cells, or HLA-A2+ mock-K32 cells, loaded with 1 μM SLLMWITQV peptide at a 10:1 T cell:K32 ratio after 6 days. (C) Proliferation and IFN-γ release after 6 days of NY-ESO-1 TCR+ T cells cultured at a 10:1 T cell:K32 ratio with HLA-A2+ BTN3A1-K32 cells previously treated, or not, with zoledronate (Zol; 1 μM), loaded with 1 μM SLLMWITQV peptide, and preincubated with 1 μg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or 103.2). (D) Specific killing of NY-ESO-1 TCR+ T cells cocultured with OVCAR3Luci cells pretreated with 1 μM zoledronate, or not, and preincubated with 1 μg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or 103.2) at a 10:1 αβ T cell:target cell ratio for 6 hours. Data are presented as the fold change relative to isotype control. (E) Cytotoxicity of immunopurified tumor-infiltrating CD3+ αβ T cells from three HGSOC samples after coculturing with matched tumor cells pretreated with 1 μM zoledronate, or not, and preincubated with 1 μg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or 103.2) at a 5:1 αβ T cell:target cell ratio for 12 hours. (F, left) Crystal structure of the CTX-2026 interaction with CD277 homodimer in space-fill and ribbon diagram. (Middle and right) Interface highlighting hydrogen bonds between CD277 and CDRH2, CDRH3, and CDRL2 of CTX-2026 (middle) or CDRH1 of CTX-2026 (right). (Bottom) Epitopes of CTX-2026 and 20.1 are shown, with common residues highlighted in red (bottom). (G) Similar to the experiment shown in (D), except immunopurified γδ T cells were used at a 5:1 γδ T cell:target cell ratio for 24 hours. (H) Similar to the experiment in (E), except immunopurified γδ T cells were used at a 5:1 γδ T cell:target cell ratio for 24 hours. (I) Relative quantity of BTN2A1 mRNA in BTN-K32 cells electroporated with CRISPR RNA targeting BTN2A1, or not. (J) IFN-γ release from immunopurified γδ T cells cocultured with BTN3A1-K32 cells, or BTN3A1-K32BTN2A1-ablated cells previously treated, or not, with zoledronate (1 μM) and preincubated with 1 μg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or 103.2) at a 5:1 γδ T cell:K32 ratio after 72 hours. (K) Proliferation and IFN-γ release of immunopurified αβ T cells cultured with OKT3-loaded (500 ng/ml) BTN3A1-K32 or BTN3A1-K32BTN2A1-ablated cells at a 10:1 αβ T cell:K32 ratio after 6 days. For all histogram panels, data are representative of three independent experiments with similar results, and data represent means ± SEM. Statistical analysis was performed as follows: for (B) and (I), two-tailed Student’s t test; for (A), (J), and (K), two-way analysis of variance (ANOVA); for (C), (D), (E), (G), (H), and (K), one-way ANOVA. ns, not significant; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Human anti-CD277 prevent BTN3A1-mediated inhibition of αβ T cells while inducing γδ T cell activation

To block the immunosuppressive activity of BTN3A1, a panel of 15 BTN3A1-reactive, full-length monoclonal antibodies were obtained by screening a combinatorial human library expressed in a yeast presentation system (26). To avoid cross-linking, antibody-dependent T cell cytotoxicity, and complement activation, these antibodies were generated on an aglycosylated human IgG1 backbone (27). Clone CTX-2026 was the most effective at restoring pMHC TCR (Fig. 2C and fig. S1F) and OKT3 activation (fig. S1, G and H) of CD4+ and CD8+ αβ T cells, and this clone advanced as the lead compound. As expected, CTX-2026 had no effect on T cell proliferation in mock-transduced (BTN3A1) K32 or K32A2 aAPCs (fig. S1, G and I). In addition, zoledronate treatment, which activates Vγ9Vδ2T cells (19), and CD277-specific Vγ9Vδ2-agonistic [clone 20.1 (23)] and antagonistic [clone 103.2 (28)] antibodies restored BTN3A1-dependent αβ T cell activation (Fig. 2C and fig. S1J). Accordingly, zoledronate and anti-CD277 also enhanced antigen-specific killing of NY-ESO-1–transduced, HLA-A2+BTN3A1+ OVCAR3 cells (NY-OVCAR3) (Fig. 2D and fig. S1K) and heightened cytotoxic elimination of BTN3A1+ primary cancer cells from three HGSOCs by autologous tumor-infiltrating lymphocytes (Fig. 2E and fig. S1L).

To gain insights into the mechanism of CTX-2026 action, we determined the crystal structure of the CTX-2026:BTN3A1 complex (fig. S2A). Notably, the F(ab) binds to the IgV domain of CD277 with a stoichiometry of 1:1. All three CDR loops of the heavy chain are involved in the paratope, with a minimal contribution from CDRL2 (Fig. 2F). We superimposed this complex with the 20.1:CD277 complex (PDB 4F9L) to gain insight into the relative binding position of 20.1 and CTX-2026 (fig. S2B). Epitope evaluation of CTX-2026 further revealed partial spatial and sequential overlap with clone 20.1 (Fig. 2F, fig. S2C, and files S1 and S2). Accordingly, in vitro, CTX-2026 (but not 103.2) redirected the cytotoxic activity of Vγ9Vδ2 T cells from multiple donors against BTN3A1+ OVCAR3 cells (Fig. 2G) or primary HGSOC cells (Fig. 2H and fig. S3A) with the same efficacy as clone 20.1 or zoledronate.

In support of other recent reports (19, 20), CRISPR-mediated ablation of BTN2A1 in BTN-K32 aAPCs (Fig. 2I and fig. S3B) abrogated antibody- and zoledronate-dependent interferon-γ (IFN-γ) production by Vγ9Vδ2 T cells (Fig. 2J). However, BTN2A1-ablated BTN-K32 aAPCs maintained their ability to suppress αβ T cell activation (Fig. 2K and fig. S3C), suggesting that the switch between the αβ-immunosuppressive and Vγ9Vδ2-immunostimulatory activities of BTN3A1 is regulated by BTN2A1 signaling.

BTN3A1 suppresses αβ T cells by preventing the segregation of CD45 from the immune synapse

To elucidate the mechanism by which BTN3A1 inhibits αβ T cell activity, we confirmed that upon TCR activation, homodimeric BTN3A1-Fc binding (fig. S3D) to the surface of activated primary T cells blunts the phosphorylation of TCR proximal signaling molecules, as at activating residues in LCKY394, Zap70Y319, and CD3ζY142 (Fig. 3A). We excluded binding to potential immunosuppressive receptors by comparing the binding of recombinant GPR174, NRP1, and NRP2 Fc proteins to BTN-K32 with that of mock-transduced aAPCs (fig. S3E). We next used BTN3A1-Fc proteins to coimmunoprecipitate the binding partner(s) of BTN3A1 on OKT3-activated primary αβ T cells from multiple donors. In three independent experiments, liquid chromatography–tandem mass spectrometry (LC-MS/MS) showed BTN3A1 coimmunoprecipitated with a complex that consistently included only four T cell proteins with an extracellular domain—CD3ε, CD3ζ, HLA-A, and the phosphatase CD45—and also the intracellular TCR signaling protein LCK and the phosphatase PTPN6 (SHP-1) (Fig. 3B and file S3). CD45 similarly consistently coimmunoprecipitated with BTN3A1 from nonactivated primary αβ T cells and CD45+ Jurkat cells (fig. S3F and file S4). By contrast, BTN3A1 did not reproducibly coimmunoprecipitate with other heavily glycosylated or abundant surface molecules, including CD44, CD5, or CD2. Accordingly, BTN3A1-Fc proteins bound to CD45+ Jurkat cells but not to CD45-ablated Jurkat cells (fig. S3G), whereas ectopic expression of CD45RA or CD45RO (fig. S3H) rescued BTN3A1-Fc binding (Fig. 3C and fig. S3I). Furthermore, in situ proximity ligation confirmed CD45-BTN3A1 interactions within ≤30 to 40 nm on the T cell surface (Fig. 3D and fig. S3J), whereas BTN3A1-Fc proteins (but not control PD-L1-Fc) pulled down CD45 from activated αβ T cells (Fig. 3E). Although both CD45RA and CD45RO were found to directly bind to immobilized BTN3A1 proteins, binding to CD45RO was significantly stronger (Fig. 3F). Notably, the extracellular IgV domain of BTN3A1 was sufficient to engage both isoforms (Fig. 3G).

Fig. 3 BTN3A1 interacts with the CD45 phosphatase, blunting proximal T cell signaling.

(A) Binding of BTN3A1-Fc to the surface of primary human T cells (left); immunoblot of purified αβ T cells after TCR cross-linking by plate-bound OKT3 in the presence of BTN3A1-Fc or control immunoglobulin G (IgG)–Fc proteins (all at 10 μg/ml) for 1 min (right). (B) LC-MS/MS readout of BTN3A1-specific pulldowns after incubation with activated αβ T cells. (C) Binding of BTN3A1-Fc to the surface of CD45 Jurkat cells or CD45 Jurkat cells with rescued expression of CD45RA or CD45RO. (D) In situ proximity ligation far red median fluorescence between BTN3A1 and CD45, single-stained controls, or detection probes alone (S. Ab), using purified primary T cells. (E) Immunoblot of CD45 after primary T cells were coated with either BTN3A1-Fc or PD-L1-Fc (10 μg/ml), lysed, and Fc protein immunopurification (IP). (F) Absorbance at 450 nm after immobilized BTN3A1 proteins (10 μg/ml) were incubated with CD45RA or CD45RO proteins (20 μg/ml) and CD45RA and CD45RO detection antibodies (20 μg/ml), or after incubation of CD45RA or CD45RO proteins (10 μg/ml) and CD45RA and CD45RO detection antibodies (20 μg/ml) without BTN3A1 immobilization (S.Ab). (G) Similar to the experiment shown in (F), except only the IgV domain of BTN3A1 was immobilized. (H) Proliferation of CD45+ and CD45-ablated primary αβ T cells cultured with BTN3A1-K32 cells or mock-K32 cells in the presence or absence of 1 μg/ml CTX-2026 or isotype control for 6 days. (I, left) Segregation of CD45 from CD3ζ in the presence of control-Fc or BTN3A1-Fc proteins (10 μg/ml) after TCR cross-linking in the presence of plate-bound OKT3 (10 μg/ml) for 3 min. (Right) Cumulative quantification of CD3ζ and CD45 colocalization in the presence of BTN3A1-Fc or control-Fc proteins is shown from five fields per condition with ~120 cells per field. Scale bars represent 2 μm. (J) Immunoblot of CD45 Jurkat cells expressing CD45ROE624R after TCR cross-linking by plate-bound OKT3 in the presence of BTN3A1-Fc or control IgG-Fc proteins (all at 10 μg/ml) for 1 min. (K) Binding (left) and median fluorescence (right) of BTN3A1-Fc proteins after primary T cells were treated overnight with 100 U/ml of PNGase F or vehicle. (L) Binding of BTN3A1-Fc proteins to CD45+ Jurkat cells after treatment overnight with 100 U/ml PNGase F or vehicle. (M) Binding of BTN3A1-Fc proteins to CD45+ Jurkat cells after treatment with the indicated concentrations of tunicamycin, or vehicle, for 72 hours. (N) Binding of BTN3A1-Fc proteins to CD45 Jurkat cells or CD45 Jurkat cells expressing CD45RO, CD45RON→D, or CD45ROΔFibr.D. (O) Absorbance measurements, similar to those for the experiment shown in (F), except N-linked glycans were removed from both CD45RA and CD45RO prior to the assay by overnight treatment with PNGase F. (P) Immunoblot assay similar to that shown in (A), except a portion of the primary T cells was treated with PNGase F overnight (100 U/ml) before the assay. (Q) Binding (left) and median fluorescence (right) of BTN3A1-Fc proteins after primary T cells were preincubated with increasing concentrations of mannan polysaccharides or vehicle. For all histogram panels, data are representative of three independent experiments with similar results and represent means ± SEM. Statistical analysis was performed as follows: for (I), two-tailed Student’s t test; for (F), (G), and (O), two-way ANOVA; for (D), (H), (K), and (Q), one-way ANOVA. ns, not significant; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

CD45-BTN3A1 interactions specifically drive αβ T cell suppression, because CRISPR ablation of PTPRC in primary αβ T cells (fig. S3K) abrogated both the inhibitory effect of BTN3A1 and the rescuing effect of CTX-2026, whereas nonablated CD45+ T cells in the same cultures were effectively suppressed and rescued (Fig. 3H and fig. S3L).

Given these findings, we hypothesized that BTN3A1 might disrupt TCR triggering by preventing the segregation of CD45 from the immune synapse. To test this, a CD3ζ–green fluorescent protein (GFP) fusion protein was generated and was used to monitor the degree of CD3ζ-CD45 colocalization after OKT3-induced TCR triggering in the presence of BTN3A1-Fc versus control-Fc. As predicted, CD45 segregated from CD3ζ after TCR activation in the presence of control-Fc (Fig. 3I). In contrast, the presence of BTN3A1 impeded the segregation of CD45 from CD3ζ in multiple independent experiments with different donors, further supporting that BTN3A1 abrogates αβ T cell responses by effectively dismantling the immune synapse (Fig. 3I and fig. S4). We observed that αβ T cells activated in the presence of BTN3A1-Fc proteins (but not PD-L1-Fc) generated CD45-specific peptides approximately double the predicted size of monomeric CD45 under nonreducing conditions (fig. S5A and file S5), which are indicative of inhibitory CD45 dimerization (29). However, whereas CD45 Jurkat cells were not sensitive to BTN3A1 suppression at TCR-proximal residues as expected (fig. S5B), CD45 Jurkat cells expressing CD45RO with an inactivating mutation in the cytoplasmic inhibitory wedge (CD45ROE624R) remained sensitive to BTN3A1 inhibition at Y394LCK, which is consistent with localized CD45 within the immune synapse inhibiting pMHC-TCR ligation independent of its protein tyrosine phosphatase potential (Fig. 3J).

BTN3A1 binds to N-mannosylated residues in CD45

To elucidate how BTN3A1 binds to different isoforms of CD45, we focused on its heavily N-glycosylated residues (30). Peptide N-glycosylase (PNGase) F–treated (N-deglycosylated) primary T cells (Fig. 3K), CD45+ Jurkat cells (Fig. 3L and fig. S5C), and CD45 Jurkat cells with ectopic expression of CD45RA and CD45RO (fig. S5, D and E) all showed significantly reduced binding to BTN3A1-Fc proteins, whereas surface level expression of CD45 remained intact (fig. S5, C and F). Similar results were obtained using the N-glycan inhibitor tunicamycin (Fig. 3M and fig. S5G), although conservative replacement of asparagine with aspartic acid in the extracellular domain of CD45RA also abrogated BTN3A1-Fc–Jurkat cell interactions (Fig. 3N and fig. S5H); these results are all indicative of an N-glycosylation–dependent mechanism. Accordingly, N-deglycosylation by PNGase F or tunicamycin decreased CD45 coimmunoprecipitated with BTN3A1-Fc from activated αβ T cells (fig. S5I), whereas N-deglycosylation of either CD45RO or CD45RA by PNGase F abrogated their binding to immobilized BTN3A1 proteins (Fig. 3O) and treatment of primary αβ T cells with PNGase F abrogated the ability of BTN3A1 to blunt TCR proximal signaling in primary T cells (Fig. 3P). Corresponding results were observed using CD45 Jurkat cells transduced or not with CD45RO and CD45RA (fig. S5B). However, PNGase F–mediated deglycosylation of BTN3A1-Fc did not abrogate binding to CD45RO (fig. S5J), indicating BTN3A1-specific N-glycan recognition.

CD45 mutants expressing only the membrane-proximal fibronectin domains in CD45 Jurkat cells restored BTN3A1-Fc binding to an even greater extent than expression of CD45RA (Fig. 3N), suggesting increased accessibility to proximal N-mannosylated oligosaccharides (30). Accordingly, mannan polysaccharides inhibited binding of BTN3A1-Fc proteins to αβ T cells in a dose-dependent manner (Fig. 3Q). Collectively, these data suggest that BTN3A1 recognizes N-mannosylated oligosaccharides within the membrane-proximal domains of CD45, which anchors dimeric CD45 near the TCR and blunts effective TCR signaling, possibly by physically blocking pMHC-TCR ligation (3134).

Anti-BTN3A1 elicit coordinated αβ and γδ T cell responses to impede growth of established human ovarian tumors in vivo

To assess the potential of targeting BTN3A1 by utilizing CTX-2026 to rescue human αβ T cell responses against advanced tumors in vivo, we implemented the NY-OVCAR3/αβ NY-ESO-1–specific TCR T cell system (25) (Fig. 4A and fig. S1, C and K). Adoptive transfer of these (γδ T cell–free) SLLMWITQC-reactive αβ T cells had only modest effects in preventing the growth of established (75 to 250 mm3) NY-OVCAR3 tumors compared to mock-transduced T cells (fig. S6A). In contrast, BTN3A1 blockade with CTX-2026 antibodies delayed malignant progression from nine different donors (in 10 independent experiments) and was more effective than zoledronate and 20.1 (Fig. 4B and fig. S6, B and C). Superior therapeutic effects elicited by CTX-2026 were associated with significant increases in the accumulation of intratumoral antigen-reactive CD8+ T cells, compared with control IgG-treated mice (Fig. 4C). CTX-2026 was more effective than the anti–PD-1 antibody nivolumab when combined with tumor-reactive T cells (Fig. 4D and fig. S6D), despite PD-L1 up-regulation by NY-OVCAR3 tumor cells in vivo (fig. S6E). Of note, tumors in all experiments were allowed to grow for at least 15 days and were treated with a single T cell injection, with significant growth delays for tumors as large as 300 mm3 at the time of adoptive transfer (Fig. 4A and fig. S4, A and B).

Fig. 4 Targeting CD277 in vivo rescues antigen--specific αβ T cell responses and leverages the cytotoxic potential of γδ T cells.

(A) Experimental schema. (B, left) Progression of NY-OVCAR3 tumors in NSG mice treated with 1.5 × 106 NY-ESO-1-TCR–transduced αβ T cells and treated every third day with zoledronate (Zol; 0.05 mg/kg; intraperitoneally (ip)] or CTX-2026, 20.1, or control IgG (5 mg/kg; ip). (Right) Quantification of tumor weight from each group after 15 days. Data represent means ± SEM with five mice per treatment group, representative of three independent experiments with similar results. (C) Absolute number of GFP+ CD8+ αβ T cells within NY-OVCAR3 tumors treated with zoledronate, CTX-2026, 20.1, or control IgG. (D) Progression of NY-OVCAR3 tumors in NSG mice treated with NY-ESO-1-TCR αβ T cells and thennivolumab, CTX-2026, or control IgG every third day (5 mg/kg; ip). Data represent means ± SEM with five mice per treatment group, representative of three independent experiments with similar results. (E) Progression of NY-OVCAR3 tumors in NSG mice treated with CTX-2026 or treated with 3 × 105 purified γδ T cells and then CTX-2026 or control IgG every third day. Data represent means ± SEM with three mice per treatment group, representative of three independent experiments with similar results. (F) Absolute number of γδ T cells within NY-OVCAR3 tumors treated with CTX-2026, 20.1, or control IgG. (G) Progression of NY-OVCAR3 tumors in NSG mice treated with purified γδ T cells and CTX-2026 or control IgG, αβ T cells with CTX-2026 or control IgG, or the combination of antigen-specific αβ T cells and autologous γδ T cells (ratio of 6:1) with CTX-2026 or control IgG. Data represent means ± SEM with three mice per treatment group, representative of three independent experiments with similar results. (H) Representative formation of cystic cavities in NY-OVCAR3 tumors treated with the combination of antigen (Ag)-specific αβ T cells, γδ T cells, and CTX-2026. Scale bars represent 3 mm. Statistical analysis was performed as follows: for (B) (left), (D), (E), (F), and (G), two-tailed Student’s t tests; for (B) (right) and (C), one-way ANOVA. *P < 0.05; **P ≤0.01; ***P ≤ 0.001.

Notably, the protective effects of CTX-2026 in vivo were not restricted to tumor-reactive αβ T cells, because γδ T cells from eight different donors also elicited tumor growth reduction, albeit only in combination with CTX-2026 antibodies (Fig. 4E and fig. S6F). Accordingly, BTN3A1 targeting resulted in a greater accumulation of Vγ9 T cells within tumor beds (Fig. 4F). However, maximal antitumor responsiveness against established tumors was only achieved upon coadministration of γδ and tumor-specific αβ T cells into NY-OVCAR3 tumor-bearing NSG mice in combination with CTX-2026 administration (Fig. 4G and fig. S6G) and was superior to zoledronate treatment (fig. S6H). This treatment provoked the formation of cystic cavities within the tumor (Fig. 4H), which indicate partial tumor rejection. Thus, targeting BTN3A1 orchestrates coordinated responses between tumor-reactive αβ T cells rescued from BTN3A1-mediated immunosuppression and cytotoxic Vγ9Vδ2 T cells that are redirected to kill BTN3A1+ tumors.

Preexisting antitumor immune responses in immunocompetent hosts are restored by anti-CD277

To confirm the antitumor effectiveness of blocking BTN3A1 in myeloid cells in immunocompetent hosts, we engineered a knock-in mouse expressing human BTN3A1 under the control of the mouse Itgax/Cd11c promoter (BTN3A1KI) (Fig. 5A). As expected, CD11c+ bone marrow–derived dendritic cells (BMDCs) from BTN3A1KI mice expressed human BTN3A1 on the cell surface (Fig. 5B). Equally important, activation of murine T cells in the presence of BTN3A1-Fc fusion proteins decreased IFN-γ release compared with controls (fig. S7A), whereas proliferation of OT-I T cells in response to SIINFEKL-pulsed CD11c+BTN3A1+ BMDCs was significantly lower than in response to BTN3A1 BMDCs from littermates (Fig. 5C and fig. S7B). Thus, although mouse CD45 has a somewhat different pattern of glycosylation near the N terminus, human BTN3A1 can also functionally suppress mouse αβ T cells. Accordingly, treatment with anti-CD277 CTX-2026 also blocked human BTN3A1-mediated suppression of mouse αβ T cells (Fig. 5D and fig. S7C).

Fig. 5 Targeting BTN3A1 results in spontaneous antitumor immunity in BTN3A1TG mice.

(A) Schematic of the CD11c-BTN3A1 construct. (B) BTN3A1 expression on BMDCs generated from wild-type (WT) C56/BL6 mice or BTN3A1KI mice. (C and D) Proliferation of OT-I T cells in the presence of WT-derived or BTN3A1KI-derived BMDCs previously pulsed with 1 nM SIINFEKL peptide (C) and in the presence of CTX-2026 antibody (D) after 72 hours. (E) Survival of BTN3A1KI mice bearing ID8-Defb29-Vegf-a peritoneal tumors treated every 5 days with zoledronate (Zol; n = 5; 0.05 mg/kg; ip), or CTX-2026 (n = 5; 5 mg/kg; ip), 20.1 (n = 5; 5 mg/kg; ip), or control IgG (n = 5; 5 mg/kg; ip), beginning 7 days after tumor challenge. (F) Frequency of CD8+ T cells among total CD3+ T cells in the ascites fluid of BTN3A1KI mice bearing ID8-Defb29-Vegf-a peritoneal tumors treated as described for (E). (G) ELISpot readout comparing IFN-γ release from CD8+ T cells isolated from BTN3A1KI mice bearing ID8-Defb29-Vegf-a peritoneal tumors at day 25, treated as for (E). (H) Frequency of γδ TCR+ T cells among total CD3+ T cells in the ascites fluid of BTN3A1KI mice, or WT C57/Bl6 mice, bearing ID8-Defb29-Vegf-a peritoneal tumors and as treated as for (E). Data represent means ± SEM of two independent experiments performed with five replicates each. (I) Survival of BTN3A1KI mice bearing ID8-Defb29-Vegf-a peritoneal tumors treated every 5 days with PD-1 neutralizing antibody (n = 8; 5 mg/kg; ip), CTX-2026 (n = 8; 5 mg/kg; ip), or irrelevant IgG (n = 8; 5 mg/kg; ip), beginning 7 days after tumor challenge. For (F) and (G), data are representative of three independent experiments with similar results. Statistical analysis was performed as follows: for (D) and (E), log-rank (Mantel-Cox) test for survival; for (F), two-tailed Student’s t test; for (G) and (H), one-way ANOVA. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

To test the role of BTN3A1 in an immunocompetent syngeneic ovarian cancer model, BTN3A1KI mice were challenged intraperitoneally with orthotopic ID8-Defb29/Vegf-a tumors, an aggressive model that responds to checkpoint inhibitors (35) and provokes the accumulation of tumor-promoting CD11c+ myeloid cells (36). Predictably, compared with control littermates, BTN3A1KI recipients accumulated BTN3A1+ dendritic cells in the ascites fluid (fig. S7D) and showed accelerated malignant progression (fig. S7E). Targeting tumor-bearing BTN3A1KI recipient mice with zoledronate extended their survival, but to a lesser extent than treatment with CTX-2026 and 20.1 antibodies (Fig. 5E), with corresponding differences in intratumoral CD8+ T cell accumulation (Fig. 5F). Consequently, the frequencies of peritoneal T cells responding to cognate tumor antigen in IFN-γ enzyme-linked immunosorbent spot (ELISpot) analyses were increased by all BTN3A1-targeting therapeutics, with the highest readout for CTX-2026 (Fig. 5G). Murine γδ T cells accumulated in the ascites fluid of BTN3A1KI tumor-bearing mice in a CTX-2026–dependent manner (Fig. 5H). As in our humanized model, targeting BTN3A1 was demonstrably more effective in delaying malignant progression than neutralizing the PD-L1/PD-1 checkpoint (Fig. 5I). Thus, targeting of BTN3A1 overcomes the highly immunosuppressive microenvironment of ovarian cancer, a disease that, thus far, has been resistant to existing checkpoint inhibitors.

DISCUSSION

We report that targeting BTN3A1 with certain human antibodies is sufficient to elicit coordinated αβ and γδ T cell antitumor responses against established tumors, and we have demonstrated that BTN3A1 targeting in validated orthotopic xenograft and syngeneic models of ovarian cancer is superior to PD-1 checkpoint therapy. Most human malignancies remain resistant to existing checkpoint inhibitors, which are aimed to rescue αβ T cell responses. Yet, γδ T cells also infiltrate multiple human cancers, where they primarily play regulatory roles (4, 37). Engaging the cooperation of these two T cell subsets could therefore extend the range of cancer patients benefiting from immunotherapy. In addition, BTN3A1 targeting was more effective than PD-1 neutralization in αβ T cells in our PD-L1+ humanized systems, and it is possible that anti-CD277 also block αβ T cell inhibition by the nearly identical BTN3A2 and BTN3A3. Because γδ T cells also express PD-1, it will be interesting to determine whether coordinated action also takes place in patients that respond to checkpoint inhibitors. Nevertheless, unlike PD-L1, BTN3A1 does not require a specific receptor to mediate its immunosuppressive activity. Rather, BTN3A1, independently of BTN2A1, inhibits αβ T cells by binding to N-mannosylated residues in human CD45, preventing its segregation from the immune synapse and promoting its dimerization, and thus abrogating effective TCR activation. At least in ovarian cancer, BTN3A overexpression in myeloid and tumor cells is associated with accelerated malignant progression; thus, it is possible that BTN3A1 targeting might be combined with other checkpoint inhibitors to enhance immunotherapeutic responses.

An interesting finding in our study is that both phosphometabolites and CTX-2026 antibodies transform BTN3A1 from an immunosuppressive to an immunostimulatory mediator. Recent work showed that phosphometabolite-driven cosignaling by BTN3A1 and BTN2A1 promotes Vγ9Vδ2 activation, whereas BTN2A1 is also required for the agonistic activity of antibody 20.1 (19). The structure of CTX-2026 bound to BTN3A1 is similar to that of mouse scFv 20.1:BTN3A complexes (23), in which both antibodies partially share an overlapping epitope. This supports the results of Adams and colleagues that agonistic antibodies modify the extracellular domains of BTN3A molecules, mimicking the effect of phosphoantigens (23). It is tempting to speculate that antibody-phosphoantigen–induced clustering separates BTN3A1 from MHC:antigen complexes, allowing robust engagement with specific αβ TCRs. However, BTN2A1 is essential for antibody-driven switching of BTN3A1 from an immunosuppressive to immunostimulatory action, suggesting that CD277 clustering could release a BTN2A1 partner additionally required for Vγ9Vδ2 activation. Perhaps these mechanisms might be effectively elicited in vivo and in situ at tumor beds as an anticancer intervention.

Supplementary Materials

science.sciencemag.org/content/369/6506/942/suppl/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S3

References (3848)

Files S1 to S7

References and Notes

Acknowledgements: We are grateful to Chemical Biology, Analytic Microscopy, Advanced CLIA Tissue Imaging, Proteomics, and Flow Cytometry Shared Resources at Moffitt Cancer Center, as well as E. Larson of HarkerBio, for exceptional support. Funding: Support for shared resources was provided by Cancer Center Support Grant (CCSG) CA076292 to H. Lee Moffitt Cancer Center. This study was supported by the National Institutes of Health (R01CA157664, R01CA124515, R01CA178687, R01CA211913, and U01CA232758 to J.R.C.-G.; R01CA184185 to P.C.R.; and R01NS114653 and R21CA248106 to J.R.C.-R.); the U.S. Department of Defense Ovarian Cancer Research Program (W81XWH-16-1-0438 and W81XWH-20-1-0191 to J.R.C.-R.), and Stand Up to Cancer (SU2C-AACR-IRG-03-16 and SU2C-AACR-PS24 to J.R.C.-R.). K.K.P. was supported by T32CA009140 and an American Cancer Society Postdoctoral Fellowship. Competing interests: J.R.C.-G. and D.I.G. are members of the External Advisory Board of Compass Therapeutics and receive consulting fees and stock options from the company. J.R.C.-G. additionally receives consulting fees from Anixa Bioscience and Leidos. A.P.-P. is the Vice President for Research & Development at Geneos Therapeutics. J.L., M.O., P.B., B.M., U.E., and M.S. were employees of Compass Therapeutics. J.R.C.-G., K.K.P., M.S., B.M., and P.B. are inventors on patent application WO2020033923A1 submitted by Compass Therapeutics LLC, The Wistar Institute of Anatomy and Biology, and H. Lee Moffitt Cancer Center; this patent covers a method for reducing CD277-mediated inhibition of αβ T cells, as well as a method for inducing or enhancing CD277-mediated γδ T cell stimulation. Data and materials availability: The crystal structure of the human BTN3A1 ectodomain in complex with the CTX-2026 Fab was deposited in wwPDB (accession code PDB ID 6XLQ). The humanized BTN3A1 transgenic mouse, as well as CD45-ablated Jurkat cells transduced with CD45RA or CD45RO, are available from J.R.C.-G. under a material transfer agreement with Moffitt Cancer Center.

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