BTN2A1 targeting reprograms M2-like macrophages and TAMs via SYK and MAPK signaling.

Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAbs), and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift toward an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T cell proliferation and interferon-gamma (IFNγ) secretion. Mechanistically, BTN2A1 engagement induced spleen tyrosine kinase (SYK) recruitment, leading to sequential SYK and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of SYK or ERK phosphorylation abolished M2 reprogramming upon BTN2A1 engagement. Our findings, derived from an analysis of macrophages from healthy donors and human tumors, underscore the pivotal role of BTN2A1 in immunosuppressive macrophage differentiation and function, offering potential applications in cancer immunotherapy.

BTN3A Targeting Vγ9Vδ2 T Cells Antimicrobial Activity Against Coxiella burnetii-Infected Cells.

Vγ9Vδ2 T cells have been reported to participate to the immune response against infectious diseases such as the Q fever caused by Coxiella burnetii infection. Indeed, the number and proportion of Vγ9Vδ2 T cells are increased during the acute phase of Q fever. Human Vγ9Vδ2 T cell responses are triggered by phosphoantigens (pAgs) produced by pathogens and malignant cells, that are sensed via the membrane receptors butyrophilin-3A1 (BTN3A1) and -2A1 (BTN2A1). Here, by using CRISPR-Cas9 inactivation in THP-1 cells, we show that BTN3A and BTN2A are required to Vγ9Vδ2 T cell response to C. burnetii infection, though not directly involved in the infection process. Furthermore, C. burnetii-infected monocytes display increased BTN3A and BTN2A expression and induce Vγ9Vδ2 T cell activation that can be inhibited by specific antagonist mAb. More importantly, we show that the antimicrobial functions of Vγ9Vδ2 T cells towards C. burnetii are enhanced in the presence of an BTN3A activating antibody. This supports the role of Vγ9Vδ2 T cells in the control of C. burnetii infection and argues in favor of targeting these cells as an alternative treatment strategy for infectious diseases caused by intracellular bacteria.

BTN2A1, an immune checkpoint targeting Vγ9Vδ2 T cell cytotoxicity against malignant cells.

The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

FHR4-based immunoconjugates direct complement-dependent cytotoxicity and phagocytosis towards HER2-positive cancer cells.

Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-α-/ß-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VH H targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VH H(T) or VH H(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VH H(T) or FHR4/VH H(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface.