Butyrophilin (BTN) family: γδ T Cell Receptor Ligands, Immunomodulators and More

Immunotherapy is an essential option for current disease treatment. However, most patients fail to respond adequately to existing immunotherapies, such as PD-1/PD-L1 or CTLA-4 antibody drugs. Current immune therapies primarily focus on the function of αβ T cells, neglecting the role of γδ T cells that have nonspecific killing functions. Butyrophilins (BTNs) are a group of transmembrane proteins that belong to the immunoglobulin (Ig) superfamily. They have a similar structure to immunosuppressive B7 family members, like CD80 and PD-L1, at the extracellular domain level. BTNs play a crucial role in reactivating the anti-tumor function of γδ T cells.

BTN3A1&BTN2A1 are involved in activating γδ T cells

γδ T cells are a minor population of T cells in the human body, mainly involved in innate immune response. Unlike αβ T cells, γδ T cells typically have limited antigen receptor diversity and can only recognize common antigenic components of pathogens.

They identify ligands using TCRγδ dimers, which include cognate antigens like stress-induced antigens, phosphorylated antigens, heat shock proteins (HSP), etc. Additionally, they can directly recognize intact polypeptides that have not been processed by antigen-presenting cells.

BTN3A1 (CD277) and BTN2A1 are members of the BTN family and belong to the Ig superfamily of transmembrane proteins. Their extracellular segments are stabilized with disulfide bonds and contain IgV and IgC domains, which are homologous to the B7 family. Furthermore, their cytoplasmic region contains a B30.2/SPRY domain. BTN proteins are involved in regulating both innate and adaptive T cell immune responses. Notably, BTN3A1 has a specific role in activating γδ T cells.

Figure 1: Schematic representation of the modular domain structure of the members of human butyrophilin family [1].

Cells infected by pathogens or tumor cells produce abnormal metabolites such as (E)-4-hydroxy-3-methyl- but-2-enyl pyrophosphate (HMBPP), dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The B30.2 structural domain of BTN3A1 can bind to these phosphonates, causing a conformational change in itself and forming a heterodimer with BTN2A1, which is then recognized by γδ T cells. This leads to the activation of γδ T cells, which induce the death of target cells through various methods, such as releasing granzymes or perforin to directly kill tumor cells, or indirectly suppressing them by secreting cytokines like IFNγ and TNFα.

BTN3A1 and BTN2A1 are key proteins that mediate the recognition of phosphoantigens by γδ T cells. Together, they can activate the most abundant subset of γδ T cells in peripheral blood, γ9δ2 T. This non-classical inside-out signaling plays a crucial role in the body's anti-infective, anti-tumor, and immunomodulatory processes.

Figure 2: Activation of γδ T cells by BTN3A1-BTN2A1 heterodimers [2].

Drug development around BTN3A1-BTN2A1

Antibodies targeting BTN3A1

BTN3A1 prevents the segregation of N-glycosylated CD45 from the immune synapse, which in turn inhibits the activation of tumor-reactive αβ T cell receptors. Antibodies targeting BTN3A1 convert BTN3A1 from an immunosuppressive molecule to an immunostimulatory molecule, inducing effector activity of αβ T cells as well as BTN2A1-dependent killing of BTN3A1-positive cancer cells by γδ T cells. Consequently, targeting BTN3A1 may mobilize αβ T cells and γδ T cells for a synergistic attack against established tumors, offering a novel therapeutic strategy for tumors that resist current immunotherapies [3].

Recent reports on BTN3 antibodies have highlighted the development of ICT01 by ImCheck Therapeutics. ICT01 is a humanized monoclonal antibody that binds to the extracellular domains of all three isoforms of BTN3A1/A2/A3. It induces the activation of non-pAg-dependent γ9δ2 T cells, enabling them to kill various types of tumor cells. Currently, ICT01 is undergoing phase II clinical trials for its effectiveness in targeting solid tumors.

Figure 3: Mechanism of action of ICT01 [4].

BTN3A1-BTN2A1 fusion protein

A recent study has developed a novel structure for the BTN3A1-BTN2A1 fusion protein. This structure is formed by combining the extracellular domains of BTN2A1 and BTN3A1 with a specifically designed CD19scfv segment. It serves as a bridge that connects γδ T cells and CD19-positive tumor cells, facilitating targeted killing by γδ T cells.

Figure 4: Bispecific "binder" containing BTN3A1-BTN2A1 heterodimeric fusion protein [5].

BTN family proteins from KACTUS

γδ T cells are among the most potent cytotoxic cells, with the ability to directly exert anti-tumor activity. Notably, they are not restricted by major histocompatibility complex (MHC), which means they do not trigger graft-versus-host reactions (GVHR), positioning them as candidates for universal cell therapy products. The therapeutic drugs related to γδ T cells, particularly those targeting BTN2A1/BTN3A1, have significant potential for development. While BTN-targeted therapies are mostly in the research phase without market-available drugs, the deepening research into BTN is expected to bring additional benefits to patients.

KACTUS provides high-quality BTN3A1, BTN2A1 and other BTN family proteins, which have undergone stringent quality testing, to facilitate the development of BTN-related targeted drugs.

Product Validation Examples

Figure 5: Human BTN3A1 & BTN2A1 can bind Anti-BTN3A1 Antibody with EC50 value of 5.9 ng/ml, as verified by ELISA.

Figure 6: Cynomolgus BTN3A1 can bind Anti-BTN3A1 Antibody with EC50 value of 10.9 ng/ml, as verified by ELISA.

Figure 7: Human BTN2A1 can bind Human CD209 protein with an affinity constant of 0.12 μM, as verified by SPR assay.

Available BTN Family Proteins

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References:

[1] Redwan EM, Al-Hejin AM, Almehdar HA, Elsaway AM, Uversky VN. Prediction of Disordered Regions and Their Roles in the Anti-Pathogenic and Immunomodulatory Functions of Butyrophilins. Molecules. 2018 Feb 4;23(2):328.

[2] Rigau M, Ostrouska S, Fulford TS, Johnson DN, Woods K, Ruan Z, McWilliam HEG, Hudson C, Tutuka C, Wheatley AK, Kent SJ, Villadangos JA, Pal B, Kurts C, Simmonds J, Pelzing M, Nash AD, Hammet A, Verhagen AM, Vairo G, Maraskovsky E, Panousis C, Gherardin NA, Cebon J, Godfrey DI, Behren A, Uldrich AP. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science. 2020 Feb 7;367(6478):eaay5516.

[3] Payne KK, Mine JA, Biswas S, Chaurio RA, Perales-Puchalt A, Anadon CM, Costich TL, Harro CM, Walrath J, Ming Q, Tcyganov E, Buras AL, Rigolizzo KE, Mandal G, Lajoie J, Ophir M, Tchou J, Marchion D, Luca VC, Bobrowicz P, McLaughlin B, Eskiocak U, Schmidt M, Cubillos-Ruiz JR, Rodriguez PC, Gabrilovich DI, Conejo-Garcia JR. BTN3A1 governs antitumor responses by coordinating αβ and γδ T cells. Science. 2020 Aug 21;369(6506):942-949.

[4] https://www.imchecktherapeutics.com/pipeline/ict01/

[5] https://www.shattucklabs.com/wp-content/uploads/2022/04/2022_AACR_Shattuck-Poster_GADLEN_Final.pdf