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192 Characterization of antibodies against BTN2A1 for Vδ2+ γδ T cell-based tumor immunotherapy
  1. Kok Fei Chan1,
  2. Simone Ostrouska1,
  3. Ranjeeta Prasad2,
  4. Daria Kurtov2,
  5. Andrew Hammet2,
  6. Marc Rigau3,
  7. Adam Uldrich3 and
  8. Andreas Behren1
  1. 1Olivia Newton-John Cancer Research Institute, and School of Cancer Medicine, La Trobe University, Heidelberg, Victoria, Australia, Melbourne, Australia
  2. 2CSL Limited at the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia, Melbourne, Australia
  3. 3Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria 3010, Australia, Melbourne, Australia


Background Human γδ T cells are ideal candidates for tumor immunotherapy because of their natural tropism for tumor microenvironment, elicit rapid innate-like immune responses upon tumor recognition and the ability to orchestrate other tumor-infiltrating immune cells for tumor cell killing.1 Our group has recently defined aspects of the mechanism of T-cell receptor (TCR) dependent activation of Vγ9Vδ2+ T cells by tumors following the presentation of phosphoantigens via the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1.2 Dysregulation of the mevalonate pathway in tumors can cause activation of Vγ9Vδ2+ T cells via phosphoantigen accumulation and induces γδ T cell chemotaxis toward tumor cells.3, 4 Most clinical studies so far have used aminobisphosphonates (to promote accumulation of phosphoantigens in cells) or synthetic phosphoantigen analogues such as bromohydrin pyrophosphate (BrHPP) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) to activate Vγ9Vδ2+ T cells in cancer patients.5–8 More recently, agonist antibodies against BTN3A (e.g., clone 20.1, ICT-01 and CTX-2026) have been identified and used as a phosphoantigen-independent approach to activate Vγ9Vδ2+ T cells for targeted cell killing.1

Methods We are currently characterizing a number of anti-BTN2A1 antibodies that can potentially be used to modulate the activity of Vγ9Vδ2+ T cells in in vitro assays.

Results We have also established a pre-clinical humanized tumor model using NOD scid gamma (NSG) mice and showed delayed tumor growth in mice that received 4 rounds of human Vδ2+ γδ T cell adoptive cell transfer in combination with clone 20.1 agonist antibody, which will allow further characterization of our anti-BTN2A1 antibodies.

Conclusions Taken together, our study has demonstrated the potential to target BTN2A1 and BTN3A1 for Vγ9Vδ2+ T cell-based cancer immunotherapy development.


  1. Chan KF, Da Gama Duarte J, Ostrouska S, Behren A. Gamma-delta T cells in the tumor microenvironment–interactions with other immune cells. Frontiers in Immunology. 2022;13:3598.

  2. Rigau M, Ostrouska S, Fulford TS, Johnson DN, Woods K, Ruan Z, et al. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science. 2020;367(6478):eaay5516.

  3. Benzaïd I, Mönkkönen H, Stresing V, Bonnelye E, Green J, Mönkkönen J, et al. High phosphoantigen levels in bisphosphonate-treated human breast tumors promote Vγ9Vδ2 T-cell chemotaxis and cytotoxicity in vivo. Cancer research. 2011;71(13):4562–72.

  4. Ashihara E, Munaka T, Kimura S, Nakagawa S, Nakagawa Y, Kanai M, et al. Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells. Biochemical and biophysical research communications. 2015;463(4):650–5.

  5. Abe Y, Muto M, Nieda M, Nakagawa Y, Nicol A, Kaneko T, et al. Clinical and immunological evaluation of zoledronate-activated Vγ9γδ T-cell-based immunotherapy for patients with multiple myeloma. Experimental Hematology. 2009;37(8):956–68.

  6. Meraviglia S, Eberl M, Vermijlen D, Todaro M, Buccheri S, Cicero G, et al. In vivo manipulation of Vγ9Vδ2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients. Clinical & Experimental Immunology. 2010;161(2):290–7.

  7. Sebestyen Z, Prinz I, Déchanet-Merville J, Silva-Santos B, Kuball J. Translating gammadelta (γδ) T cells and their receptors into cancer cell therapies. Nature Reviews Drug Discovery. 2020;19(3):169–84.

  8. Yazdanifar M, Barbarito G, Bertaina A, Airoldi I. γδ T cells: the ideal tool for cancer immunotherapy. Cells. 2020;9(5):1305.

Ethics Approval ‘This study was approved by Australian Red Cross for the isolation of human Vδ2+ γδ T cells from healthy donors’ peripheral blood mononuclear cells, agreement number: 21–07VIC-09.’

‘This study was approved by Austin Health Animal Ethics Committee for adoptive cell transfer of human Vδ2+ γδ T cells into NSG mice, AEC Reference number: A2020/05661.’

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