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1362 Agonistic 4–1BB IgM antibodies enhance CD8+ T cell proliferation and proinflammatory cytokine secretion in vitro and in vivo
  1. Tasnim Kothambawala,
  2. Thin Wai,
  3. Poonam Yakkundi,
  4. Natalia L dos Santos,
  5. Adam Smith,
  6. Gary Bolton,
  7. Aditya Bharti,
  8. Balaji Banoth,
  9. Deepal Pandya,
  10. Vidhya Bai Krishnoji Rao,
  11. Elizabeth Perez,
  12. Sachi Rahman,
  13. Paul Hinton,
  14. Yuan Cao,
  15. Harrison Horwitz,
  16. Dean Ng,
  17. Umesh Muchhal,
  18. Bruce A Keyt,
  19. Angus M Sinclair,
  20. Hyewon Phee and
  21. Beatrice Wang
  1. IGM Biosciences, Mountain View, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background 4–1BB is an inducible costimulatory receptor expressed on activated T and natural killer cells. 4–1BB activation triggers an NFkB signaling cascade that results in effector cell proliferation and cytokine production. Since receptor multimerization is required for 4–1BB activation, we hypothesized that multivalent IgM antibodies would be effective 4–1BB agonists.

Methods Anti-4–1BB IgM and IgG antibodies were generated by inserting the same variable domains into an IgM or IgG heavy chain framework and co-expressing with light chain and J chain for IgM. Binding affinity was determined using surface plasmon resonance and binding to 4–1BB expressing cells was evaluated by flow cytometry. 4–1BB signaling was evaluated using reporter cells. Primary T cells were labeled with Cell Trace Violet, treated with anti-CD3 and 4–1BB agonists, and proliferation and IFNγ secretion were assessed by flow cytometry and MSD. In vivo pharmacodynamic studies were performed in human PBMC-engrafted NSG mice (graft versus host disease (GVHD) model) and in human 4–1BB knock-in mice implanted with MC38 tumors. Immune profiling in the blood, spleen, and tumor was assessed by flow cytometry and cytokine analysis by MSD.

Results The 4–1BB IgM and IgG antibodies evaluated had different binding affinities and ability to compete ligand. Although IgG and IgM antibodies bound similarly to cells, IgMs were significantly more potent than IgGs in reporter assays. Anti-4–1BB IgMs were also more effective at enhancing CD8+ T cell proliferation and IFNγ secretion in vitro. In a GVHD model, anti-4–1BB IgM was superior to IgG at expanding CD8+ T cells and these cells exhibited increased proliferative and cytotoxic capacity, evidenced by Ki67 and granzyme B expression. Anti-4–1BB IgM also reduced the number of total and activated FoxP3+ regulatory T cells compared to IgG. IFNγ and TNFα were also increased in the serum following treatment with anti-41BB IgM treatment. In an MC38 model in human 4–1BB knock-in mice, 4–1BB agonist antibodies increased CD8+ T cells and the CD8+/Treg ratio within the tumor. In the blood and spleen, 4–1BB agonists activated CD8+ and CD4+FoxP3- T cells and increased their proliferation. Granzyme B expression was also increased in CD8+ and CD4+FoxP3- T cells from the blood and in CD8+ T cells from the spleen following 4–1BB agonist treatment.

Conclusions Agonist 4–1BB IgM antibodies can efficiently crosslink 4–1BB to drive CD8+ T cell proliferation and cytokine production in vitro and in vivo. These studies suggest a potential opportunity for the development of agonistic IgMs targeting 4–1BB.

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