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208 T-SIGn vector-mediated antigen “spray painting” and tumor microenvironment (TME) reprogramming to enable CAR-T cell targeting of solid tumors
  1. Maria Stella Sasso,
  2. Rachel Bergin,
  3. Manuela Zonca,
  4. Eva Vainiute,
  5. Tae Hyun Jang,
  6. Zahra Oubihi,
  7. Meg Snowden,
  8. Alice Muntzer,
  9. Carla Cerqueira,
  10. Rochelle Lear,
  11. Katy West and
  12. Brian Champion
  1. PsOxus Therapeutics Ltd, Abingdon, UK


Background Multiple barriers in the tumor microenvironment (TME) have hampered development of CAR-T cell therapies for solid tumors. These challenges include an immunosuppressive TME, poor trafficking of CAR-T cells to the tumor and shortage of highly expressed tumor-specific antigens. We recently demonstrated that Tumor-Specific Immuno-Gene (T-SIGn) viral vectors encoding immunostimulatory cytokines, costimulators and chemokines can reprogram the TME towards a pro-inflammatory phenotype resulting in a markedly increased therapeutic efficacy of CAR-T cells in a A549 human tumor xenograft and lung metastasis model.1 Here we further explored the potential of the T-SIGn platform in combination with CAR-T cell therapy by developing and characterizing a T-SIGn viral vector that simultaneously expresses immunostimulatory cytokines/chemokines and a secreted bispecific protein incorporating a CAR-T cell target antigen. This secreted ligand binds to (“spray paints”) tumor cells to enable recognition by CAR-T cells.

Methods We used a T-SIGn virus (NG-1125) expressing a secreted anti-HER2-CD19 fusion protein (saH2-19), as a model “spray paint” antigen, encoded together with IFNa and CXCL9 as example cytokine/chemokines. In vitro, human tumor cell lines were used to assess the ability of T-SIGn viruses to induce tumor-specific expression and activity of saH2-19 as CAR-T cell target antigen. We quantified T-SIGn vector-encoded CD19 expression on tumor cell surfaces using flow cytometry and CAR-T mediated killing via xCELLigence. In vivo, expression of CD19 fusion protein and transferred CD19 CAR-T cells in tumors were assessed by flow cytometry analysis and immunohistochemistry of A549 tumor xenografts.

Results Using in vitro human cell culture models, the NG-1125 vector led to efficient expression of saH2-19 on tumor cell surfaces, both on vector-infected and non-infected or non-permissive cells. This enabled effective antigen-specific tumor cytotoxicity by CD19-specific CAR-T cells. Using an in vivo human A549 lung tumor xenograft model adoptively transferred with human CD19-specific CAR-T cells, NG-1125 induced tumor-specific CD19 expression on both vector infected and non-infected cells (demonstrating antigen “spray painting”) together with accumulation of activated T cells. This accumulation of T cells was not seen with a vector only expressing the saH2-19 transgene.

Conclusions Together, our data provide a proof of concept that T-SIGn vectors can be designed to deliver TME-modifying immunomodulators together with “spray paint” antigens that effectively enable tumor cell recognition and destruction by CAR-T cells specific for target antigens not endogenously expressed by tumors. Further studies are exploring the full potential of this “spray painting” approach to enable CAR-T cell therapy for of solid tumors.


  1. Sonzogni O, Zak DE, Sasso MS, Lear R, Muntzer A, Zonca M, West K, Champion BR, Rottman JB. T-SIGn tumor reengineering therapy and CAR T cells synergize in combination therapy to clear human lung tumor xenografts and lung metastases in NSG mice. Oncoimmunology 2022; 11.

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