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364 Antigen abundance and TCR avidity impact T cell-mediated tumor recognition in novel B16F10 ACT model
  1. Jenna Newman1,
  2. John Finnigan1,
  3. Andrew Ishizuka2,
  4. Geoffrey Lynn2,
  5. Alexander Rubinsteyn3,
  6. Timothy O’Donnell1,
  7. Jeffrey Hammerbacher4 and
  8. Nina Bhardwaj1
  1. 1Icahn School of Medicine at Mount Sinai, New York, NY, USA
  2. 2Avidea Technologies, Washington, USA
  3. 3University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
  4. 4Medical University of South Carolina, Charleston, SC, USA


Background Adoptive cell transfer (ACT) of neoantigen-reactive CD8+ T cells has had some success in the clinic; however, mouse models recapitulating neoantigen-reactive CD8+ T cell ACT have been limited, especially in poorly immunogenic models such as the murine melanoma model B16F10. Further, direct comparison of neoantigen-reactive CD8+ T cell ACT versus ACT utilizing T cells reactive against overexpressed-self or heteroclitic tumor-associated antigen (TAA) peptides has been lacking. To address these gaps, we developed a model system to study neoantigen- and TAA-reactive CD8+ T cell ACT in parallel.

Methods Whole exome sequencing and RNA sequencing were employed to predict neoantigens present in B16F10. C57BL/6 mice were then administered charge-modified TLR7/8 conjugate vaccines targeting neoantigenic peptides predicted to elicit T cell responses. Vaccination against neoepitopes and previously characterized TAA epitopes elicited neoantigen- or TAA- reactive CD8+ T cells and modest tumor growth control; T cell receptors were isolated from neoantigen- and TAA-reactive CD8+ T cell clones. To develop an ACT model, we conducted CRISPR/Cas9-mediated knockdown of endogenous TCR and subsequent transduction (g-retrovirus encoding neoantigen- or TAA-reactive TCRs) in murine CD8+ T cells. T cells were expanded in vitro for use in downstream in vitro and in vivo applications.

Results Peptide stimulation in vitro of neoantigen- and TAA-reactive T cells revealed wide ranges of 1) specificity (vs. cross-reactivity to wild type peptide), and 2) avidity for cognate peptide. Neoantigen- and TAA-reactive T cells were able to recognize B16F10 cells in vitro, with the most robust recognition (readout:% T cells IFNg+) when target antigen is highly expressed by tumor cells. Ability of neoantigen- or TAA-reactive T cells to kill B16F10 in vitro was strongly dependent upon both tumor antigen expression and T cells’ TCR avidity. Similarly, reduction of tumor growth in vivo required both high tumor antigen expression and transfer of high avidity neoantigen- or TAA-reactive CD8+ T cells.

Conclusions To conclude, we have created a novel model of neoantigen- and TAA-reactive ACT in immunotherapy-refractory B16F10 melanoma. Our data suggest that antigen abundance and TCR avidity are parameters that influence ACT efficacy; future research will be conducted to dissect the individual and summative contributions of these parameters and translate this knowledge towards improving ACT design in the clinic.

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