Theo Lorenzini

T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has been successfully translated to clinical practice for the treatment of B cell malignancies. The suppressive microenvironment of many malignancies is a bottleneck preventing treatment success of CAR T cells in a broader range of tumours. Among others, the immunosuppressive metabolite adenosine is present in high concentrations within many tumours and dampens anti-tumour function of immune cells and consequently therapeutic response. METHODS: receptor antagonist AB928/etrumadenant on CAR T cell cytokine secretion, proliferation, and cytotoxicity. Using phosphorylation-specific flow cytometry, we evaluated the capability of AB928 to shield CAR T cells from adenosine-mediated signalling. The effect of orally administered AB928 on CAR T cells was assessed in a syngeneic mouse model of colon carcinoma. RESULTS: We found that immunosuppressive signalling in CAR T cells in response to adenosine was fully blocked by the small molecule inhibitor. AB928 treatment enhanced CAR T cell cytokine secretion and proliferation, granted efficient cytolysis of tumour cells in vitro and augmented CAR T cell activation in vivo. CONCLUSIONS: Together our results suggest that combination therapy with AB928 represents a promising approach to improve adoptive cell therapy.

We have isolated and mapped the rHuEPO epitopes for three noncompeting anti-EPO monoclonal antibodies (MoAbs). The MoAb 9G8A recognizes a linear epitope that includes amino acids 13, 16, and 17. MoAb F12 recognizes a conformational epitope that includes amino acids 31 through 33, 86 through 91, and 138. MoAb D11 recognizes a conformational epitope that includes amino acids 64 through 78 and 99 through 110. MoAb D11 neutralizes rHuEPO activity which suggests that its epitope may contain the receptor binding domain. Analysis of the effect of mutations on folding allowed the identification of buried residues, alpha-helical, and non alpha-helical regions. This data along with epitope mapping data of anti rHuEPO monoclonals was used to model rHuEPO protein structure. A model consistent with the data is a 4-helix bundle with short and long interconnecting loops.

We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability.