We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4 C Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted Tcell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNg, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4C and CD8 C T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.
Background: The functional role and clinical utility of measuring PD-1, LAG-3 and TIM-3 in NSCLC tumor tissue remain poorly understood even though corresponding clinical trials with blocking agents are ongoing. We analyzed the expression of these targets in association with key functional immune metrics and outcome after treatment with PD-1 axis blockers in human NSCLC. Methods: We performed CyTOF on immune cell suspensions from 20 primary human NSCLCs to map the distribution of PD-1, LAG-3 and TIM-3 and explore their function. We analyzed RNA levels of these markers in TCGA NSCLC datasets and their association with CD4/CD8 mRNA and mutational burden. Using multiplex quantitative immunofluorescence (QIF) we measured the levels of CD3, PD-1, LAG-3 and TIM-3 in 66 pre-treatment tumor samples from NSCLC patients receiving PD-1 axis blockers in 2 independent cohorts (Cohort #1, Yale, N ¼ 42 cases [Training set] and cohort #2, Cleveland Clinic/University of Navarra, N ¼ 24 [Validation set]. Results: In primary NSCLCs, PD-1 was predominantly expressed on T-and NKT cells. LAG-3 expression was higher in CD8 þ , CD4 þ CD25 þ FOXP3 þ and NKT cell subsets, but low/absent in antigen-presenting cells (APCs). TIM-3 was broadly expressed in adaptive and innate immune cells, with the highest levels in APCs. Expression of all 3 markers in T-cells was associated with lymphocyte activation (CD69/HLA-DR), effector function (Granzyme-B) and proliferation (Ki-67). LAG-3-expressing T-cells showed higher association with early activation and effector function than TILs expressing PD-1 or TIM-3. In TCGA, PD-1 and LAG-3 transcripts strongly correlated with CD8, while TIM-3 was associated with CD4 mRNA. There was limited association between the markers and tumor mutational burden. In pre-treatment specimens from both cohorts of patients treated with PD-1 blockers, elevated LAG-3 but not PD-1 or TIM-3 protein were significantly associated with shorter OS. Conclusions: PD-1, LAG-3 and TIM-3 show variable expression and are associated with T-cell activation and effector function in NSCLC. Elevated T-cell LAG-3 in baseline tumor samples predicts primary resistance to PD-1-axis blockers.
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