Generation of TGFßR2(-1) neoantigen-specific HLA-DR4-restricted T cell receptors for cancer therapy.

BACKGROUND: Adoptive transfer of patient's T cells, engineered to express a T cell receptor (TCR) with defined novel antigen specificity, is a convenient form of cancer therapy. In most cases, major histocompatibility complex (MHC) I-restricted TCRs are expressed in CD8+ T cells and the development of CD4+ T cells engineered to express an MHC II-restricted TCR lacks behind. Critical is the choice of the target antigen, whether the epitope is efficiently processed and binds with high affinity to MHC molecules. A mutation in the transforming growth factor ß receptor 2 (TGFßR2(-1)) gene creates a frameshift peptide caused by the deletion of one adenine (-1) within a microsatellite sequence. This somatic mutation is recurrent in microsatellite instable colorectal and gastric cancers and, therefore, is a truly tumor-specific antigen detected in many patients. METHODS: ABabDR4 mice, which express a diverse human TCR repertoire restricted to human MHC II molecule HLA-DRA/DRB1*0401 (HLA-DR4), were immunized with the TGFßR2(-1) peptide and TGFßR2(-1)-specific TCRs were isolated from responding CD4+ T cells. The TGFßR2(-1)-specific TCRs were expressed in human CD4+ T cells and their potency and safety profile were assessed by co-cultures and other functional assays. RESULTS: We demonstrated that TGFßR2(-1) neoantigen is immunogenic and elicited CD4+ T cell responses in ABabDR4 mice. When expressed in human CD4+ T cells, the HLA-DR4 restricted TGFßR2(-1)-specific TCRs induced IFNy expression at low TGFßR2(-1) peptide amounts. The TGFßR2(-1)-specific TCRs recognized HLA-DR4+ lymphoblastoid cells, which endogenously processed and presented the neoantigen, and colorectal cancer cell lines SW48 and HCT116 naturally expressing the TGFßR2(-1) mutation. No MHC II alloreactivity or cross-reactivity to peptides with a similar TCR-recognition motif were observed, indicating the safety of the TCRs. CONCLUSIONS: The data suggest that HLA-DR4-restricted TCRs specific for the TGFßR2(-1) recurrent neoantigen can be valuable candidates for adoptive T cell therapy of a sizeable number of patients with cancer.

The role of CD4 T cells in rejection of solid tumors.

The focus in cancer immunotherapy has mainly been on CD8 T cells, as they can directly recognize cancer cells. CD4 T cells have largely been neglected, because most cancers lack MHC II expression and cannot directly be recognized by CD4 T cells. Yet, tumor antigens can be captured and cross-presented by MHC II-expressing tumor stromal cells. Recent data suggest that CD4 T cells act as a swiss army knife against tumors. They can kill cancer cells, if they express MHC II, induce tumoricidal macrophages, induces cellular senescence of cancer cells, destroy the tumor vasculature through cytokine release and help CD8 T cells in the effector phase. We foresee a great future for CD4 T cells in the clinic, grafted with tumor antigen specificity by T cell receptor gene transfer, either alone or in combination with engineered CD8 T cells.

Effective NY-ESO-1-specific MHC II-restricted T cell receptors from antigen-negative hosts enhance tumor regression.

Adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells is a promising approach in cancer therapy but needs improvement for more effective treatment of solid tumors. While most clinical approaches have focused on CD8+ T cells, the importance of CD4+ T cells in mediating tumor regression has become apparent. Regarding shared (self) tumor antigens, it is unclear whether the human CD4+ T cell repertoire has been shaped by tolerance mechanisms and lacks highly functional TCRs suitable for therapy. Here, TCRs against the tumor-associated antigen NY-ESO-1 were isolated either from human CD4+ T cells or from mice that express a diverse human TCR repertoire with HLA-DRA/DRB1*0401 restriction and are NY-ESO-1 negative. NY-ESO-1-reactive TCRs from the mice showed superior recognition of tumor cells and higher functional activity compared with TCRs from humans. We identified a candidate TCR, TCR-3598_2, which was expressed in CD4+ T cells and caused tumor regression in combination with NY-ESO-1-redirected CD8+ T cells in a mouse model of adoptive T cell therapy. These data suggest that MHC II-restricted TCRs against NY-ESO-1 from a nontolerant nonhuman host are of optimal affinity and that the combined use of MHC I- and II-restricted TCRs against NY-ESO-1 can make adoptive T cell therapy more effective.

Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity.

For thymic selection and responses to pathogens, T cells interact through their αß T cell receptor (TCR) with peptide-major histocompatibility complex (MHC) molecules on antigen-presenting cells. How the diverse TCRs interact with a multitude of MHC molecules is unresolved. It is also unclear how humans generate larger TCR repertoires than mice do. We compared the TCR repertoire of CD4 T cells selected from a single mouse or human MHC class II (MHC II) in mice containing the human TCR gene loci. Human MHC II yielded greater thymic output and a more diverse TCR repertoire. The complementarity determining region 3 (CDR3) length adjusted for different inherent V-segment affinities to MHC II. Humans evolved with greater nontemplate-encoded CDR3 diversity than did mice. Our data, which demonstrate human TCR-MHC coevolution after divergence from rodents, explain the greater T cell diversity in humans and suggest a mechanism for ensuring that any V-J gene combination can be selected by a single MHC II.

Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation.

Similar to T-helper (Th) cells, CD8(+) T cells also differentiate into distinct subpopulations. However, the existence of IL-9-producing CD8(+) T (Tc9) cells has not been elucidated so far. We show that murine CD8(+) T cells activated in the presence of IL-4 plus TGF-ß develop into transient IL-9 producers characterized by specific IFN-γ and IL-10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL-associated transcription factors T-bet and Eomesodermin. Similarly to the CD4(+) counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL-9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2-associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2-dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.