Developing Novel Nanobodies Against the 5-Methylcytosine Dioxygenases TET2

The Ten-eleven Translocation (TET) family of dioxygenases mediate DNA demethylation by oxidating 5-methylcytosine in the mammalian genome. Among the three TET family members, TET2 is frequently mutated in hematological disorders, including age-related clonal hematopoiesis and hematological malignancies. Previous reports suggest that loss-of-function mutations of TET2 lead to increased self-renewal and myeloid bias of hematopoietic stem and progenitor cells (HSPCs) in both mouse and human studies, suggesting its tumor-suppressive role. On the other hand, TET2 depletion in myeloid or lymphoid cells enhances their immune function in the tumor microenvironment and remarkably augments the efficacy of anti-tumor immunotherapy. Indeed, loss of TET2 enables antigen-independent CAR-T cell clonal expansion and improves tumor infiltration. Therefore, there remains a clinical need to harness TET2 in the hematopoietic system in a tunable manner to enhance anti-cancer immunity and suppress tumor cell growth while limiting the risk of uncontrolled proliferation.

Developing nanobodies as therapeutics has emerged as a promising avenue, offering minimal toxicity and facile delivery. A nanobody is about one-tenth of the size of a full-size antibody (~15 kDa), ranking it among the smallest known antigen-binding modules. The unique structures of complementarity determining regions (CDRs), along with their smaller molecular size, allow nanobodies to penetrate deeper and more rapidly into target cells or tissues. Additionally, the high homology of nanobodies to the human family III heavy chain domain results in low immunogenicity and high tolerance, making them ideal candidates for translational therapeutic development.

In this study, we developed a set of nanobodies against human TET2. We validate the specific recognition of nanobodies to human TET2 based on cell-based imaging and biochemical immunoprecipitation approaches. Randomized mutagenesis was performed in CDRs to further improve the binding affinity between nanobodies and TET2. The specific recognition between optimized nanobodies and TET2 were further evaluated using GST-tagged recombinant protein. We then further fused the nanobodies with modular domains derived from the proteasomal degradation machinery to enable specific degradation of TET2 protein in both HEK293T and Jurkat T cell lines. We will apply this nanobody-based degradation tool in human CD19 CAR-T cells to explore its potential to increase CAR-T efficacy and enhance tumor killing. In summary, the development of nanobodies against TET2 provides a novel approach to modulate endogenous TET2 protein level and activity, setting the stage for precise epigenetic regulation in both HSPCs and immune cells to benefit cell-based therapies.

No relevant conflicts of interest to declare.