Evading host immune surveillance is one of the hallmarks of cancer. Immune checkpoint therapy, which aims to eliminate cancer progression by reprogramming the antitumor immune response, currently occupies a solid position in the rapidly expanding arsenal of cancer therapy. As most immune checkpoints are membrane glycoproteins, mounting attention is drawn to asking how protein glycosylation affects immune function. The answers to this fundamental question will stimulate the rational development of future cancer diagnostics and therapeutic strategies.

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing 4-1BB as a therapeutic target for immune-related disorders, less is known about the pattern of its intracellular behaviors and regulations. It has been previously demonstrated that 4-1BB is heavily modified by N-glycosylation; however, the biological importance of this modification lacks detailed elucidation. Through biochemical, biophysical, and cell-biological approaches, we systematically evaluated the impact of N-glycosylation on the ligand interaction, stability, and localization of 4-1BB. We hereby highlighted that N-glycan functions by preventing the oligomerization of 4-1BB, thus permitting its membrane transportation and fast turn-over. Without N-glycosylation, 4-1BB could be aberrantly accumulated intracellularly and fail to be sufficiently inserted in the membrane. The N-glycosylation-guided intracellular processing of 4-1BB serves as the potential mechanism explicitly modulating the “on” and “off” of 4-1BB through the control of protein abundance. Our study will further solidify the understanding of the biological properties of 4-1BB and facilitate the clinical practice against this promising therapeutic target.

4‐1BB [tumor necrosis factor receptor superfamily (TNFRSF9), CD137) is a critical immune stimulator that sustains T cell activity and antitumor immune response. The strategy to eliminate cancers by agonistically targeting 4‐1BB is under clinical investigation. As a protein expressed in an inducible manner, 4‐1BB is under tight control on both transcription and translation levels to maintain its homeostasis. So far, the mechanisms underlying the transcriptional activation of 4‐1BB have been well‐interpreted; however, it remains inexplicit how 4‐1BB is regulated on the protein level. In this study, we presented experimental evidence supporting that 4‐1BB, especially the heavily N‐glycosylated (mature) form, is polyubiquitinated and subjected to the ubiquitin‐proteasomal system for degradation. By performing proximity‐dependent biotin identification screening coupled with biochemical assays, we identified that F‐box/LRR‐repeat protein 20 acts as the E3 ligase that promotes the polyubiquitination of 4‐1BB at the intracellular domain. Our data provided mechanistic insight into 4‐1BB regulation on the protein level by unmasking, for the first time, a posttranslational mechanism governing 4‐1BB abundance in cells. The findings of this study could potentially guide the development of 4‐1BB–targeted therapy for cancers as well as other immune disorders.

Biotherapeutics have demonstrated remarkable therapeutic efficacy across a wide range of diseases; however, the desired clinical outcomes of biotherapeutics are sometimes hindered by the development of antidrug antibodies (ADAs). Traditionally, ADA monitoring relies heavily on ligand-binding assays (LBAs) such as enzyme-linked immunosorbent assays (ELISA) and electrochemiluminescence assays (ECLIA). Although effective, these methods require multiple washing and incubation steps, extending the data generation time to several hours or even days. In contrast, homogeneous immunoassays offer a promising alternative due to their "mix-and-read" feature, which significantly reduces operational complexity, time, and cost compared to conventional LBAs. Luciferase complementation represents an invaluable tool for visualizing protein-protein interactions in vitro, in cellulo, and in vivo. In this study, we explored the feasibility of rapid ADA detection using a wash-free homogeneous assay based on a split nanoluciferase (SplitNluc) biosensor system. Through case studies involving diverse therapeutic modalities, including antibody-drug conjugate (ADC), Fc-fusion protein, Fc-less multidomain biotherapeutic (MDB), and single-domain antibody (SdAb), we demonstrated that the SplitNluc platform is a versatile and efficient tool for ADA assay, offering ideal sensitivity, high precision and broad adaptability to various modalities. In summary, the SplitNluc assay represents an innovative and valuable platform for expedited and robust ADA detection. Moreover, our study highlighted the broader potential of luminescent biosensor-based approaches in advancing bioana-lytical workflows.

The emergence of anti-drug antibodies (ADAs) poses a major obstacle in the clinical development of therapeutic proteins (TPs) such as monoclonal antibodies and their derivatives. While standard multitiered ADA assays and neutralizing antibody assays offer valuable insights into the humoral immunogenicity risks of TPs, they are not sufficient to provide in-depth knowledge such as ADA epitope specificities. For complex multidomain biotherapeutics (MDBs), ADAs targeting individual domains can elicit distinct pharmacological effects. Therefore, it is crucial to implement straightforward and reliable methodologies to deconvolute ADA epitope profiles of MDBs. Herein, we report a case study using domain specificity analysis, linear peptide scanning and bioinformatic B cell epitope prediction to unveil the clinical ADA epitope landscape of TAK-186, a multidomain T cell engager that has been discontinued from clinical development. By applying this workflow, we observed strong domain specificity variability among patient samples. Furthermore, the data showed that many patients demonstrated evolved ADA epitope specificities throughout the course of the treatment. Several potential linear epitopes were identified subsequently through experimental and computational approaches. Overall, we presented in this study a practical strategy to elucidate and potentially mitigate the immunogenicity liabilities of complex biotherapeutics.