Delivery of antigenic peptides to antigen presenting cells (APCs) such as dendritic cells (DCs) using monoclonal antibodies (mAbs) is an attractive approach to evoke antigen-specific T cell activation and improve drug efficacy. Peptide linkage to mAbs has previously been achieved through genetic fusion, chemical conjugation, nano-engineered platforms and high affinity peptides. In this study, we have developed a flexible antibody-peptide linking technology using oppositely charged coiled coil domains to non-covalently link peptides to mAbs. The technology comprises (1) an anti-CD40 mAb connected with negatively charged E domains and (2) an immunogenic OVA peptide (SIINFEKL) from ovalbumin used as a model antigenic peptide fused with positively charged K domains. Combining these constructs leads to the formation of complexes that can be targeted to CD40 expressed on cells. Proof of concept antibody constructs connected with E domains generated from transient expressions exhibited good manufacturability, binding, and stability attributes comparable to a control mAb. Also, optimal repeat lengths for coiled-coil oligomerization domains were identified in these studies. Binding kinetics studies showed that connecting E domains to mAbs do not impede Fc gamma and neonatal receptor interactions. Additionally, formation of stable complexes capable of binding CD40 expressing cells was demonstrated in vitro. In vivo functionality evaluations showed that treatment of human CD40 transgenic mice with complexes elicited expansion of OVA peptide-specific CD8+ T cells and potent antitumor effects superior to peptide monotherapies. Overall, these findings demonstrate that the technology has great potential for application as an in vivo tool for antigenic peptide delivery.

MYC provides a rheostat linking cell growth and division. Deregulation of MYC drives transformation in aggressive B-cell neoplasms, often accompanied by BCL2-mediated apoptotic protection. We assess how MYC and BCL2 deregulation impacts on the ability of human B cells to complete plasma cell (PC) differentiation. As B cells differentiate, MYC deregulation has little impact on the regulatory circuitry controlling B-cell identity. Induction of transcriptional regulators BLIMP1 and IRF4 remains intact and accompanies loss of B-cell surface markers. However, such differentiating cells develop an aberrant surface phenotype with reduced expression of phenotypic markers of differentiation. Although functional antibody secretion is established, enforced MYC expression dampens the expression of secretory programmes associated with PC differentiation. Accompanying this, diverse changes in the expression of genes related to translation and metabolism are observed. The establishment of this aberrant differentiated state depends on MYC homology box II. This dependence is profound and resolves to residue W135.
© 2025 Vardaka et al.

Immune checkpoint therapy has transformed cancer treatment, yet efficacy and safety challenges persist. Selectively inhibiting tumor-infiltrating regulatory T cells (Ti-Tregs) while enhancing CD8+ T cell function are complementary strategies in cancer immunotherapy. Here, we engineered a bispecific antibody, FRP303, targeting 4-1BB and CCR8, which are co-expressed on a highly immunosuppressive subset of Ti-Tregs. In vivo, FRP303 outperformed monoclonal antibodies in CT26 and MC38 colorectal tumors and poorly immunogenic B16F10 melanoma. Treatment with FRP303 reduced Ti-Treg frequency, increased CD8+ T cell infiltration, and elevated antitumor cytokines IFN-γ and TNF-α. Safety assessments showed FRP303 does not disrupt immune homeostasis in peripheral tissues or induce significant hepatotoxicity. Moreover, FRP303 demonstrated strong synergistic effects when combined with a PD-1 antibody. In summary, FRP303 mediated anti-tumor activity through a dual mechanism involving the selective depletion of Ti-Tregs and the enhancement of CD8+ T cell function, offering a promising strategy for cancer immunotherapy.
© 2025 The Authors.

Persistent chronic inflammation is a hallmark of ankylosing spondylitis (AS), with cytotoxic T cells (CTLs) increasingly implicated in its pathogenesis. Ordinarily, T cell exhaustion follows sustained, persistent T cell activation to limit collateral tissue damage. Using mass cytometry and single-cell RNA sequencing (scRNA-seq), we identified a clonally expanded CTL subset in AS synovial fluid that expresses inhibitory receptors (PD-1, TIGIT, LAG-3) yet retains its effector capacity to express granzymes, perforin, TNF-α, and IFN-γ. Gene expression profile of this CTL subset shows the downregulation of canonical exhaustion markers. At the protein level, TOX, a critical transcription factor regulating CTL exhaustion, is downregulated in PD-1+TIGIT+LAG-3+CTLs. In-silico trajectory analyses suggest that these cells may differentiate into other effector CTL subsets. Our findings reveal a checkpoint-expressing CTL population in AS that resists exhaustion and retains an activated, effector phenotype. We propose that failure to undergo exhaustion may be a fundamental mechanism sustaining AS chronic inflammation.
© 2025 The Authors.

Immunotherapy has revolutionized cancer treatment, yet understanding immunotherapy resistance mechanisms remains challenging. Here, a CRISPR cas9 screening in vivo and an RNA-sequencing for clinical immunotherapy resistance BC samples identified enolase 1 (ENO1) as a potent regulator of anti-PD-L1 treatment efficacy. Investigation of clinical BC samples demonstrated a correlation between ENO1 overexpression and immune evasion in BC, evidenced by reduced CD8+ T cell infiltration and resistance to anti-PD-L1 therapy. Increased CD8+ T cell infiltration and function were indicative of antitumor immunity, which was elicited by ENO1 knockdown, which also suppressed carcinogenesis. Single-cell RNA sequencing demonstrated that wild-type (WT) and ENO1 knockout (KO) tumors have different immune cell compositions with the latter preferring an immunostimulatory microenvironment. Mechanistically, ENO1 regulated CD8+ T cell function and tumor-associated macrophage (TAM) polarization via the SPP1-ITGA4/ITGB1 pathway in the TME. Importantly, genetic and pharmacological inhibition of ENO1 sensitizes tumors to anti-tumor immunity and synergizes with anti-PD-L1 therapy. The results highlight tumor-intrinsic ENO1 as a critical regulator of tumor immune evasion in BC. Targeting ENO1 enhance the efficacy of immune checkpoint blockade therapy by promoting antitumor immunity.
© 2025. The Author(s).