Immunotherapy using aPD-1 monoclonal antibodies represents a highly promising approach in cancer immunotherapy. However, due to the lack of response or recurrence observed in most patients, its efficacy against tumor immunity remains relatively limited. Shapir Itai et al. [1] demonstrated that the interaction between conventional type I dendritic cells (cDC1) and T cells is crucial for an effective aPD-1-mediated antitumor response. Consequently, this study developed a bispecific dendritic cell–T cell conjugate (BiCE), which facilitates physical interactions between PD-1+ T cells and cDC1s. BiCE-based therapy promotes the formation of active cross-talk between dendritic cells and T cells in both tumors and their draining lymph nodes. In vivo experiments, single-cell analyses, and physical interaction assays all indicate that tumors and their draining lymph nodes treated with BiCE exhibit distinct and superior immune reprogramming effects compared with conventional aPD-1 therapy. By bridging immune cells, BiCE enhances the cellular circuits and communication pathways essential for effective antitumor immunity, thereby providing novel strategies for cancer immunotherapy.

Figure 1: Mechanism of antitumor activity of the bispecific DC–T cell conjugate (BiCE)

Background and structure of bispecific DC-T cell conjugates (BiCEs):

The experimental results of this study highlight the importance of the interaction between PD-1+ T cells and cDC1 during PD-1 inhibitor therapy. Given that cDC1 represents a relatively rare immune cell population within the tumor microenvironment (TME), the low probability of T-cell–cDC1 interaction may be one of the factors limiting the ability of anti-PD-1 therapy to elicit an effective T-cell-mediated immune response. Consequently, this study developed a bispecific antibody (bsAb) designed to promote direct physical interaction between cDC1 and PD-1+ T cells in both the tumor site and its draining lymph nodes, thereby facilitating anti-tumor immune activation. This bispecific antibody (bsAb) is termed the Bispecific DC–T Cell Binder (BiCE). The BiCE adopts a natural IgG-like bispecific format, wherein one Fab fragment binds to PD-1 and blocks its interaction with PD-L1, while the other Fab fragment binds to the surface marker CLEC9A (DNGR1) on cDC1 (Figure 2). CLEC9A was selected for binding due to its highly specific expression pattern on cDC1 in both murine and human models.

Figure 2: Structure of the bispecific DC–T cell conjugate (BiCE)

Characterization of bispecific DC–T cell conjugates (BiCEs):

This study also designed a monovalent aPD-1 bispecific antibody (PD-1/IC), in which the CLEC9A-binding arm was replaced with an isotype control antibody, serving as a PD-1 blockade control comparable to the BiCE format that does not involve binding to cDC1. The Fc fragment of the BiCE was mutated to avoid interaction with Fcγ receptors (FcgRs), thereby preventing the activation of Fc effector functions and the unnecessary depletion of target cells. After preparation and purification, the BiCE exhibited a homogeneous heterodimeric form, retaining the expected binding specificity of both constituent antibodies (Figure 3A–C). Compared with the parental aPD-1 monoclonal antibody, the BiCE demonstrated lower PD-1 binding affinity (IC50 values of 3.54 mg/mL versus 1.50 mg/mL), which is consistent with expectations, as bispecific antibodies (bsAbs) employ a monovalent binding mode, whereas monoclonal antibodies (mAbs) utilize a bivalent binding mode. The BiCE was capable of simultaneously binding to both targets and promoting dose-dependent coupling between PD-1+ and CLEC9A+ cells (Figure 3D–E). Furthermore, the BiCE facilitated the formation of dose-dependent T cell–dendritic cell (DC) bi-cellular complexes in murine splenocytes, demonstrating its ability to effectively connect targeted cells (Figure 3F–I).

Figure 3: Characterization results of the bispecific DC–T cell conjugate (BiCE)

BiCE forms activated DC–T cell complexes.

To evaluate the ability of BiCE to form T-cell–dendritic cell (DC) synapses at the in vivo target site, this study analyzed B16F10 melanoma tumors and their tumor-associated lymph nodes (dLNs) in mice injected with BiCE at multiple time points (Figure 4A). The results demonstrated that 24 hours after injection of the PD-1/CLEC9A BiCE, the frequency of T-cell–cDC1 dual-cell complexes was increased in the dLNs; however, this phenomenon was not observed when mice were injected with an aPD-1 bsAb control (a format lacking the cDC1-binding arm, i.e., PD-1/IC) (Figure 4B). At this time point, in the dLNs of mice injected with BiCE, both the frequency of cDC1s (Figure 4C) and the proportion of these cells forming dual-cell complexes with T cells (Figure 4D) were increased, indicating that BiCE enhances the proportion of physical interactions between T cells and DCs by expanding the cDC1 pool.

Figure 4: BiCE enables characterization of activated DC–T cell conjugates.

BiCE exhibits potent antitumor activity.

To validate the therapeutic efficacy of BiCE-mediated dendritic cell–T cell interactions, this study employed corresponding treatment regimens in three aggressive tumor models: lung large cell carcinoma (LLC), B16F10 melanoma, and AT3 triple-negative breast cancer (TNBC). BiCE therapy (versus the control group using a PD-1/IC bispecific antibody) significantly reduced the growth rates and tumor volumes in LLC and B16F10 models (Figures 5A–B). Under these experimental conditions, the AT3 model showed no response to BiCE therapy (Figure 5C). The study also evaluated the efficacy of conventional PD-1 blockade therapy (where the original PD-1 monoclonal antibody was cloned into the BiCE format) across these models; results demonstrated that both LLC and AT3 models were refractory to aPD-1 therapy, whereas in the B16F10 model, the therapeutic effect achieved with aPD-1 was comparable to that observed with BiCE (Figures 5D–F).

Figure 5: Assessment of BiCE's anti-tumor activity

Analysis of the tumor microenvironment (TME) revealed that, compared with the untreated control group, mice receiving aCD40 monoclonal antibody therapy (an immunotherapy approach that targets dendritic cells (DCs) by inducing a cDC1/CD8 response) exhibited elevated levels of CD8+ T cells and cDC1 in the tumor-associated lymph nodes (dLNs) within their AT3 tumors; furthermore, the levels of CD8+ T cells in the AT3 tumors of mice treated with aCD40 monoclonal antibody were significantly higher than those observed in the untreated control group (Figures 6A–B). The combination therapy of aPD-1 inhibitor with aCD40 monoclonal antibody did not demonstrate any synergistic effect in inhibiting tumor growth beyond that achieved by monotherapy with aCD40 monoclonal antibody. In contrast, mice receiving BiCE treatment showed a significant reduction in tumor volume compared with those treated with aCD40 monoclonal antibody alone (Figure 6C). In both LLC and B16F10 tumor models, the combination of BiCE with aCD40 monoclonal antibody also elicited a similar synergistic effect (Figures 6D–F).

Figure 6: Assessment of the synergistic anti-tumor activity between BiCE and aCD40

brief summary ：

This study integrates advanced single-cell multi-omics technologies with antibody engineering techniques to propose a novel concept in the field of immunotherapy: achieving physical interactions between immune cells through engineered approaches to enhance anti-tumor immunity. It is anticipated that extensive research efforts focused on analyzing cellular network characteristics across cohorts involving human tumors, autoimmune diseases, and neurodegenerative disorders will further enhance the capability to develop a wider range of immune conjugates, thereby providing new insights and data support for future immunotherapies targeting these diseases. Taik Bio (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform leveraging phage display and yeast display technologies; this platform provides global clients with high-quality preliminary candidate sequence discovery services, as well as integrated one-stop solutions—including supporting downstream bispecific antibody design and preparation, in vitro validation (encompassing various customized validation protocols such as protein-level and cell-level binding validation), and in vivo pharmacodynamic evaluation (via animal model experiments and efficacy assessment)—thereby offering robust technical support for clients' scientific research projects.

References ：

[1] Shapir Itai Y, Barboy O, Salomon R. et al. .Bispecific dendritic-T cell engager potentiates anti-tumor immunity. Cell, 187, 375-389.e18（2024）