The design and optimization of a trispecific with conditional CD28 engagement and obligate cis T cell binding will be discussed, including enhanced and sustained T cell functionality and antitumor activity compared to bispecific cell engagers and safety in in vitro and in vivo models. The transferability to diverse targeting applications including 2+1+1, pMHC targets, and logic-gated designs will be shared, along with highlights from the ZW209 program, a DLL3, CD3 and CD28 targeting trispecific.

We established several routes for the design of trispecific engagers with desired biophysical and functional properties. The strategy to discover and design heavy chain only binders paired with a common light chain will be highlighted. Alternatively, two-in-one antibodies were established, where two different targets are recognized individually by the VL and the VH domain followed by amendment of additional binding modules to obtain symmetric trispecifics. Design examples for different formats and applications will be presented.

Multispecific antibodies open new avenues of exploration within the ADC field. By targeting multiple tumor antigens, one common route of tumor escape through antigen loss can be avoided. Multispecifics also allow targeting of tumor matrix components alongside tumor antigens to assist in addressing tumors where the ECM prevents access to the tumor. Furthermore, biparatopics as well as conditional binding and internalization strategies can be utilized to enhance safety and efficacy.

We have developed induced-proximity platforms to modulate surface receptor activity by recruiting endogenous enzymes. Our approach selectively inhibits immune checkpoint and oncogenic receptor signalling through targeted phosphatase recruitment, while conversely enhancing inhibitory receptor function via kinase recruitment. Additionally, we are mapping general principles that drive effective receptor dimerization at the cell surface, enabling modulation of receptor complexes not naturally associated. These novel strategies offer new insights into receptor signal control, opening opportunities to therapeutically rewire cellular communication in cancer and inflammation therapeutically.

Clustering antibodies present a compelling approach to treat diseases driven by defective signaling pathways, but their discovery has been limited by the difficulty of identifying epitopes that successfully trigger receptor signaling. Using a combination of physics-aware deep learning and experimental approaches, we successfully generated bispecific agonist antibodies against ALK1 and BMPRII that activate the signaling complex and treated HHT pathologies in various mouse models. Our approach has shown to be applicable across diseases and targets.

Bispecific antibody mixtures provide a novel means to achieve controlled receptor agonism of T cell cytokine and co-stimulatory pathways. In contrast to native cytokines or monoclonal antibodies, which are constrained by systemic toxicity, this approach enables localized and selective pathway engagement to drive potent anti-tumor immunity. These findings demonstrate how bispecific antibodies can safely unlock immune mechanisms that have previously been inaccessible for therapeutic intervention.

Tetravalent bispecific antibodies (BsAbs) represent an emerging class of immunotherapeutics designed to enhance functional avidity and dual-target engagement compared with conventional bivalent formats.

We conducted a rigorous, side-by-side comparison of T-cell engager antibodies incorporating distinct co-stimulatory signals (4-1BB, CD28, and OX40) using a consistent structural framework and affinity profile. We highlight that distinct T-cell costimulation signals exert diverse effects on T-cell response dynamics. Specifically, we observed that TriTCE-4-1BB and TriTCE-CD28 costimulation preferentially expanded effector memory T cells and increased the presence of CD4+ T cells more than TriTCE-OX40. Additionally, this finding underscores the critical role of co-stimulatory signals in enhancing T-cell metabolic fitness, equipping them to withstand the demands of repeated antigen exposure in the tumor microenvironment.

Prodrug-activating chain exchange (PACE) approaches apply two inactive antibody derivatives that, upon co-accumulation on tumor cells, reconstitute prodrug functionality. Single-chain Fab PACE harbors functional Fc domains to improve pharmacokinetic properties, and places the effector prodrug domains into linker-connected Fab arms which reduces the risk of nonspecific prodrug activation. Examples for this approach include Her2-targeted proTCBs that activate CD3-binding functionalities, as well as dual prodrug approaches that conditionally engage CD3 and CD28 on target cells.

We developed pH-selective CD28xVISTA bispecific antibodies that conditionally co-stimulate T cells within the acidic, myeloid-rich tumor microenvironment. By engaging VISTA on tumor-associated myeloid cells, our lead bispecific drives localized CD28 activation and enhances anti-tumor immunity. Functional studies demonstrated potent, VISTA-dependent T cell activation and proliferation without systemic cytokine release, indicating a favorable safety profile. This conditional activation approach broadens the immunotherapy toolbox, providing a tumor-selective co-stimulatory mechanism that may synergize with PD-1 blockade or T cell–redirecting biologics. Our findings highlight a novel therapeutic platform enabling safe, localized immune modulation for solid tumors.

T cell engagers (TCEs) are highly potent immunotherapeutic drug modalities. However, their broad application is constrained by on-target, off-tumor toxicity and cytokine release syndrome (CRS), resulting in a narrow therapeutic index. We present here the development of a conditional, dual-antigen targeting trispecific TCE (TriMab) that integrates a synapse-gated design with affinity-tuned binding arms to achieve AND-gated tumor selectivity. This approach enhances target discrimination and mitigates off-tumor activation while maintaining potent anti-tumor activity. Collectively, our work establishes synapse-gated, dual-targeting trispecifics as a next-generation framework for engineering safer and more precise T cell therapeutics.