What Trispecific T-cell engager (TriTE) are being developed?

Introduction to Trispecific T-cell Engagers (TriTEs)

Definition and Mechanism of Action
Trispecific T-cell engagers, or TriTEs, are engineered multivalent antibody constructs that extend the concept of bispecific T-cell engagers (BiTEs) by incorporating a third binding domain. In their design, one domain is typically directed to a T-cell–specific marker such as CD3, while the other two domains target distinct epitopes on tumor cells or incorporate a costimulatory signal (e.g., CD28 or CD137) meant to boost T-cell activation. This tripartite binding mechanism facilitates a more controlled and potent induction of immune cell–mediated cytotoxicity, ensuring that T cells are brought into close proximity with cancer cells and receive a robust activation stimulus. Mechanistically, TriTEs form a cytolytic synapse between the T cell and the tumor cell, enhancing the efficiency of target cell lysis while simultaneously reducing the potential for off-tumor toxicities by requiring multi-antigen recognition before activation occurs.

Historical Development and Evolution
The evolution of T-cell engagers began with the emergence of BiTEs, such as blinatumomab, which redirected T cells toward malignant cells via dual specificity. However, while BiTEs have demonstrated impressive anti-leukemic activity, their application to solid tumors has been limited by factors such as suboptimal tumor cell engagement and adverse effects (e.g., cytokine release syndrome and T-cell exhaustion). Over time, researchers recognized that adding a third specificity could provide additional control over T-cell activation. Early preclinical studies showed that trispecific formats might enable selective targeting of two distinct tumor-associated antigens (TAAs) or combine tumor targeting with costimulation, overcoming limitations in efficacy and safety observed with bispecific constructs. This evolution has been driven by the need for enhanced tumor specificity in heterogeneous tumor microenvironments as well as the desire to reduce systemic toxicity by ensuring that activation signals are delivered only in the presence of multiple tumor markers.

Current TriTEs in Development

Leading TriTEs in Preclinical and Clinical Trials
Recent advances in TriTE design have led to the development of several innovative constructs now undergoing preclinical testing and early-phase clinical trials. One prominent example is a trispecific T-cell engager that simultaneously targets DLL3, MUC17, or CLDN18.2 on tumor cells and engages critical cluster of differentiation markers—CD3, CD28, or CD137—on T cells. This design aims to ensure not only tumor recognition but also provides ancillary costimulatory signals necessary for optimal T-cell activation.

Another notable construct is described in a paper that details a TriTE consisting of a CD3-specific single-chain variable fragment (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. This particular TriTE has been engineered for the treatment of colorectal cancer, with preclinical data indicating that the tandem construct can bind its three cognate antigens simultaneously, trigger robust T-cell–mediated cytotoxicity, and exhibit a 100-fold increase in potency in vitro compared to controls targeting single antigens.

Moreover, innovative nanobody-based TriTEs have been developed. One strategy involved creating a bispecific nanobody-based T-cell engager (Nb-BiTE) against fibroblast activation protein (FAP) and subsequently fusing an anti-PD-1 nanobody to it to produce an Nb-TriTE. This construct not only demonstrated high binding affinity and selective tumor killing in vitro but also suppressed tumor growth and improved survival in multiple mouse xenograft models of solid tumors—with the added advantage of overcoming tumor-mediated immunosuppression.

Another TriTE under investigation targets dual immune checkpoints. In one study, researchers developed a trispecific T cell engager capable of simultaneously binding to PD‑L1 and HLA‑G while engaging T cells. This design is particularly innovative because it aims to direct T cells specifically toward tumor cells expressing these immune checkpoint molecules, thereby circumventing some of the resistance mechanisms that limit the efficacy of single-agent checkpoint inhibitors.

Furthermore, a trispecific T-cell engager targeting CD19 has been developed for hematological malignancies. This TriTE is designed to bind CD19 on cancer cells and simultaneously engage T cells, leading to dose-dependent activation and expansion of T cells, which in turn mediates potent cytotoxicity against CD19-positive tumor cells. Detailed in vitro and in vivo studies have shown that this construct can promote significant T-cell proliferation and effective target cell lysis at nanomolar concentrations.

Clinical pipelines are also being populated by companies advancing Tri-specific platforms. For instance, Harpoon Therapeutics is leveraging its proprietary Tri-specific T cell Activating Construct (TriTAC®) platform to develop candidates such as HPN424, HPN536, HPN217, and HPN328, which target various antigens including prostate-specific membrane antigen (PSMA), mesothelin, BCMA, and DLL3 respectively. These candidates are in various phases of clinical studies aimed at treating solid tumors and hematologic malignancies, with early-phase data suggesting effective engagement of patient T cells to confer antitumor effects.

Companies and Research Institutions Involved
A robust network of biotechnology companies and academic research institutions is heavily involved in the development of TriTEs.
- Harpoon Therapeutics is a leading company pioneering the TriTAC platform. Their portfolio includes candidates addressing multiple tumor types, with clinical programs not only in hematologic malignancies but also solid tumors.
- Merus has entered into collaborations, as highlighted by strategic partnerships with Gilead to explore novel dual tumor-associated antigens and to further exploit their patented Triclonics® platform for trispecific antibody development.
- Merck and Eyebiotech (EyeBio) have also advanced trispecific therapies, with the acquisition of EyeBio incorporating a tetravalent trispecific antibody named RestoretTM (EYE103) in their pipeline for the treatment of conditions such as diabetic macular edema and neovascular age-related macular degeneration (NVAMD).
- Other companies, including AbbVie and Genmab, have shown interest in T cell engager technology and multispecific approaches, expanding the scope to cover both hematological and solid tumor indications.
- Numerous academic laboratories worldwide, often in collaboration with these companies, are focusing on refining the molecular design, improving the affinity balance among the binding domains, and addressing the pharmacokinetic challenges associated with TriTE constructs.

Challenges in TriTE Development

Scientific and Technical Challenges
Despite their promising potential, the development of TriTEs is not without significant scientific and technical hurdles. One of the foremost challenges is the molecular complexity inherent in these constructs. Optimizing the binding affinities of three distinct domains—one for the T cell (frequently CD3) and two for tumor-associated targets or costimulatory receptors—requires a delicate balance. If any binding activity is too strong relative to the others, it could lead to non-specific T cell activation or suboptimal tumor cell engagement.

Manufacturability is another pressing technical challenge. The tri-specific format, often larger and more complex than conventional antibodies or BiTEs, can pose difficulties in protein folding, stability, and yield during production. Ensuring that the final product is stable and remains functionally active over extended periods or within different storage and delivery conditions is critical for clinical success. Moreover, issues of tumor penetration are magnified with larger or more complex molecules, meaning that TriTEs must be engineered to maintain a favorable biodistribution profile to ensure effective localization to tumor sites.

Preclinical model optimization and the translation of in vitro findings to in vivo contexts present additional challenges. Robust functional screening systems are required to validate that TriTEs not only bind their targets with high specificity but also trigger the desired T-cell activation and cytotoxicity in a clinically relevant manner. Studies have shown that even minimal off-target activation can lead to detrimental side effects, underscoring the importance of precise engineering and rigorous screening.

Regulatory and Safety Concerns
The introduction of a third specificity, while promising enhanced efficacy, also raises pertinent questions regarding safety and regulatory oversight. Multifunctional biologics such as TriTEs could potentially induce cytokine release syndrome (CRS) or other immune-related adverse events if T-cell activation occurs systemically rather than being confined to the tumor microenvironment. Regulatory bodies require robust evidence that these molecules have a sufficiently wide therapeutic window—the range between an effective and a toxic dose—and that any off-target effects can be minimized.

Moreover, tri-specific constructs must undergo extensive regulatory scrutiny due to their novel design. Clinical trials must be designed to carefully monitor not only the antitumor efficacy but also a spectrum of potential immune toxicities or other adverse events. Long-term safety data must ultimately be ascertained to ensure that sustained immune activation does not lead to deleterious autoimmune consequences or other complications in patients. Regulatory challenges also extend to the manufacturing and quality control processes, which must meet stringent standards to ensure consistency and purity across production batches.

Potential Applications and Impact

Therapeutic Areas Targeted
The potential applications of TriTEs span a broad range of therapeutic areas, primarily in oncology but also potentially in other fields where precise immune modulation is required. In oncology, the versatility of the TriTE format shows promise for both hematological malignancies and solid tumors. For instance, TriTEs designed to target CD19 are under development for B-cell malignancies, while constructs directed against antigens such as EGFR, EpCAM, DLL3, mesothelin, and BCMA are being explored for colorectal cancer, lung cancer (including small cell lung cancer), multiple myeloma, and prostate cancer.

In addition, a novel tetravalent trispecific antibody known as RestoretTM (EYE103) is being evaluated for the treatment of ocular conditions such as diabetic macular edema (DME) and neovascular age-related macular degeneration (NVAMD). This highlights the potential for TriTEs to extend beyond oncology and into diseases where localized immune stimulation may produce therapeutic benefits. Moreover, the possibility of targeting dual immune checkpoints through trispecific designs—for example, binding PD‑L1 and HLA‑G—opens new avenues for overcoming tumor immune escape mechanisms, particularly in cancers that are refractory to traditional checkpoint inhibition therapies.

Case Studies and Clinical Outcomes
Several case studies and preclinical trial outcomes underscore the promise of TriTEs. In colorectal cancer models, a TriTE comprising a CD3-specific scFv flanked by anti-EGFR and anti-EpCAM VHH domains demonstrated markedly increased potency. In vitro experiments revealed that bivalent bispecific targeting of HCT116 cells resulted in up to a 100-fold improvement in potency compared to targeting single antigens, and in vivo studies showed significant prolongation of survival with this construct.

Nanobody-based TriTEs have also yielded encouraging results. In one study, an Nb-TriTE designed by fusing an anti-PD-1 nanobody to a FAP-targeting Nb-BiTE not only activated T cells robustly in vitro but also suppressed tumor growth in several mouse models without the toxicity observed in control groups. These studies are particularly important as they demonstrate the feasibility of using smaller, more stable nanobody formats to create effective trispecific constructs that overcome some inherent limitations of larger molecules.

Furthermore, trispecific antibodies targeting dual immune checkpoints such as PD‑L1 and HLA‑G have shown early promise in augmenting cytotoxic T-cell responses in preclinical models of non‑small cell lung cancer (NSCLC). These constructs are designed to harness T-cell activity specifically in the presence of tumors expressing both targets, thereby reducing systemic side effects and enhancing local antitumor activity.

Clinical outcomes from early-phase trials with candidates such as those developed on the TriTAC® platform (e.g., HPN217, HPN424, HPN536, and HPN328) are awaited, and their future data will be crucial in determining the therapeutic impact of trispecific T cell engagers. Preliminary reports suggest that these agents offer improved T-cell activation profiles and favorable safety margins in early dosing studies, paving the way for more extensive clinical investigations.

Future Directions and Innovations

Emerging Trends in TriTE Technology
Looking ahead, several emerging trends indicate that trispecific T-cell engagers will continue to evolve. One notable trend is the increasing incorporation of costimulatory domains into TriTE constructs. By including binding sites for additional T-cell costimulatory receptors such as CD28 or CD137, future TriTEs may further enhance T-cell expansion, persistence, and cytotoxic function. This approach aims to overcome one of the primary challenges observed with bispecific constructs—insufficient T-cell activation, especially in the immunosuppressive tumor microenvironment.

Another key trend is the utilization of nanobody technology in trispecific formats. The smaller size and enhanced tissue penetrability of nanobodies allow for the creation of more compact TriTEs with improved pharmacokinetic profiles, which may lead to better tumor infiltration and reduced systemic exposure. Recent studies have demonstrated that nanobody-based TriTEs not only exhibit robust in vitro activity but also provide significant antitumor efficacy in animal models, making them a promising technology for clinical translation.

Advances in protein engineering and computational modeling are also propelling the field forward. Multiscale simulations and quantitative systems pharmacology (QSP) models are increasingly being used to design and optimize multispecific biologics. These computational approaches enable the optimization of binding affinities and the prediction of intercellular interactions, helping researchers fine-tune the structural and functional aspects of TriTEs before moving into clinical studies.

In addition, there is growing interest in combining TriTEs with other therapeutic modalities. For example, pairing TriTEs with immune checkpoint inhibitors or adoptive T-cell therapies may produce synergistic effects, enhancing overall response rates in patients who have historically been nonresponsive to monotherapies. Such combination strategies are particularly appealing in the context of “cold” tumors, which exhibit low immunogenicity and minimal T-cell infiltration but might be rendered susceptible through targeted T-cell redirection.

Research and Development Opportunities
The promise of trispecific T-cell engagers opens up a broad array of research and development opportunities. One avenue is the identification of novel tumor-associated antigens (TAAs) that are uniquely or overly expressed in malignant tissues. This specificity is critical for ensuring that TriTE-mediated T-cell activation is restricted to cancer cells, thereby minimizing off-tumor effects. Collaborative efforts between biotech companies and academic institutions are already underway to map the antigenic landscape of various cancers and to integrate these findings into TriTE design.

Another opportunity lies in refining the manufacturing processes and improving the stability of these complex molecules. As manufacturing challenges are addressed, scalability and consistency in production will improve, accelerating clinical translation. Innovations in bioprocess engineering, including advanced cell lines for protein expression and refined purification techniques, are essential components of this development pipeline.

Furthermore, there is significant potential for optimizing the dosing regimens and routes of administration for TriTEs. Preclinical studies have revealed that dosing concentration, timing, and the route of administration can drastically influence therapeutic outcomes. Research focused on pharmacokinetic and pharmacodynamic parameters will be vital to ensure that TriTEs achieve an optimal balance between efficacy and safety in patients. Quantitative systems pharmacology models provide an exciting tool for predicting patient-specific responses and tailoring treatment regimens accordingly.

Finally, capitalizing on next-generation sequencing and high-throughput screening methods will allow for the rapid evaluation of various TriTE formats. The development of “function-first” approaches that rely on direct assessment of T-cell activation and cytotoxicity in response to candidate molecules—without the need for extensive purification—is another promising strategy that can shorten the cycle time from discovery to clinical testing. This is particularly important in a rapidly evolving therapeutic landscape where speed to market can determine competitive advantage and patient access.

Conclusion
Trispecific T-cell engagers (TriTEs) represent a significant leap forward in the field of immuno-oncology, offering the potential to overcome many of the limitations associated with bispecific T-cell engagers. By incorporating three distinct binding domains, TriTEs are designed to recruit T cells more effectively, target tumor cells with dual antigen recognition or combine tumor targeting with costimulation, and thereby enhance antitumor efficacy while minimizing off-target effects.

Historically evolving from the successes and shortcomings of BiTEs, TriTEs have undergone rapid development over the past several years. Preclinical studies have demonstrated their superior potency and specificity, as seen in constructs targeting combinations of EGFR, EpCAM, DLL3, mesothelin, BCMA, and CD19. The incorporation of nanobody technology and the integration of costimulatory signals, such as CD28 or CD137 binding, further emphasize the advanced design strategies being employed.

Several leading candidates are currently in preclinical and early clinical development. Notable among these are TriTEs being developed through proprietary platforms such as Harpoon Therapeutics’ TriTAC®, which includes candidates like HPN424, HPN536, HPN217, and HPN328, alongside innovative approaches that target dual immune checkpoints or exploit nanobody formats for better tissue penetration. In parallel, strategic alliances and collaborations among biotech companies (e.g., Merus with Gilead, Merck with Eyebiotech) and academic research institutions are accelerating the pace of innovation in this area.

Despite tremendous promise, the development of TriTEs faces significant scientific, technical, regulatory, and safety challenges. Balancing the affinities of three binding domains, ensuring manufacturability, achieving efficient tumor penetration, and mitigating the risk of systemic cytokine release are all critical challenges that researchers are actively addressing through innovative design strategies and advanced computational modeling. Regulatory hurdles require that these novel constructs demonstrate a favorable safety profile and clear clinical benefit, which underscores the need for robust preclinical and clinical trial data.

The potential applications of TriTEs are vast, with therapeutic areas spanning various hematological malignancies and solid tumors such as colorectal cancer, lung cancer, prostate cancer, and multiple myeloma. Some constructs are even being developed for non-oncologic conditions like diabetic macular edema and neovascular age-related macular degeneration, highlighting the versatility and broad therapeutic applicability of this technology. Preclinical case studies have provided compelling evidence for the efficacy of TriTEs—not only in terms of enhancing T-cell activation and tumor cell lysis but also in improving overall survival in animal models.

Looking into the future, emerging trends suggest that optimization of TriTE structure will continue to benefit from advances in protein engineering, nanobody technology, and computational design. The integration of costimulatory domains and combinatorial approaches with other immunotherapies, such as checkpoint inhibitors and adoptive cell therapies, is likely to yield even more potent therapeutic options. In addition, improvements in the manufacturing processes, dosing strategies, and preclinical screening methods will further expedite the translation of TriTEs from the laboratory to the clinic.

In summary, the current landscape of trispecific T-cell engager development is both dynamic and promising. The extensive efforts from various research groups and industry leaders are converging on the design and clinical testing of TriTEs that are expected to deliver a potent and selective antitumor response. As ongoing research addresses the existing scientific, technical, and regulatory challenges, TriTEs are poised to become a transformative modality in the fight against cancers and potentially other diseases. The future of TriTE technology lies in its ability to precisely harness the immune system for targeted therapy, thus opening up new horizons in personalized and effective cancer treatment.