What are the different types of drugs available for Trispecific T-cell engager (TriTE)?

Introduction to Trispecific T-cell Engagers (TriTEs)

TriTEs represent an emerging class of immunotherapeutics that build upon the concept of bispecific T-cell engagers (BiTEs) by adding a third specificity. This additional binding domain expands their functional versatility and therapeutic window, allowing them to engage multiple antigens or costimulatory receptors simultaneously. In essence, TriTEs are engineered antibody constructs that “bridge” T cells and tumor cells while incorporating an extra modality to improve specificity, potency, or safety. Over the past decade, T-cell engaging antibodies have revolutionized immuno-oncology, and the evolution from bispecific to trispecific formats is driven by the need to overcome limitations such as tumor heterogeneity, immune escape, and suboptimal T-cell activation.

Definition and Mechanism of Action

Trispecific T-cell engagers (TriTEs) are defined as antibody-based molecules that simultaneously bind to three different epitopes. One arm typically binds to the T-cell receptor complex, most commonly targeting CD3, which recruits and activates T cells. The other two arms are used for tumor targeting or for providing additional costimulatory signals (such as through CD28 or CD137) that augment the activation and persistence of T cells. Some TriTEs are designed to engage two distinct tumor-associated antigens (TAAs) ensuring enhanced selectivity—by requiring tumor cells to express both antigens for effective lysis—and thereby limit off-target toxicities. Other TriTE formats incorporate costimulatory receptor binding, such as to CD28, to further potentiate T-cell activation and overcome anergy, which is especially useful in cases where tumor antigen density is low and in circumventing immune escape mechanisms.

Mechanistically, the simultaneous engagement of three epitopes establishes a potent immunologic synapse between the T cell and tumor cell. This formation not only brings T cells into close proximity to the malignant target but also ensures that multiple activation signals are delivered concurrently, leading to robust T-cell activation, proliferation, and cytolytic activity. In preclinical and early clinical studies, TriTEs have demonstrated improved tumor cell killing, enhanced T-cell expansion, and the ability to overcome some resistance mechanisms inherent in solid tumors.

Historical Development and Clinical Significance

The concept of T-cell engager antibodies dates back several decades, with the development of bispecific formats paving the way for clinical successes, notably with agents like blinatumomab. However, while these agents provided a strong proof-of-concept by redirecting T cells against malignant cells, their application was limited by factors such as short half-life, systemic toxicities, and tumor antigen escape. In response, the field has witnessed a progressive evolution toward multi-specific platforms.

Trispecific constructs emerged as a logical next step in design—a response to the challenges observed with simpler bispecific molecules. By harnessing a third binding domain, investigators aimed to add functionalities such as dual tumor antigen targeting or incorporation of costimulatory signals. These advancements are clinically significant because they hold the promise of achieving higher efficacy with reduced toxicity, particularly in solid tumors where antigen heterogeneity poses a substantial challenge. In summary, TriTEs not only build on the established successes of earlier T-cell engagement strategies but also open up new avenues for targeting an even broader range of tumor antigens and for modulating immune responses more precisely.

Types of TriTE Drugs

The landscape of TriTE drugs is diverse, with variations based on their design, target specificity, and additional engineered functions. Although several TriTE constructs remain in developmental or early clinical phases, the classifications can be approached from several perspectives: regulatory status (approved versus investigational), and mechanistic differences in the binding domains.

Currently Approved TriTE Drugs

Currently, the majority of T-cell engagers approved in clinical practice are bispecific in nature, while truly trispecific agents are largely in the clinical development or investigational stage. As of now, no TriTE drug has yet received full market approval solely on the basis of its trispecific format. However, the preclinical and early clinical results are encouraging and suggest that it is only a matter of time before one or more TriTE drugs gain regulatory approval.

The clinical successes of bispecific T-cell engagers have set a high benchmark and illustrate the necessity for enhanced tools that address limitations such as antigen escape and lack of optimal T-cell costimulation. TriTE drugs, by virtue of their third binding domain, are anticipated to provide a more refined therapeutic index through dual tumor targeting or costimulatory potentiation, potentially offering superior efficacy with mitigated off-target effects. The transition from bispecific to trispecific platforms is a response to both mechanistic insights and the need for improved clinical outcomes, and clinical trials are underway that may soon bring these agents into the market.

Investigational TriTE Drugs in Clinical Trials

A number of investigational TriTE agents are in various phases of clinical trials, targeting a wide range of malignancies—from solid tumors to hematological cancers. These investigational drugs leverage different design approaches:

1. Dual Tumor Antigen Targeting TriTEs:
One notable investigational approach involves the design of TriTE molecules with a central CD3-binding scFv flanked by two distinct tumor antigen-targeting domains. For instance, a TriTE designed for colorectal cancer targets epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) simultaneously, thereby ensuring that only tumor cells expressing both markers are targeted. This dual targeting enhances selectivity and potentially circumvents the issue of antigen loss that is common in tumor evolution. The specificity provided by the dual targeting mechanism also contributes to improved in vitro potency and better in vivo survival benefits in preclinical models.

2. Costimulatory-Enhanced TriTEs:
Another investigative strategy employs the incorporation of costimulatory receptors into the TriTE format. In these designs, one of the tumor-targeting arms may be replaced or augmented by a binding domain that targets costimulatory receptors such as CD28, CD137, or other members of the cluster of differentiation family. Such constructs aim to overcome the limitations of mere T-cell recruitment by providing additional signals that promote T-cell activation, proliferation, and sustained cytotoxicity even in immunosuppressive tumor microenvironments. A patent document describes trispecific molecules that bind to tumor antigens like DLL3, MUC17, or CLDN18.2, and in addition recruit T cells via CD3 while also engaging a costimulatory receptor (e.g., CD28 or CD137), thereby integrating multiple immune activation pathways.

3. TriTEs with Risk Mitigation Modules:
One of the challenges in T-cell engager development is the risk of cytokine release syndrome (CRS) and other immune-mediated toxicities. In response, some investigational TriTE formats incorporate additional functionalities to mitigate these risks. For example, the TriTECM (Tri-specific T-cell Engager with Cytokine Modulation) combines a CD3-binding element, dual tumor-targeting arms (such as binding EGFR and PD-L1) and an anti-interleukin 6 receptor (IL-6R) module to dampen excessive cytokine release while still maintaining robust T-cell activation. This design shows promise in enhancing the therapeutic window by controlling adverse inflammatory responses.

4. Nanobody-Based TriTEs:
In addition to traditional antibody fragment-based designs, nanobody-based TriTEs are being explored. Nanobodies are small, single-domain antibodies that offer advantages such as improved tissue penetration, stability, and ease of engineering. A nanobody-based trispecific T-cell engager (Nb-TriTE) has been developed that not only recruits T cells but also overcomes tumor-mediated immunosuppression by fusing an anti-PD-1 nanobody to a bispecific T-cell engager construct targeting fibroblast activation protein (FAP). In preclinical models, this Nb-TriTE has induced potent T-cell activation and tumor-specific cytotoxicity while reducing immune evasion, making it a promising avenue for solid tumor treatment.

5. CD38/CD3xCD28 TriTEs for Hematologic Malignancies:
Specific TriTE designs are also being evaluated for hematologic malignancies such as relapsed/refractory multiple myeloma (RRMM). For example, investigational constructs that incorporate CD38 targeting with a CD3-binding domain and a CD28 costimulatory arm have shown preclinical efficacy in overcoming resistance to anti-CD38 monoclonal antibodies. Preclinical studies demonstrated that these TriTEs could restore T-cell functionality and engage T cells in a manner less reliant on antigen density and NK cell activity, potentially benefiting patients with diminished CD38 expression and impaired NK cell function following previous therapies.

These investigational TriTEs are at various stages of clinical testing, with extensive preclinical data underscoring their potential. The diversity of designs—from dual tumor-targeting formats to those enriched with costimulatory and risk-mitigation features—reflects the multifaceted approach researchers are taking to maximize therapeutic efficacy and minimize adverse effects.

Mechanistic Variations among TriTE Drugs

The distinct mechanistic variations across TriTE platforms can be primarily categorized based on the nature of the third specificity. These variations are critical because they determine not only the efficacy of tumor cell lysis but also the safety profile and clinical usability of the molecule.

1. Dual Tumor Antigen Recognition:
In dual tumor antigen targeting TriTEs, two separate arms bind to distinct surface antigens on cancer cells. This strategy increases tumor selectivity by ensuring that only cells expressing both antigens are targeted, thereby reducing the probability of off-tumor effects. For solid tumors that typically show heterogeneous antigen expression, this approach may limit immune escape by requiring dual-antigen co-expression for T-cell engagement. The simultaneous binding to two TAAs also improves the formation of the immunological synapse between the T cell and tumor cell, which is critical for effective cytotoxicity.

2. Costimulatory Signal Incorporation:
Variations of TriTEs that include an arm binding to a costimulatory receptor (for example, CD28) provide the additional activation signals necessary for overcoming T cell exhaustion or suboptimal activation. This design is especially important in tumor environments with high levels of immunosuppressive signals that can blunt T-cell responses. By adding a costimulatory element, the TriTE not only brings T cells in proximity to tumor cells but also ensures that T cells receive the full activation cascade required for proliferation and cytokine production. Costimulatory-enhanced TriTEs can generate stronger and more sustained tumor-specific immune responses.

3. Modulation of Cytokine Release:
Another mechanistic variant includes modules for risk mitigation, particularly targeting the cytokine release cascade. For instance, the TriTECM design integrates an IL-6 receptor-binding domain alongside the CD3-binding and tumor antigen targeting arms. This addition helps modulate the inflammatory response triggered by T-cell activation, thereby reducing the risk of cytokine release syndrome (CRS) while retaining effective killing of the tumor cells. The balance between robust immune activation and controlled cytokine release is vital for translating strong antitumor activity into a safe therapeutic profile.

4. Integration of Nanobody Technology:
Nanobody-based TriTEs are unique due to their compact, highly stable nature that can facilitate better tissue penetration and potentially lower immunogenicity. The small size of nanobodies allows for more flexible linking formats, which may be used to combine tumor antigen recognition with T-cell engagement and immunomodulatory functions. The nanobody-based approach further expands the repertoire of antigens that can be targeted and provides opportunities for engineering constructs that are minimal yet highly active in engaging T cells.

5. Antigen Density and Microenvironment Considerations:
Finally, some TriTEs are being engineered with an eye toward the microenvironment dynamics of the tumor. By combining a T-cell binding domain with two different tumor antigen recognition moieties or by incorporating aspects that target both tumor cells and components of the immunosuppressive stroma (for example, molecules targeting fibroblast activation protein), these TriTEs are designed to be effective in complex solid tumor architectures. This mechanistic alignment helps to overcome challenges related to antigen density variability and improves the overall therapeutic index by ensuring that T-cell activation happens predominantly at the tumor site.

Clinical Applications and Efficacy

TriTE drugs are primarily being developed for use in oncology, where precise T-cell targeting against tumor cells is paramount. The clinical applications cover both solid tumors and hematologic malignancies, with preclinical studies and early trial results showing promising efficacy. The inclusion of dual tumor targeting and costimulatory engagement has been correlated with improvements in both cytotoxicity assays and overall survival in experimental models.

Therapeutic Indications

The therapeutic indications for TriTE drugs are broad and are being explored in several clinical settings:

1. Solid Tumors:
Given the challenge of heterogeneity in antigen expression among solid tumors, TriTEs that incorporate dual tumor antigen recognition have gained attention. For example, the TriTE designed for colorectal cancer targets both EGFR and EpCAM, leveraging the co-expression of these antigens to improve tumor selectivity and to limit off-target activity. The dual targeting mechanism potentially abrogates tumor escape due to antigen loss and ensures a robust immune engagement in complex tumor microenvironments.

2. Hematologic Malignancies:
While bispecific T-cell engagers have already established a foothold in hematologic cancers such as acute lymphoblastic leukemia (ALL), TriTEs are being developed to address resistance mechanisms that emerge after repeated antibody exposure. One investigational TriTE format targets CD38 along with CD3 and incorporates a costimulatory domain (CD28) for the treatment of relapsed/refractory multiple myeloma. This approach is particularly valuable for patients who exhibit reduced expression of CD38 and impaired natural killer (NK) cell function due to prior therapies.

3. Overcoming Immune Resistance:
By offering costimulatory signals via an added receptor binding domain, TriTEs may also be applied in settings where the tumor microenvironment is highly suppressive. For instance, in tumors that upregulate inhibitory ligands or exhibit high levels of checkpoint proteins (e.g., PD-L1), TriTEs that incorporate an anti-PD-1 or other immunomodulatory domain can effectively reinvigorate T cells even in the face of resistance mechanisms. This strategy is under investigation for various solid tumors where immune escape is a clinical challenge.

4. Combination with Other Therapeutic Modalities:
The modular design of TriTEs also permits their combination with other immunotherapies—such as checkpoint inhibitors or adoptive cell therapies—leading to synergistic effects. The potential for TriTEs to serve as “off-the-shelf” therapeutics that can be used in combination protocols expands their applicability across a range of cancers, potentially improving response rates and durability of responses.

Clinical Trial Outcomes

Preclinical studies and early-phase clinical trials of TriTE drugs have provided insights into their potential clinical efficacy:

1. Improved T-cell Activation and Proliferation:
Investigational TriTE constructs have demonstrated the ability not only to recruit T cells but also to enhance their proliferation. For instance, studies on CD33-targeted TriTEs have shown that inclusion of the trispecific format resulted in stronger T-cell expansion compared to associated bispecific formats, which is critical for sustained anti-tumor responses. Enhanced expansion is often correlated with improved in vitro cytotoxicity and better in vivo outcomes in xenograft models.

2. Potent Cytotoxicity and Dose-Dependent Killing:
TriTEs that have been designed to simultaneously engage dual tumor antigens have shown highly specific, dose-dependent cytotoxicity against target cancer cells. This specificity is especially important in preclinical models, where dual antigen recognition has translated to up to 100-fold improvement in cell killing potency for double-positive cells compared to single antigen-positive cells. This potent cytotoxic effect underlines the rationale for targeting tumors with heterogeneous antigen expression.

3. Efficacy with Costimulation:
Constructs incorporating costimulatory domains such as CD28 have further demonstrated improved efficacy. By providing both T-cell engagement (through CD3 binding) and an extra activation signal (via CD28), these TriTEs have been shown to reduce tumor growth in animal models and have improved T-cell-mediated killing. Such dual signaling not only promotes immediate cytotoxicity but also contributes to the generation of memory T-cell subsets that could provide long-term antitumor immunity.

4. Reduction of Adverse Effects:
Early safety data from investigational studies also suggest that TriTEs incorporating cytokine modulation modules (such as those binding IL-6R) can achieve a favorable balance between robust T-cell activation and the reduction of cytokine-associated toxicities, such as CRS. This balance is vital for improving patient tolerability and expanding the therapeutic window of these agents.

Overall, clinical trial outcomes in preclinical models indicate that these drugs have the potential to be more effective than their bispecific predecessors, particularly in tumors where antigen heterogeneity and immune suppression are significant hurdles.

Challenges and Future Directions

Despite the promising data, several challenges remain in the development and clinical application of TriTE drugs. Addressing these challenges is critical to fully harnessing the potential of trispecific platforms and ensuring that these therapeutics can transition successfully from bench to bedside.

Current Challenges in TriTE Development

1. Manufacturing Complexity and Stability:
The engineering of trispecific molecules involves more complex production processes than their bispecific counterparts. Linking three distinct binding domains into a single functional molecule requires careful design to ensure proper folding, stability, and activity. Issues such as protein aggregation and inconsistent yields may pose significant hurdles during scale-up, manufacturing, and quality control.

2. Pharmacokinetics and Biodistribution:
The molecular size and format of TriTEs influence their half-life and biodistribution. Since many T-cell engagers are relatively small molecules with rapid clearance, achieving a balance between adequate tissue penetration and a sufficiently prolonged half-life is a significant challenge. Modifications such as the inclusion of Fc-fragment, albumin-binding domains, or nanobody-based designs could help overcome these issues, but they also require rigorous optimization to avoid compromising activity.

3. Safety and Cytokine Release:
T-cell engagers inherently carry the risk of triggering cytokine release syndrome (CRS) due to robust T-cell activation. Incorporating additional activation domains, as seen in TriTEs, might exacerbate this risk if not properly regulated. While constructs like TriTECM aim to mitigate this by adding IL-6R binding modules, fine-tuning the balance between efficacy and safety remains challenging. This necessitates extensive preclinical safety studies and cautious dose escalation in early-phase trials.

4. Tumor Heterogeneity and Antigen Escape:
Tumor heterogeneity remains a central challenge for targeted immunotherapies. Even with dual antigen targeting, tumors may downregulate one or both target antigens under immune pressure, leading to treatment resistance. Overcoming antigen escape requires designing TriTEs that can target multiple markers simultaneously or in combination with treatments that increase antigen expression on tumor cells.

5. Optimizing Costimulatory Signaling:
While adding a costimulatory binding domain (such as CD28) to TriTEs has shown promise in enhancing T-cell activation, the intensity and duration of costimulatory signals need to be carefully balanced. Excessive costimulation may lead to off-target effects or exhaustion of T cells, whereas inadequate costimulation could result in suboptimal therapeutic responses. Determining the optimal configuration for costimulatory engagement is an ongoing area of research.

6. Immunogenicity and Off-Target Effects:
Given their engineered nature, TriTE molecules may elicit antidrug antibody responses that could neutralize their activity or lead to adverse immune reactions. The design of the molecule—particularly when incorporating non-human components such as camelid single-domain antibodies (nanobodies)—must account for potential immunogenicity. Strategies to humanize these constructs are under active investigation to improve their clinical safety profile.

Future Research and Development Prospects

1. Innovative Molecular Designs:
Future research is likely to focus on the continued evolution of TriTE designs. Next-generation constructs may incorporate more sophisticated modular components, including tunable costimulatory signals and integrated safety off-switches to mitigate toxicity. The adoption of novel linking technologies and scaffolds will likely improve molecular stability and expand the design space for creating multifunctional T-cell engagers.

2. Personalized Medicine Approaches:
Advances in genomics and proteomics are paving the way for more personalized approaches to cancer therapy. In the context of TriTEs, selecting target antigens based on individual tumor profiles and adjusting the TriTE format accordingly could result in more effective and patient-specific treatments. Biomarker-driven patient stratification will be crucial in optimizing the use of these agents in clinical trials and, eventually, routine practice.

3. Combination Therapies:
The future of TriTE drug development may lie in combination with other immunotherapeutic agents such as checkpoint inhibitors, adoptive cell therapies (CAR-T cells), and conventional chemotherapy. These combination strategies could provide synergistic benefits, enhancing immune activation while minimizing resistance mechanisms. Trial designs that incorporate TriTEs together with agents targeting different aspects of tumor biology are anticipated to offer improved outcomes.

4. Enhanced Preclinical Models and Quantitative Systems Pharmacology (QSP):
To better predict clinical responses and optimize dosing regimens, researchers are increasingly turning to quantitative systems pharmacology (QSP) models that integrate multiple layers of biological complexity. These models, which have been effectively applied in studies of trispecific T-cell engagers in multiple myeloma, will be instrumental in guiding clinical trial design, predicting optimal dosing ranges, and understanding the impact of tumor–immune cell interactions. Improvements in preclinical models will likely reduce translational gaps between animal studies and human clinical outcomes.

5. Risk Mitigation Strategies:
New strategies to minimize adverse effects remain a high priority. Refinements in the design of TriTEs—such as incorporation of elements that modulate cytokine release or provide controlled activation—will be critical. In particular, the development of platforms such as prodrug TriTEs that remain inactive until they reach the tumor microenvironment holds promise for reducing systemic toxicity while preserving antitumor activity.

6. Regulatory and Manufacturing Innovations:
As TriTEs transition from investigational to potentially approved therapeutic agents, regulatory pathways and manufacturing processes must adapt. Streamlined production methods, along with improved characterization and quality control procedures, will facilitate the large-scale manufacturing of these complex biologics. Regulatory agencies are also evolving their frameworks to better evaluate multi-specific therapeutics, which will help expedite the clinical development of TriTEs.

Conclusion

In summary, trispecific T-cell engagers (TriTEs) are an innovative evolution in immunotherapy that builds upon the foundation set by bispecific antibodies. They exploit three distinct binding domains—typically engaging the T-cell receptor (CD3), a tumor antigen or dual TAAs, and an additional costimulatory receptor such as CD28 or CD137—to enhance the formation of an immunologic synapse, improve T-cell activation, and increase specificity for tumor cells.

We have identified several mechanistic variations among TriTE drugs. Some investigational TriTEs focus on dual tumor antigen recognition, thereby enhancing selectivity and reducing off-target effects. Others incorporate costimulatory signals, which not only enhance T-cell activation but also potentially overcome immune resistance in hostile tumor microenvironments. Moreover, innovative designs that incorporate risk-mitigating modules—such as IL-6 receptor targeting to dampen cytokine release—are currently under investigation. Nanobody-based TriTEs further underscore the diversity of design approaches, offering potential advantages in tissue penetration and stability. Although no TriTE has yet received full market approval, several promising candidates are under active clinical investigation for solid tumors and hematologic malignancies, including relapsed/refractory multiple myeloma and colorectal cancer.

The clinical applications of TriTEs are broad, targeting a range of malignancies where precise tumor recognition and potent T-cell activation are crucial. Early clinical trial outcomes have demonstrated improved T-cell activation, proliferation, and highly specific tumor cell killing, all of which are encouraging indicators of their therapeutic potential. However, substantial challenges—ranging from manufacturing complexity, pharmacokinetics, safety (notably cytokine release syndrome), to tumor antigen heterogeneity—remain to be addressed.

Looking forward, future research is expected to focus on innovative molecular design, personalized medicine approaches, combination therapy strategies, and refined preclinical models to fully realize the promise of TriTEs. Advances in quantitative systems pharmacology and regulatory innovations will also play key roles in guiding these developments. Ultimately, the successful integration of these strategies may lead to the next generation of highly effective, safe, and personalized cancer immunotherapies.

In conclusion, the different types of drugs available for Trispecific T-cell engagers highlight a diverse and rapidly evolving field. Researchers and clinical investigators are exploring a range of formats—from dual tumor antigen targeting to costimulatory-enhanced to risk-mitigated constructs—all of which aim to overcome the limitations seen with earlier T-cell engager formats. These innovative approaches hold the potential to significantly improve the therapeutic index, reduce adverse effects, and ultimately broaden the application of immunotherapies in both solid and hematologic malignancies. The next few years are poised to be transformative as these investigational TriTEs progress through clinical trials and, potentially, into approved use, fundamentally altering the paradigm of cancer treatment.