What Trispecific antibody are being developed?

Introduction to Trispecific Antibodies
Trispecific antibodies are an advanced class of engineered antibody therapeutics that are designed to simultaneously bind three distinct antigens or epitopes. Unlike traditional monoclonal antibodies that recognize a single target or bispecific antibodies that engage two targets, trispecifics leverage a multi‐targeting approach to enhance specificity, add additional functionalities, and overcome emerging resistance mechanisms by engaging multiple biological pathways in a single molecule. Their design embodies the convergence of antibody engineering, molecular biology, and immunotherapy, enabling novel mechanisms of action that include redirection of immune effector cells, simultaneous blockade of multiple signaling pathways, and even combination strategies that mix immune cell activation with tumor antigen targeting.

Definition and Mechanism of Action
At their core, trispecific antibodies are defined by their ability to bind three individual targets concurrently. This multifunctionality affords them several potential advantages. First, when one arm binds a tumor antigen, a second arm can recruit an immune cell such as a T cell (via CD3 engagement) while the third arm may deliver a costimulatory signal or block an alternate tumor escape pathway (for example, by targeting checkpoint molecules or alternative growth factor receptors). For instance, some trispecific constructs are being designed to recruit both T cells and natural killer (NK) cells to the tumor microenvironment. One study described trispecific antibody formats termed “T cell and NK cell engagers” (TaKEs) which, after their construction, showed the ability to bind cancer cells, T cells, and NK cells, while demonstrating robust cancer growth inhibitory effects that were comparable to or exceeded that of certain bispecific antibodies. This multidimensional engagement is hypothesized to increase the efficiency of tumor cell eradication by overcoming limitations such as the heterogeneity in immune effector cell populations. Furthermore, trispecific antibodies can be engineered via various formats such as full-length IgG-like molecules with additional antigen-binding fragments, single-chain variable fragments (scFv), or even VHH-based constructs that present favorable drug-like properties including increased stability, solubility, and manufacturability.

Historical Development and Evolution
The evolution of antibody therapeutics has been marked by progressive enhancements in specificity and functionality, beginning with monoclonal antibodies, advancing to bispecific formats, and now moving to trispecific constructs. Historically, monoclonal antibodies represented a breakthrough in targeted therapy, however, their “one target – one response” paradigm soon proved inadequate for complex diseases like cancer where redundancy and compensatory pathways are common. Building upon the clinical success of bispecific antibodies (evidenced by early products that engaged T cells to kill tumor cells), researchers began exploring trispecific formats to integrate additional mechanisms—such as the provision of both activating and costimulatory signals—to further enhance antitumor efficacy and reduce the potential for tumor escape. Early proofs-of-concept were established in preclinical studies, wherein trispecific antibodies demonstrated the ability to engage multiple receptor targets simultaneously, evidenced by studies targeting combinations such as epidermal growth factor receptors together with T cell receptors and NK cell markers. Furthermore, innovative platforms that allowed for orthogonal Fab assembly or common light chain technologies have enabled scientists to overcome technical hurdles in creating stable, manufacturable trispecific constructs. As our understanding of tumor immunology and resistance mechanisms has evolved, so too have the design strategies underlying trispecific antibodies, marking a clear evolution from the simpler formats of earlier decades to today’s highly engineered and multifunctional molecules.

Current Trispecific Antibodies in Development
Modern drug development programs have embraced trispecific antibodies as a promising approach to tackle the unmet needs in various disease indications, particularly in oncology and infectious diseases. Several research groups and biopharmaceutical companies are actively developing trispecific antibody candidates, often using a variety of engineering strategies to maximize their potential efficacy and safety.

Leading Research and Development Companies
A number of global and regional pharmaceutical companies and biotech start‐ups are contributing to the advancement of trispecific antibodies:

- AbbVie and Simcere Zaiming: AbbVie, a leader in hematologic malignancies, has partnered with Simcere Zaiming to develop a humanized trispecific antibody (SIM0500) aimed at relapsed or refractory multiple myeloma (MM). This candidate targets three antigens: G-protein-coupled receptor class 5 member D (GPRC5D), CD3, and B-cell maturation antigen (BCMA). This strategic collaboration aims to harness the antitumor benefits of simultaneous targeting, potentially leading to a product with an improved therapeutic window and widespread market opportunities.
- Sanofi: In addition to its work in oncology, Sanofi has invested in trispecific antibodies for other indications such as HIV/AIDS. A landmark study demonstrated that trispecific antibodies engineered to target three distinct HIV epitopes were highly effective in suppressing virus replication and showed superior breadth in neutralization compared to previous monoclonal and bispecific approaches. While this candidate is distinct from the cancer-focused ones, it reinforces the versatility of the trispecific platform.
- Merus: Utilizing their proprietary Triclonics platform, Merus is one of the pioneers in generating trispecific antibodies. The company has reached proof-of-concept candidates that are currently moving towards early-phase clinical evaluation. Their pipeline is highlighted by collaborations with industry partners like Gilead Sciences, which aims to discover novel trispecific T-cell engagers with robust immune activation profiles.
- Johnson & Johnson: The trispecific antibody JNJ-79635322A, which targets three as yet undisclosed antigens, has been developed with the aim of modulating the tumor microenvironment and enhancing immune-mediated antitumor responses. This example underscores the growing trend among large pharmaceutical companies to include trispecific formats in their late-stage preclinical and early clinical development pipelines.
- Affimed: Although traditionally known for bispecific modalities, companies like Affimed are expanding their efforts by exploring trispecific formats that can significantly widen the therapeutic window by better distinguishing between tumor cells and healthy tissues.

Collectively, these efforts across multiple companies highlight a competitive and collaborative environment, with a consistent focus on integrating multiple signaling mechanisms into a single therapeutic entity to maximize antitumor efficacy while mitigating potential side effects.

Clinical Trials and Pipeline Status
While trispecific antibodies are still largely in the early phases of clinical development, there is a robust pipeline indicating high expectations for these next-generation molecules:

- Phase I/II Clinical Trials: Most trispecific antibodies under development are currently confined to early-phase clinical trials. Candidates such as SIM0500 for multiple myeloma are undergoing Phase I studies in the US and China. Similarly, diverse trispecific constructs targeting key oncogenic receptors (e.g., combinations of Eph receptors as seen in a candidate targeting EphA2, EphA4, and EphB4) have demonstrated promising preclinical efficacy and are expected to enter early-phase human trials.
- Pipeline Diversity: The pipeline is characterized not only by the variety of targeted antigens but also by the different engineering formats. Some candidates are designed to recruit both T cells and NK cells simultaneously, thereby addressing the heterogeneity of immune cell populations within tumors. Others combine tumor target engagement with immune costimulation and blockade of escape pathways, which may eventually allow for a reduction in combination therapies where multiple monoclonal antibodies are administered separately.
- Regulatory Outlook: Although no trispecific antibody has received regulatory approval yet, industry projections suggest that the first such novel therapeutic could be available in the market as early as the mid-2020s. Given the considerable investments from major pharmaceutical companies, it is clear that trispecific antibodies represent an attractive innovation with the potential to significantly impact clinical practice in oncology and beyond.

Applications and Therapeutic Areas
Trispecific antibodies are being developed for a range of therapeutic applications, with oncology being the most prominent, but with emerging interest in other fields such as autoimmune diseases and viral infections.

Oncology
The primary application of trispecific antibodies lies in cancer therapy, owing to several attractive mechanisms:

- Enhanced Immune Cell Engagement: By binding to a tumor antigen and concurrently engaging receptors on T cells (e.g., CD3) and NK cells (e.g., CD16), trispecific antibodies have been designed to effectively recruit and activate multiple effector cell types simultaneously. This dual immune cell engagement not only results in more robust cytotoxic responses but also may overcome limitations posed by the heterogeneous infiltration of immune cells within solid tumors.
- Targeting Multiple Tumor-Associated Antigens: Some trispecific candidates, for example, the one engineered to concurrently block and induce internalization of Eph receptors (EphA2, EphA4, and EphB4), target multiple growth or survival pathways on cancer cells. Such multi-targeting pode help prevent tumor escape via compensatory signaling pathways, thus possibly resulting in improved patient outcomes.
- Potential Combination with Other Modalities: The integration of trispecific antibodies into combination immunotherapy regimens has high potential. Their multifaceted design not only allows for direct tumor cell killing but also can synergize with other immunotherapeutic agents, including checkpoint inhibitors, to further enhance the overall antitumor immune response.
- Preclinical Efficacy in Various Indications: Preclinical models have shown that trispecific antibodies can lead to significant tumor growth inhibition and in some cases complete remissions that were previously unachievable with bispecific or monospecific therapies alone. These promising results have ignited several clinical development programs in hematologic malignancies, solid tumors, and even rare, aggressive cancers which have limited treatment options.

Autoimmune Diseases
While the majority of trispecific antibody development has been focused on oncology, their ability to modulate multiple immune pathways simultaneously also suggests potential applications in autoimmune diseases. Although less extensively explored compared to cancer, the following perspectives are noteworthy:

- Dual Regulation of Immune Cell Activation: In autoimmune conditions, aberrant immune activation is a key pathogenic mechanism. A trispecific antibody that could simultaneously engage an inhibitory receptor, block pro-inflammatory cytokines, and modulate autoreactive cell populations might offer unprecedented precision in restoring immune homeostasis.
- Preclinical Modeling and Early Research: Some early-stage studies have begun to investigate whether trispecific approaches can be applied to modulate immune responses in autoimmune diseases. However, most of the available literature in our current references is skewed toward oncology. Future research may lead to the adaptation of trispecific platforms initially designed for cancer immunotherapy towards treating autoimmune disorders.
- Potential to Minimize Off-Target Effects: By virtue of its ability to target three specific antigens simultaneously, a trispecific antibody has the potential to achieve a more refined targeting of diseased tissues while sparing healthy tissues. If successful, such an approach could prove especially beneficial for autoimmune diseases where systemic immunosuppression poses significant risks.

Challenges and Future Directions
The development of trispecific antibodies, despite its promising potential, is not without significant challenges. Addressing these challenges is key for their successful transition from bench to bedside.

Technical and Manufacturing Challenges
Producing a trispecific antibody poses several technical hurdles beyond those encountered with bispecific formats:

- Molecular Complexity: The addition of a third specificity significantly increases the structural complexity of the antibody molecule. This can lead to issues with correct folding, assembly, and overall stability. For example, ensuring that all three binding domains have optimal affinity and are presented in the right spatial orientation is a formidable bioengineering challenge.
- Manufacturability and Expression Yields: The production process for trispecific antibodies is generally more complicated. Manufacturing requires advanced recombinant technologies to maintain homogeneity and high expression yields, while preventing formation of aggregates or misassembled products. Techniques such as employing orthogonal Fab interfaces or using a common light chain approach have been adopted to tackle these challenges, but further optimization is needed.
- Pharmacokinetics and Stability: Achieving favorable pharmacokinetics is another hurdle. The addition of multiple binding domains may impact serum half-life and in vivo distribution, creating a balance between prolonged efficacy and potential off-target toxicity.
- Risk of Immunogenicity: As with all engineered biological molecules, trispecific antibodies run the risk of inducing anti-drug antibodies (ADAs). The increased number of epitopes and structural alterations compared to native antibodies can trigger an immune response, potentially compromising the efficacy and safety of the therapeutic.
- Analytical Challenges: From a regulatory and clinical development perspective, the characterization, analysis, and quality control of these molecules are more complex compared to monoclonal or bispecific antibodies. Advanced biophysical and biochemical techniques must be employed to ascertain that each binding domain is functional and that the overall molecule behaves as expected in formulation and during storage.

Potential Future Developments
Looking forward, several strategies and innovations are anticipated to further improve trispecific antibody technology:

- Refinement of Engineering Platforms: Continued development of engineering platforms such as Triclonics (employed by Merus) and various VHH-based formats are expected to yield trispecific antibodies with improved developability, stability, and reduced immunogenicity. The evolution of computational structural modeling and screening methods will likely play a central role in designing fully optimized molecules that meet both therapeutic and manufacturability criteria.
- Enhanced Immune Engagement Strategies: Future trispecific antibodies may incorporate even more sophisticated immune modulation strategies. For example, further refinement of dual immune cell engagers that simultaneously recruit T cells and NK cells may allow for enhanced precision in tumor cell lysis and overcome tumor microenvironment resistance. Likewise, novel costimulatory or checkpoint blockade functionalities may be integrated into the design to provide balanced immune activation.
- Broadening the Therapeutic Landscape: Beyond oncology and the emerging foray into autoimmune diseases, trispecific antibodies have the potential to be applied in various other therapeutic areas such as infectious diseases. The successful demonstration of a trispecific antibody for HIV by Sanofi highlights the platform’s versatility, and similar strategies may be applicable for other infectious agents or even neurological diseases.
- Combination Therapies and Personalized Medicine: In the context of precision medicine, trispecific antibodies may eventually be used in combination with other therapeutic modalities, such as small molecule inhibitors or cellular therapies (e.g., CAR T-cell therapy), to create synergistic regimens that are tailored to the individual patient’s tumor antigenic profile. As our understanding of the tumor microenvironment and immune signatures expands, future trispecific constructs might be custom-designed to target a patient’s specific molecular aberrations.
- Regulatory and Clinical Evolution: With the maturation of the clinical pipelines and accumulation of clinical efficacy data, regulatory agencies are likely to refine guidelines for multispecific antibodies. This cultural shift may facilitate accelerated development and eventual regulatory approval of trispecific antibodies, paving the way for their use as standard-of-care therapies in oncology and potentially other disease areas.

Conclusion
In summary, trispecific antibodies represent a significant next-generation advancement in the field of therapeutic antibodies. They are defined by their ability to simultaneously engage three distinct targets, thereby offering enhanced specificity, improved immune cell recruitment, and the potential to better overcome tumor resistance mechanisms. The historical progression from monoclonal to trispecific antibodies reflects an evolutionary leap driven by the need for superior therapeutic efficacy in complex diseases such as cancer. Leading companies such as AbbVie, Sanofi, Merus, Johnson & Johnson, and Affimed are at the forefront of developing various trispecific antibody candidates, with several molecules already in early-phase clinical trials. These constructs are primarily being developed for oncology, with promising preclinical results indicating that simultaneous recruitment of T cells, NK cells, and blockade of multiple tumor-associated antigens can result in significant tumor growth inhibition and enhanced cell killing. Although applications in autoimmune diseases are still under exploration, the underlying technology holds promise for multiple therapeutic areas.

From a technical perspective, the design and manufacture of trispecific antibodies remain challenging due to their intrinsic molecular complexity, manufacturing yields, stability, pharmacokinetics, and the potential immunogenicity issues that may arise. However, innovations in antibody engineering platforms, computational modeling, and quality assessment methods are progressively addressing these obstacles. Future developments are anticipated to further refine immune engagement strategies, broaden the therapeutic applications beyond oncology, and enable personalized combination therapies, all while meeting stringent regulatory requirements.

In conclusion, while trispecific antibodies have not yet received regulatory approval, their ongoing development and early clinical trial data suggest that they may soon fill critical therapeutic gaps in cancer treatment and potentially other challenging disease areas. Their multifaceted mode of action, combined with advanced engineering capabilities, positions them as a promising modality for achieving improved clinical outcomes and enhanced patient benefit. Continued research into optimizing these molecules, addressing manufacturing challenges, and understanding clinical efficacy will ultimately determine their role in the future of precision medicine.