What Trispecific killer cell engager (TriKE) are being developed?

Introduction to Trispecific Killer Cell Engagers (TriKEs)

Definition and Mechanism of Action
Trispecific killer cell engagers (TriKEs) are an advanced class of engineered immunotherapeutics designed to harness the power of natural killer (NK) cells for targeted cancer therapy. In their design, TriKEs are composed of three functional components:
A binding domain that targets CD16, which is a prominent activating receptor on NK cells,
A cytokine moiety, typically an interleukin-15 (IL-15) crosslinker, that not only promotes NK cell activation but also drives expansion and persistence in vivo, and
A targeting module that specifically recognizes a tumor-associated antigen (TAA) expressed on the surface of cancer cells.

The combinatorial effect of these three domains is to form an immunologic synapse. This precise molecular interaction facilitates both direct cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) and durable NK cell expansion to maintain an effective antitumor response. The integration of IL-15, in particular, is fundamental because it ensures that the NK cells receive a self-sustaining proliferative signal while being armed with antigen specificity.

Overview of TriKEs in Immunotherapy
TriKEs represent an evolution of earlier bispecific killer cell engagers (BiKEs) that only targeted CD16 on NK cells and a tumor antigen. The addition of the IL-15 moiety in TriKEs has been a critical modification, resulting in superior NK cell activation, enhanced proliferation, and increased in vivo persistence relative to BiKEs. This improvement addresses one of the major limitations of NK cell therapies: their typically short lifespan and limited expansion after adoptive transfer. Consequently, TriKEs are positioned as a promising off-the-shelf immunotherapeutic alternative that not only redirects NK cell cytotoxicity towards malignancies but also overcomes several challenges of other cell-based therapies such as CAR-T cells. From hematologic cancers like acute myeloid leukemia (AML) to solid tumors expressing unique antigens, TriKEs present a modular platform adaptable to various indications.

Current TriKEs in Development

Major Companies and Research Institutions
A number of companies and research institutions have been at the forefront of developing TriKEs. Among the most prominent is GT Biopharma, which has extensively built and refined its TriKE® platform over recent years. This platform integrates advanced protein engineering techniques—including the use of scFvs (single chain variable fragments) and even camelid nanobodies for enhanced targeting specificity—with IL-15 cytokine delivery to NK cells. The University of Minnesota has also played a significant role as a collaborative partner, offering foundational research expertise and intellectual property that underpins the TriKE technology.

Beyond GT Biopharma, several academic groups and biotech companies are exploring alternative configurations of TriKEs. Some researchers are investigating TriKEs that substitute conventional scFvs with nanobodies, aiming to improve structural stability, reduce immunogenicity, and enhance tissue penetration. These academic collaborations are vital, as they contribute not only to the clinical development of current TriKE candidates but also fuel the innovation pipeline by exploring new antigen combinations and engineering strategies.

Key TriKEs in Clinical Trials
The TriKEs currently being developed span a variety of cancer targets and clinical indications. Several key examples include:

CD16/IL-15/CD33 TriKE (GTB-3550):
This is one of the most advanced TriKE constructs in clinical development, primarily targeting AML and related myeloid malignancies. GTB-3550 has been shown to enhance NK cell activation, proliferation, and cytotoxicity against CD33-positive targets. In early-phase clinical trials, GTB-3550 has demonstrated robust NK cell expansion and significant antileukemic activity without inducing Cytokine Release Syndrome (CRS) or other severe toxicities.

CD16/IL-15/CD19 and CD16/IL-15/CD22 TriKEs:
These constructs are designed to target B-cell malignancies by binding to key antigens such as CD19 and CD22. Recent studies have demonstrated that the CD16/IL-15/CD19 TriKE is capable of rescuing NK cell functionality from patients with chronic lymphocytic leukemia (CLL) and improving killing efficiency against CD19-positive targets. Similarly, designs incorporating CD22 alongside CD19 show promise by providing dual targeting for greater specificity and reduced likelihood of antigen escape.

CD16/IL-15/HER2 TriKE (CAM1615HER2):
This TriKE targets HER2-positive solid tumors, such as certain breast and ovarian cancers. Preclinical evidence shows that CAM1615HER2 amplifies NK cell degranulation and cytokine production, even enhancing NK cell function within the challenging tumor microenvironment found in ovarian cancer. In vivo xenograft models have demonstrated significant tumor growth inhibition, highlighting the potential of this TriKE candidate for treating HER2-positive carcinomas.

PSMA-Targeted TriKE:
For prostate cancer, a PSMA (prostate-specific membrane antigen) targeted TriKE has been developed. This candidate engages NK cells via CD16 while delivering IL-15 and binding PSMA on prostate cancer cells. Studies show that this construct not only improves NK cell activation and proliferation but also maintains cytotoxicity even under conditions of hypoxia and in the presence of drug-resistant prostate cancer cell lines.

B7H3-Targeted TriKE:
Designed to address both solid tumors and hematologic malignancies, the B7H3 TriKE utilizes an anti-B7H3 scFv or dual camelid nanobody and has demonstrated broad applicability. For example, GTB-5550 is a TriKE candidate that specifically targets B7H3-positive cells, including those in mesothelioma, ovarian, and multiple myeloma indications. The results indicate selective NK cell expansion and enhanced degranulation responses, thereby validating its potential as a pan-tumor antigen engager.

Other Investigational TriKEs:
In addition to these primary candidates, there are investigational TriKE designs that substitute or add additional functionalities. Some approaches involve integrating checkpoint-blocking scFvs (targeting molecules like PD-1, KIR, or NKG2A) into the TriKE structure to further modulate NK cell activity and overcome tumor-induced immunosuppression. These newer strategies likely pave the way for the development of trispecific molecules that can be tailored to the immunosuppressive microenvironment of different cancers.

Mechanisms and Applications

Mechanisms of Action in Cancer Therapy
TriKEs function through a multi-pronged mechanism that leverages both targeting precision and immune cell activation:

Targeted Engagement of NK Cells:
TriKEs bind with high affinity to CD16 on NK cells. This interaction triggers a potent activation signal that leads to rapid NK cell degranulation, the release of cytolytic granules, and ensuing tumor cell apoptosis. This mechanism ensures that NK cells are correctly and strongly activated at the tumor site.

IL-15–Mediated NK Cell Expansion and Survival:
The pivotal IL-15 moiety provides a direct stimulatory signal to NK cells, enhancing not only their immediate cytotoxic function but also their proliferation and long-term persistence. This intrinsic cytokine signal is essential because it reduces the need for ex vivo expansion of NK cells, making TriKEs effective as an off-the-shelf therapy.

Dual Tumor Antigen Recognition:
The third binding domain of TriKEs can be engineered to recognize a specific tumor-associated antigen – such as CD33, CD19, HER2, PSMA, or B7H3 – ensuring selective targeting of malignant cells while sparing normal tissues. This dual specificity minimizes off-tumor toxicity and heightens the precision of the immune response.

Formation of the Immunologic Synapse:
By bridging NK cells to tumor cells, TriKEs form a specialized immunologic synapse where the close proximity of effector and target cells guarantees efficient cell killing. This synapse is integral for efficient ADCC, as the concentrated engagement ensures that the cytolytic machinery of the NK cell is fully activated in a tumor-directed manner.

Potential Applications in Other Diseases
While cancer therapy is the primary focus for TriKE development, the underlying mechanism—especially the capacity to expand and activate NK cells—suggests potential applications in other diseases:

Infectious Diseases:
Studies have shown that NK cells play an important role in controlling viral infections such as HIV-1. Some research groups are considering the use of TriKE-like molecules (or BiKE variants) to target infected cells by binding viral antigens along with the NK cell activating receptor CD16, thereby enhancing the clearance of virus-infected cells. Although still in early research stages, this strategy could be adapted to manage chronic viral infections.

Immunomodulatory Therapies:
By fine-tuning NK cell activity, TriKEs could also have applications in autoimmune conditions or even transplant rejection settings, where modulation of immune cell activity is required. The selective stimulation of NK cells might help restore balance in immune responses disrupted in such disorders. Nevertheless, most efforts thus far are concentrated in on oncology, where the benefits of targeted cell-mediated cytotoxicity are profound.

Challenges and Future Directions

Development Challenges
Despite the promise and early clinical success, several challenges persist in the development and clinical translation of TriKEs:

Antigen Escape and Heterogeneity:
Tumor antigen heterogeneity remains a significant challenge. Some cancers may downregulate or mutate the targeted antigens, leading to immune escape. To address this, some TriKEs are being designed to recognize multiple epitopes or multiple antigens simultaneously in order to mitigate antigen loss.

Manufacturing Complexity and Stability:
The intricate structure of TriKEs, especially those incorporating cytokine moieties and multiple binding regions, can lead to manufacturing and stability challenges. Maintaining consistency in protein folding, bioactivity, and in vivo stability are critical in ensuring that these complex molecules can be reliably produced at scale.

Tumor Microenvironment and Immunosuppression:
Many solid tumors are characterized by an immunosuppressive microenvironment (TME) that impairs NK cell infiltration and function. Although IL-15 delivery within TriKEs helps overcome some of these challenges, overcoming inhibitory factors such as TGF-β, MDSCs, and checkpoint molecules remains a formidable hurdle.

Safety Profile and Cytokine-Related Toxicity:
Although TriKEs have shown a favorable safety profile in initial trials—with reduced incidence of cytokine release syndrome compared to T cell–related therapies—they still require close monitoring to fine-tune cytokine delivery so as to maximize therapeutic benefit while minimizing adverse effects.

Regulatory and Clinical Trial Optimization:
As the technology evolves, regulatory pathways and clinical trial designs need to be adapted to accommodate the novel mechanisms and complex pharmacodynamics of TriKEs. This involves careful planning of adaptive trial designs and dose-escalation studies to ensure that the optimal therapeutic window is identified.

Future Prospects and Research Opportunities
Looking ahead, several promising directions can be anticipated in the development of next-generation TriKEs:

Integration of Checkpoint Blockade:
Future TriKE designs may incorporate additional checkpoint blockade domains (e.g., PD-1, NKG2A inhibitors) into their structure. This integration can further enhance NK cell cytotoxicity, especially against tumors with robust suppressive signals in the TME, potentially leading to TriKEs that function more effectively as combination therapies.

Expanding the Range of Targetable Antigens:
Research is ongoing to expand the repertoire of tumor-associated antigens that can be targeted by TriKEs. This includes new targets in hematologic malignancies (e.g., CLEC12A) as well as a broader range of surface markers in solid tumors such as mesothelin, EpCAM, and HER3. The incorporation of dual or even multi-antigen binding domains could reduce the chance of antigen escape and enhance specificity.

Nanobody-Based TriKEs and Alternative Formats:
With the advantages of nanobodies – smaller size, increased tissue penetration, and reduced immunogenicity – emerging TriKE formats are exploring the substitution of scFv modules with nanobodies to enhance overall clinical efficacy. This approach may lead to more stable and effective molecules with improved biodistribution profiles.

Combination with Other Immune Modulatory Agents:
Future clinical strategies may combine TriKEs with other immunotherapeutic treatments such as checkpoint inhibitors, oncolytic viruses, or even traditional chemotherapy to create a multi-pronged assault on tumors. Such combination therapies could harness synergistic mechanisms that not only boost NK cell activity but also prime the overall adaptive immune response.

Personalized and Adaptive Therapies:
In the era of precision medicine, there is increasing interest in tailoring TriKE therapy according to individual tumor antigen profiles and patient immune status. Adaptive clinical trials and biomarker-guided studies are likely to inform the selection of the most appropriate TriKE for each patient, thereby maximizing clinical benefit and minimizing adverse effects.

New Indications Beyond Oncology:
While the current focus remains on cancer treatment, preclinical research hints at the potential for TriKE-based strategies in infectious diseases and other immune-mediated conditions. Expanding the use of these molecules beyond oncology could open new therapeutic frontiers and provide alternatives in diseases where traditional treatments have failed.

Conclusion
In summary, Tri-specific killer cell engagers (TriKEs) epitomize a transformative approach in immunotherapy by combining targeted NK cell activation, IL-15–mediated expansion, and precise tumor antigen recognition. The TriKEs currently under development include the CD16/IL-15/CD33 TriKE (GTB-3550) primarily targeting AML and high-risk myelodysplastic syndromes, CD16/IL-15/CD19/CD22 TriKEs for B-cell malignancies, CD16/IL-15/HER2 TriKE for HER2-positive solid tumors, PSMA-targeted TriKE for prostate cancer, and B7H3-targeted TriKE for a variety of solid tumors such as mesothelioma and ovarian cancer. These innovative constructs are the focus of active research and early-phase clinical trials by major companies like GT Biopharma, in collaboration with key academic institutions such as the University of Minnesota.

Mechanistically, TriKEs function by engaging NK cells through CD16, delivering IL-15 to drive NK cell proliferation and survival, and targeting specific tumor-associated antigens to ensure selective cytotoxicity. This multifaceted mechanism has resulted in promising clinical data showing enhanced NK cell activation, improved in vivo persistence, and significant antitumor efficacy across various malignancies. Nonetheless, the development of TriKEs is not without challenges. Issues such as tumor antigen heterogeneity, manufacturing complexity, safety concerns related to cytokine activity, and the inhibitory effects of the tumor microenvironment need to be rigorously addressed. Moreover, the integration of adaptive trial designs and biomarker-driven patient selection will be crucial in optimizing the clinical translation of these novel therapeutics.

Looking forward, the prospects for TriKEs are exceedingly bright. Future research is already identifying opportunities to incorporate checkpoint blockade elements, expand the range of targetable antigens through dual or multi-specific designs, and refine production techniques via nanobody-based formats. Additionally, the potential application of TriKEs in non-oncologic diseases highlights their versatility as immunotherapeutic agents. As ongoing trials progress and additional preclinical data emerge, TriKEs are poised to revolutionize the field of immunotherapy by offering more durable, potent, and safer treatment options for a wide array of cancers and potentially other diseases.

In conclusion, the development of TriKEs represents a significant advancement in targeted immunotherapy. By combining effective NK cell engagement with sustained cytokine-driven proliferation and robust tumor antigen specificity, TriKEs have the potential to overcome many of the limitations of current immunotherapies. The ongoing research, supported by preclinical studies and early clinical trials, suggests that TriKEs may soon provide clinicians with powerful new tools not only for treating hematologic malignancies such as AML and CLL but also for addressing various solid tumors. The future of TriKE-based therapies lies in their continued evolution—refining their design, expanding their applications, and ultimately improving patient outcomes through precision immune targeting.