What are the different types of drugs available for Bispecific killer cell engager (BiKE)?

Introduction to Bispecific Killer Cell Engagers (BiKEs)
Bispecific killer cell engagers, or BiKEs, are a class of immunotherapeutics designed to harness the innate cytotoxic potential of natural killer (NK) cells by directly engaging them with target cells, such as malignant or virus‐infected cells. These agents achieve this by incorporating two antigen recognition domains into a single molecular entity. One domain is specific for CD16 (FcγRIII), a receptor abundantly expressed on NK cells that mediates antibody‐dependent cell‐mediated cytotoxicity (ADCC), and the other domain is specific for a target antigen present on the malignant cell or pathogens. In this way, BiKEs function to bridge NK cells to target cells, thereby triggering NK cell activation, degranulation, and the subsequent lysis of the target.

Definition and Mechanism of Action
At its core, a BiKE is essentially a bispecific antibody fragment where each binding domain has a distinct role. One part binds to the CD16 receptor on NK cells, crucial for mediating ADCC, and the other binds to a specific antigen on the target cell, which could be a tumor-associated antigen (TAA) or a pathogen protein. This dual binding not only brings NK cells into close proximity with the target cell but also leads to the formation of an activating immune synapse. This synapse results in signaling cascades that trigger the release of lytic granules, comprising perforin and granzymes, and the production of proinflammatory cytokines essential for robust immune responses against the target.

Historical Development and Significance
The concept of using engineered antibodies to redirect immune effector cells began with monoclonal antibody therapies. The initial iterations focused mainly on neutralization or triggering immune responses solely via Fc receptor engagement. However, as researchers began to understand the limitations of monoclonal antibodies—especially their inability to overcome certain tumor immune evasion strategies—a push emerged for engaging cytotoxic immune cells more directly. The development of BiKEs represented a significant advancement, as these molecules not only improve target specificity but also potentiate the innate killing mechanisms of NK cells. Over the past several years, advancements in genetic engineering and protein design have led to the emergence of various BiKE formats and, subsequently, tri-specific killer cell engagers (TriKEs) that incorporate additional moieties such as cytokines to further optimize efficacy.

Types of BiKE Drugs
The different types of BiKE drugs can be broadly categorized based on their molecular design, targeted antigens, and any additional modifications that enhance their pharmacological profile.

Classification Based on Targets
BiKE drugs are mainly distinguished by the surface target antigen they are designed to recognize on the pathological cell. This classification includes, but is not limited to, the following categories:

1. Hematological Malignancy Targets:
- CD33-Targeting BiKEs: In pediatric and adult acute myeloid leukemia (AML), CD33 is a well-characterized marker. A representative example is the CD16×CD33 BiKE which has been shown to boost NK cell cytotoxicity against CD33+ AML blasts. NK cells stimulated with these BiKEs demonstrated increased degranulation and cytokine production, highlighting their potential in treating AML and biphenotypic acute lymphoblastic leukemia (ALL).
- CD19-Targeting BiKEs: Although primarily known in the context of T-cell engagers for ALL, some bispecific constructs have also been designed for targeting CD19 when employing NK cells. These BiKEs attempt to harness NK cell-mediated cytotoxicity in B-cell malignancies, complementing approaches such as CAR-T therapies.

2. Solid Tumor Targets:
- Epithelial Cell Adhesion Molecule (EpCAM) and Other TAAs: For solid tumors, where the immune environment is more complex and immune infiltration can be limited, BiKEs have been developed to target markers such as EpCAM or other overexpressed antigens. Despite challenges in efficient tumor penetration and the immunosuppressive tumor microenvironment (TME), these BiKEs aim to leverage NK cell activity within solid tumor contexts.

3. Infectious Disease Targets:
- Viral Antigens: The inherent design of BiKEs is not limited to oncology. For instance, one of the early innovations in the field includes anti-HIV BiKEs, where the molecule comprises a CD16-binding domain and a soluble receptor or ligand domain specific for HIV-infected cells. This design potentially enables NK cell–mediated clearance of HIV-infected T cells whilst improving biodistribution in lymphoid tissues, where HIV replication predominantly occurs.

4. Customized or Dual-Targeting Approaches:
- In some cases, researchers have engineered BiKEs to target two different antigens simultaneously on the tumor cell surface, thereby enhancing specificity and reducing off-target toxicity. These dual-targeted BiKEs represent a more refined approach in overcoming tumor antigen heterogeneity and potential escape variants.

Examples of BiKE Drugs in Development or Approved
Several BiKE constructs have been developed over the years with promising preclinical and early clinical data.

1. CD16×CD33 BiKE:
This construct is among the most studied BiKEs for hematological malignancies. In preclinical studies, primary NK cells from both healthy donors and leukemic patients showed increased degranulation and production of key cytokines upon stimulation with the CD16×CD33 BiKE, indicating its potential to restore or augment NK cell functions in AML.

2. Anti-HIV BiKE:
An innovative application of the BiKE platform is in targeting HIV-1. In one study, an anti-HIV-1 BiKE was developed by fusing a CD16A-binding antibody domain to an engineered, soluble human CD4. This construct specifically activates NK cells in the vicinity of HIV-infected T cells, promoting effective clearance of infected cells with favorable in vivo biodistribution properties owing to its relatively small molecular size.

3. TriKEs – An Advanced Form of BiKE:
Although strictly speaking TriKEs incorporate an additional specificity by adding an IL-15 moiety, they are built on the foundational concept of BiKEs. The addition of IL-15 not only engages NK cells but also provides a stimulatory signal for their expansion and persistence. For instance, the CD16/IL-15/CD33 TriKE has been demonstrated to significantly enhance NK cell cytotoxicity against CD33+ targets, thereby supporting its application in AML and high-risk hematologic malignancies. Early-phase clinical trials indicate that such TriKEs can drive robust NK cell proliferation whilst maintaining a favorable safety profile.

4. BiKEs for Solid Tumors:
Although the majority of BiKE research has focused on hematological malignancies, there is emerging research on BiKEs designed for solid tumors. These constructs often require modifications—such as half-life extension or alterations in binding affinity—to improve their tumor penetration and overcome the TME-induced immunosuppression. Researchers are exploring various antigen targets, such as EpCAM or other overexpressed proteins on solid tumor cells, to harness NK cell activity in these contexts.

5. Patent-Disclosed Platforms:
Several patents have described novel bispecific binding molecules that can be classified as BiKEs. These patents detail methods for designing bispecific or multispecific agents that can be tethered to diagnostic or therapeutic payloads. For example, patents describe methods for targeting cells for diagnosis and therapy using bispecific binding complexes, as well as platforms for the production and delivery of multispecific binding agents that have direct applications in harnessing NK cell activity. These platforms underscore the ongoing innovation in developing BiKE-related therapies.

Clinical Applications of BiKE Drugs
BiKE drugs are not only a research curiosity—they have clear translational potential across various therapeutic indications. Both hematological malignancies and infectious diseases have seen encouraging preclinical responses with BiKE-based approaches. In addition, there is a burgeoning interest in extending the utility of BiKEs toward solid tumors.

Therapeutic Areas
1. Hematological Malignancies:
In diseases such as AML and biphenotypic ALL, BiKEs such as the CD16×CD33 construct have shown promise by restoring compromised NK cell function. In preclinical models, treatment with these BiKEs has resulted in significant increases in NK cell activation markers, augmentation in degranulation, and enhanced cytokine secretion, all of which translate into improved clearance of malignant blasts. This is particularly important given that NK cell deficiency or dysfunction is a common feature in advanced leukemias.

2. Infectious Diseases – Focus on HIV:
Given the challenges of eliminating latent HIV reservoirs, the specific design of anti-HIV BiKEs provides a novel therapeutic angle by directing NK cells to HIV-infected cells. The smaller molecular size of these constructs facilitates excellent tissue penetration, especially within lymphoid tissues, which are known sites of chronic HIV replication. Such targeted therapies could potentially be integrated with current antiretroviral regimens to enhance overall viral clearance.

3. Solid Tumors:
The clinical application of BiKEs to solid tumors is more challenging due to issues such as the immunosuppressive TME and limited immune cell infiltration. Nonetheless, modifications to the basic BiKE structure—such as half-life extension and modifications to binding affinities—are being explored to promote effective tumor targeting. The aim is to transform immunogenic “cold tumors” into “hot tumors” that are amenable to NK cell-mediated cytotoxicity.

4. Combination Therapies:
BiKEs are also being investigated in combination with oncolytic viruses or other immunomodulatory agents. For instance, a combination of virus-induced tumor debulking with BiKE-mediated NK cell activation represents a promising strategy to overcome resistance in solid tumors. This synergistic approach not only targets tumor cells directly but also amplifies the anti-tumor immune response.

Case Studies and Clinical Trials
Case studies and early clinical trials have provided valuable insights into the efficacy and tolerability of BiKE therapies.

- AML and Biphenotypic ALL Trials:
Preclinical studies with the CD16×CD33 BiKE have shown that NK cells derived from both healthy volunteers and leukemic patients recover their cytotoxic function when treated with BiKEs. These studies observed enhanced NK cell degranulation and increased secretion of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) upon exposure to leukemic blasts, highlighting the potential for these agents to be used therapeutically in relapsed or refractory AML and ALL cases.

- Early-Phase Trials with TriKEs:
In addition to BiKE monotherapeutics, early-phase clinical trials involving TriKEs—which add an IL-15 moiety to the bispecific format—have demonstrated improved NK cell expansion and persistence, along with enhanced anti-leukemic activities. In phase I/II trials for high-risk hematological malignancies, the CD16/IL-15/CD33 TriKE showed promising trends regarding safety and efficacy, with some patients achieving stable disease and even remission.

- HIV-Focused Studies:
Although still in preliminary stages, early preclinical evaluations of anti-HIV BiKEs have provided evidence that these constructs can activate NK cells and lead to significant cytotoxicity against HIV-1-infected T cells. The potential for these agents to be combined with other antiviral strategies is currently under investigation.

- Combination Immunovirotherapy:
An innovative approach reported in recent studies involves the simultaneous use of BiKEs with oncolytic viruses. This combination was shown to enhance NK cell activation, as evidenced by increased expression of activation markers and cytotoxic molecules. Early in vitro studies with MV-BiKE (measles virus encoding a BiKE) have demonstrated that such strategies may lead to localized, intratumoral accumulation of the therapeutic agent while reducing systemic exposure and toxicity.

Challenges and Future Directions
Despite the promise of BiKEs and TriKEs, there are scientific, clinical, and translational challenges that must be addressed.

Current Limitations and Challenges
1. Pharmacokinetics and Biodistribution:
One of the primary challenges with many bispecific constructs, including BiKEs, is their relatively short half-life and suboptimal biodistribution. Because these molecules are relatively small, they may be rapidly cleared from circulation, necessitating modifications such as PEGylation or fusion to Fc domains to improve serum half-life without compromising bioactivity.

2. Tumor Microenvironment (TME):
The complex TME associated with many solid tumors poses significant hurdles. High interstitial pressure, the presence of immunosuppressive cytokines, and regulatory cell populations can limit the infiltration and activity of NK cells. Therefore, modifications to BiKEs designed for solid tumors may need to include strategies to overcome these barriers, such as local delivery systems or incorporation of cytokine support.

3. Antigen Heterogeneity and Escape:
Tumor cells often display antigen heterogeneity and may downregulate target antigens under selective pressure from targeted therapies such as BiKEs. To address this, dual-targeted or multi-targeted BiKE derivatives are under investigation. Moreover, the potential for the development of antigen-negative variants demands combination strategies or adaptable targeting platforms.

4. Immunogenicity and Safety:
As with all biologics, there is a potential for immunogenicity. The formation of anti-drug antibodies (ADAs) can neutralize therapeutic activity or lead to adverse reactions. Fine-tuning the humanization of the scFv domains and optimizing manufacturing processes are critical to minimize this risk.

5. Manufacturing and Scalability:
The complexities of producing bispecific or trispecific molecules at scale, with the requisite quality and consistency, present significant manufacturing challenges. Ensuring that the expression, purification, and formulation processes meet regulatory standards is crucial as these drugs move from preclinical models to larger-scale clinical trials.

Future Research and Development Trends
The future of BiKEs is closely tied to ongoing innovations in protein engineering, immunology, and clinical trial design. Several trends are shaping the next generation of BiKE therapies:

1. Enhanced Engineering of BiKEs/TriKEs:
Future directions include the incorporation of additional functional moieties, such as cytokines (e.g., IL-15 in TriKEs) or costimulatory ligands, into the BiKE format. These modifications are aimed at not only bridging the NK cell with the target but also stimulating NK cell proliferation and mitigating exhaustion. New designs also explore higher valency formats, which may provide improved binding avidity and effector function.

2. Combination Regimens:
BiKEs could be combined with other immunomodulatory agents, oncolytic viruses, or even checkpoint inhibitors to create synergistic therapies. Such combination regimes might address the limitations of BiKEs, particularly in solid tumors, by both debulking the tumor and stimulating localized immune responses.

3. Personalized Medicine Approaches:
With advances in tumor profiling and biomarker-driven treatment strategies, BiKE development may benefit from more personalized approaches. Biomarker assays and genomic data can help identify patients most likely to benefit from BiKE-based therapies. Personalized dosing strategies and adaptive clinical trial designs, informed by model-informed drug development (MIDD) techniques, are likely to become integral in advancing these therapies from bench to bedside.

4. Addressing Manufacturing Challenges:
Innovations in cell therapy process development, which have been critical for the success of other biologics, are likely to be adapted to the production of BiKEs. Improved expression systems, the use of robust purification protocols, and enhanced formulation strategies will help scale these therapies while ensuring consistent product quality.

5. Expanding the Target Landscape:
Research is ongoing to identify and validate novel tumor-associated antigens as future targets for BiKEs. With the discovery of new cancer biomarkers and the better characterization of disease-specific antigens, the applications of BiKEs can be broadened both in hematology and in solid tumor oncology. The expansion into new therapeutic areas such as infectious diseases further highlights the versatility of the BiKE platform.

Conclusion
In summary, the therapeutic landscape for Bispecific Killer Cell Engagers (BiKEs) is rapidly evolving. These agents are engineered antibody constructs that bridge NK cells to their target cells via distinct binding domains—one for the NK cell receptor CD16 and one for a specific antigen on the target cell. The different types of BiKE drugs available are mainly categorized based on their target antigens, with notable distinctions between those aimed at hematological malignancies (e.g., CD16×CD33 BiKEs for AML and ALL) and those designed for solid tumors or viral targets such as HIV. Advanced formats like TriKEs, which integrate additional cytokine support via IL-15, represent further evolution of the BiKE platform, addressing issues of NK cell persistence and functional activation.

Clinically, BiKEs have shown promise in treating diseases such as AML, where impaired NK cell function is a significant barrier to effective therapy, and early-phase clinical trials as well as preclinical studies provide encouraging data regarding their efficacy and safety. While challenges remain—from pharmacokinetic limitations and TME-induced immunosuppression to manufacturing complexities and antigen escape—the future of BiKE-based therapies is buoyed by continuous innovation.
Future trends emphasize enhanced molecular designs, such as multivalent and multifunctional constructs, combination treatment regimens, personalized therapeutic strategies driven by biomarkers, and improved manufacturing processes to enable scalability and consistency.

Overall, BiKEs offer a versatile, targeted approach that could potentially revolutionize cancer immunotherapy and treatment modalities for infectious diseases. Their ability to selectively recruit and activate NK cells positions them as a promising adjunct or alternative to traditional therapies. As research continues, integrating insights from clinical pharmacology, immunoengineering, and personalized medicine will be critical to advancing BiKEs from promising preclinical models to robust clinical applications.

In conclusion, the different types of drugs available for Bispecific Killer Cell Engagers span from traditional BiKE constructs—typically involving dual scFv domains that target CD16 on NK cells and a selected antigen on target cells—to advanced formats like TriKEs that incorporate additional cytokine signals such as IL-15. These drugs are being developed for a variety of clinical indications, including hematological malignancies, solid tumors, and infectious diseases, with ongoing research addressing current limitations and defining future applications. The continued evolution of BiKE engineering, coupled with robust clinical validation and strategic combination therapies, is poised to significantly impact the field of cancer and infectious disease therapeutics in the near future.