What Bispecific T-cell Engager (BiTE) are being developed?

Introduction to Bispecific T-cell Engagers (BiTEs)

Definition and Mechanism of Action
Bispecific T-cell engagers (BiTEs) are a unique class of recombinant antibodies engineered to simultaneously bind to T cells and tumor cells. These molecules typically consist of two single‐chain variable fragments (scFvs) linked by a flexible peptide chain. One of the scFvs is designed to engage the CD3 receptor, a critical part of the T-cell receptor complex on T cells, whereas the other binds to a specific tumor-associated antigen on the surface of malignant cells. By physically redirecting T cells to tumor cells, BiTEs facilitate the formation of an immunological synapse, thereby triggering T-cell activation, proliferation, and the release of cytotoxic granules (such as perforin and granzymes) that ultimately lead to targeted tumor cell lysis. This mechanism is independent of classical major histocompatibility complex (MHC) antigen presentation and costimulatory signals, allowing BiTEs to engage even “resting” T cells in a polyclonal fashion.

Historical Development and Evolution
The conceptual basis for BiTE technology emerged from earlier attempts to harness the immune system for cancer therapy. Although the idea of recruiting T cells to kill tumor cells was proposed decades ago, initial bispecific antibody constructs faced challenges in protein engineering, stability, and manufacturing. The breakthrough came with the development and subsequent FDA approval of blinatumomab, a BiTE targeting CD19 on B cells, which demonstrated remarkable efficacy for the treatment of B-cell acute lymphoblastic leukemia (B-ALL). Since then, advances in recombinant DNA technologies, protein engineering techniques, and improved conjugation chemistries have significantly expanded the BiTE field. Innovations such as extended half-life molecules, dual-specific bivalent constructs (dbBiTEs), and even nanobody-based BiTEs represent evolutionary steps that enhance tumor targeting, improve biodistribution, and mitigate toxicity concerns. This evolution has been driven by the need to overcome inherent limitations of early BiTEs, such as their small size and rapid renal clearance, which previously necessitated continuous infusions.

Current BiTEs in Development

Leading BiTE Candidates
Several leading BiTE candidates, derived from both academic and industrial research, are demonstrating promising preclinical and clinical profiles. One of the most established BiTEs is blinatumomab, which targets CD19 and CD3 and has set the stage for subsequent developments. In addition to blinatumomab, a range of novel BiTE constructs are currently undergoing development:

- CD33/CD3 BiTEs for AML: Research has been directed toward generating BiTEs that target CD33 on myeloid cells and leukemic blasts, particularly in the context of acute myeloid leukemia (AML). Preclinical ex vivo studies have demonstrated that CD33/CD3 BiTEs can robustly redirect T cell-mediated cytotoxicity against AML cells.

- Dual‐specific, Bivalent BiTEs (dbBiTEs): Innovations in construct design have led to the development of dbBiTEs. For example, intact antibodies against tumor antigens (such as CEA) have been conjugated with anti-CD3 antibodies via specific hinge-region click chemistry, resulting in a bivalent construct with improved in vitro cytotoxicity and in vivo tumor targeting, as demonstrated by PET imaging and redirected T-cell therapy.

- Nanobody‐Based BiTEs (Nb-BiTEs): An emerging area involves the utilization of nanobodies—small, single-domain antibody fragments—for BiTE construction. These constructs exploit the excellent tissue penetration, high specificity, and low immunogenicity of nanobodies. For instance, a CD105-CD3 Nb-BiTE construct has been developed targeting CD105, a well‐documented solid tumor antigen, for enhanced immunotherapy against solid tumors.

- Extended Half-Life BiTEs: Next-generation BiTEs are being engineered to overcome the short serum half-life of traditional scFv-based formats. Strategies include the incorporation of Fc domains or other molecular modifications to prolong in vivo persistence, thereby reducing dosing frequency and potentially improving patient outcomes.

- Trispecific and Combination Molecule Constructs: Recent developments have also explored combining BiTE technology with additional binding domains—for instance, adding a co-stimulatory component such as CD28 to enhance T-cell activation (often referred to as SMITE bispecifics). These approaches may allow a single molecule to deliver both the T-cell engaging signal and an additional stimulatory signal, ultimately resulting in a more robust anti-tumor response.

Each of these candidates embodies an effort to refine the BiTE platform for both hematological and solid tumor malignancies by addressing limitations encountered with first-generation products. The designs are becoming more complex, integrating advancements from various fields such as antibody engineering, chemical conjugation, and even cell-based therapies.

Targeted Antigens and Tumor Types
The choice of tumor-associated antigens (TAAs) is fundamental for the specificity and clinical success of BiTEs. Different BiTEs are being designed to target a wide array of antigens across diverse tumor types:

- CD19 for Hematologic Malignancies: Blinatumomab is the prototypical BiTE that targets CD19, an antigen expressed on B cells, and has been effectively used to treat relapsed and refractory B-ALL.

- CD33 for Acute Myeloid Leukemia (AML): Several BiTEs under development target CD33, which is expressed on AML cells. The rationale is to harness T cells to eliminate malignant myeloid cells, although challenges such as off-tumor toxicity must be carefully addressed.

- BCMA for Multiple Myeloma: BiTE constructs targeting B-cell maturation antigen (BCMA) are in development for the treatment of multiple myeloma, providing an option that bypasses intracellular escape mechanisms. Early clinical trials are demonstrating promising responses, albeit with challenges such as cytokine release syndrome (CRS).

- Solid Tumor Targets:
- EGFRvIII and Her2/neu: BiTEs targeting mutated or overexpressed antigens such as EGFRvIII have been investigated primarily in glioblastoma and solid tumors.
- PSMA in Prostate Cancer: Pasotuxizumab, a BiTE targeting prostate‐specific membrane antigen (PSMA) and CD3, is being evaluated in clinical trials, showing dose‐dependent clinical activity in prostate cancer.
- ROR1: BiTEs targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1) are being developed for a range of solid tumors including pancreatic cancer and ovarian cancer, exploiting the broad expression of ROR1 on multiple malignancies.
- EpCAM, CEA, and DLL3: Additional candidates include BiTEs that target epithelial cell adhesion molecule (EpCAM), carcinoembryonic antigen (CEA), or delta-like protein 3 (DLL3), thereby offering therapeutic potential for gastrointestinal, lung, and other solid tumors.
- CD105: The nanobody-based BiTE (Nb-BiTE) approach targeting CD105, which is significantly expressed in many solid tumor types, is particularly promising due to its potential for deep tumor penetration and reduced systemic toxicities.

Across these targeted antigens, the diversity in tumor types—from hematological malignancies such as B-ALL and AML to solid tumors including prostate cancer, pancreatic cancer, and glioblastoma—illustrates the platform’s versatility. The evolution of BiTE technology is thus driven by tailoring the antigen specificity to the unique molecular landscape of different cancers.

Clinical Trials and Efficacy

Ongoing and Completed Clinical Trials
Clinical trials have been pivotal for translating BiTE technology from bench to bedside. The clinical investigations span early-phase studies to larger confirmatory trials, which assess both the safety and efficacy of these agents:

- Blinatumomab Trials: As the first approved BiTE, blinatumomab’s clinical trial history serves as a foundation and proof-of-concept demonstration for the platform. Clinical trials with blinatumomab in relapsed or refractory B-ALL have shown significant clinical remissions, leading to its accelerated approval by the FDA and subsequent integration into standard-of-care protocols.

- CD33-Targeting BiTEs for AML: Several phase I trials are underway for CD33-targeting BiTEs in AML patients. These studies are exploring the dosing, safety profiles, and early efficacy outcomes by administering the BiTE as a continuous intravenous infusion due to its short half-life. The approach has shown robust T cell-mediated cytotoxicity in preclinical models, which has laid the groundwork for translational studies.

- Extended Half-Life and IgG-Like BiTEs: Clinical trials are now being designed to evaluate the extended half-life BiTE constructs that incorporate modifications such as Fc domains. Teclistamab and talquetamab—IgG-like BiTEs targeting specific TAAs in multiple myeloma—are now in advanced stages of clinical testing. Talquetamab, in particular, has demonstrated a 70% overall response rate in a heavily pre-treated multiple myeloma population, prompting its expedited development.

- Nanobody-Based BiTE Trials: Early-phase studies aimed at evaluating the efficacy and safety of nanobody-based BiTEs (such as the CD105-CD3 Nb-BiTE) for solid tumors have been proposed. Results in animal models have been promising, with these agents demonstrating superior tumor cell lysis and improved tumor targeting compared to conventional scFv-based BiTEs.

- Combination Therapies: There is growing interest in combining BiTEs with other therapeutic modalities. For example, trials exploring the use of oncolytic viruses engineered to express BiTEs (viro-BiTE therapy) are underway. These approaches not only provide localized production of the BiTE within the tumor but also leverage the inherent immunostimulatory effects of oncolytic viral infection, potentially enhancing T cell infiltration and overall anti-tumor immunity. Additionally, combinatorial approaches with CAR-T cells and checkpoint inhibitors are being investigated to overcome resistance mechanisms and optimize anti-tumor responses.

- Trispecific Constructs and SMITEs: Early-stage trials and preclinical studies are also assessing the feasibility of trispecific molecules or simultaneous multiple interaction T-cell engagers (SMITEs) that incorporate additional binding domains, such as those mediating CD28 costimulation alongside CD3 engagement. These constructs are anticipated to address limitations related to insufficient T-cell activation observed with conventional BiTEs.

Efficacy and Safety Outcomes
The clinical performance of BiTEs reflects both their tremendous potential and the challenges inherent in redirecting polyclonal T cell activity:

- Efficacy in Hematological Malignancies: Blinatumomab has consistently shown significant clinical efficacy in B-ALL, achieving complete remissions in a substantial proportion of both pediatric and adult patients. Its success has catalyzed the exploration of BiTEs for other hematologic targets, such as CD33 in AML and BCMA in multiple myeloma, where early-phase trials provide encouraging signs of efficacy. However, a subset of patients remains unresponsive or eventually relapses, highlighting inherent resistance mechanisms.

- Safety Profiles and Toxicity: One of the known adverse effects associated with BiTE therapy is cytokine release syndrome (CRS), a consequence of massive T-cell activation. Neurological toxicities have also been observed, underscoring the importance of careful dosing and administration. Novel BiTE constructs with extended half-life and improved molecular design are being engineered to minimize systemic exposure and reduce the risk of such toxicities. Early clinical data with IgG-like BiTEs (which offer a more favorable pharmacokinetic profile) suggest improved tolerability and manageable safety profiles.

- Solid Tumor Challenges: In solid tumors, the microenvironment poses additional barriers for BiTE efficacy. Although preclinical studies indicate that BiTEs targeting antigens like EGFRvIII, PSMA, and ROR1 can effectively mediate T cell cytotoxicity, early clinical investigations reveal that factors such as poor tumor penetration and immunosuppressive stromal barriers may limit responses. Combination therapies, such as the integration of BiTE secretion with oncolytic virus platforms, are being evaluated specifically to enhance local T cell activation within the tumor milieu.

- BiTE Modifications for Improved Outcomes: The development of nanobody-based formats, dual-specific bivalent constructs (dbBiTEs), and trispecific derivatives aims to enhance efficacy by increasing tumor targeting precision, T-cell activation potency, and sustained anti-tumor immune responses. Preclinical data for these innovative constructs indicate that they can elicit more potent and durable cytotoxic effects compared to conventional formats.

Overall, clinical trials to date affirm that BiTEs offer a powerful means of harnessing the immune system against cancer, though careful evaluation of dosing, toxicity, and combination strategies remains critical. These outcomes, derived from both hematologic and solid tumor studies, serve as the basis for ongoing optimization and future clinical trial designs.

Challenges and Future Directions

Technical and Clinical Challenges
Despite the promising data, several technical and clinical challenges continue to limit the full potential of BiTE technology:

- Short Serum Half-Life and Rapid Clearance: The small molecular weight of conventional BiTEs leads to rapid renal clearance, necessitating continuous infusion regimens in clinical settings. This not only complicates administration but also increases the risk for systemic toxicities. Engineering modifications such as Fc conjugation or the use of IgG-like scaffolds are being pursued to overcome this limitation.

- On-Target Off-Tumor Toxicity: Many tumor-associated antigens are not exclusively expressed on malignant cells; they may also be present on normal tissues. This can result in unintended toxicities and limit the therapeutic window of BiTEs. For instance, while CD19 is largely restricted to the B cell lineage (allowing for tolerable B cell depletion when targeted), other antigens such as CD33 are expressed on multiple myeloid cells, posing a significant challenge.

- Tumor Microenvironment (TME) Barriers: The efficiency of BiTEs in solid tumors is also compromised by the immunosuppressive TME, which includes dense stroma, inhibitory cytokines, and poor T cell infiltration. These factors can lead to suboptimal T cell activation and limited BiTE-induced cytotoxicity. Novel delivery systems, such as oncolytic virus-mediated expression of BiTEs, seek to overcome these obstacles by concentrating the therapeutic agent within the tumor and inducing local inflammation.

- Manufacturing and Stability Issues: The production of bispecific antibodies, particularly when involving multiple variable regions or engineered modifications, presents challenges in ensuring proper folding, consistent expression, and prevention of aggregation. Advances in conjugation technology (e.g., click chemistry) and the use of robust scaffolds such as nanobodies are aimed at mitigating these issues and achieving higher yields in production.

- Immune Exhaustion and Resistance Mechanisms: Continuous T cell activation by BiTEs can eventually lead to T cell exhaustion, reducing the overall therapeutic effect. In addition, antigen loss or modulation by tumor cells can result in treatment resistance. Combining BiTE therapy with immune checkpoint inhibitors or costimulatory agents (e.g., through SMITEs) is one strategy being explored to counteract these mechanisms.

- Clinical Management of Adverse Events: The cytokine release syndrome and neurotoxicities associated with BiTE-induced T cell activation require careful clinical management. Strategies such as stepwise dosing, prophylactic corticosteroid administration, and patient monitoring are crucial in mitigating these adverse events.

Future Prospects and Research Directions
The future of BiTE development is bright, with a number of promising avenues for further research and clinical optimization:

- Extended Half-Life Constructs: Continued efforts to develop BiTEs with extended serum half-lives through Fc-fusion or IgG-like scaffolding should result in more convenient dosing schedules and improved therapeutic indices. These advancements may reduce the need for continuous infusions and lower overall systemic toxicity.

- Nanobody-Based Platforms: The use of nanobody-based BiTEs (Nb-BiTEs) is a burgeoning area of research. Owing to their small size, high stability, and excellent tissue penetration, nanobodies may provide superior tumor targeting and reduced immunogenicity. The successful preclinical demonstration of a CD105-CD3 Nb-BiTE underscores the potential of this approach for solid tumors.

- Multispecific and Dual-Function Molecules: Beyond conventional bispecific constructs, there is increasing interest in developing trispecific molecules or SMITEs that incorporate additional functional domains. For example, engaging a costimulatory receptor (such as CD28) alongside CD3 can provide a more robust T-cell activation signal, ultimately leading to enhanced cytotoxicity. Integrating cytokine domains (e.g., IL-15) further represents a strategy to sustain T cell expansion and improve efficacy.

- Combination Therapies: The integration of BiTEs with other immunotherapeutic strategies—such as CAR-T cells, oncolytic virus therapy, or immune checkpoint inhibitors—is an exciting frontier. For example, oncolytic viruses engineered to express BiTEs can create a localized “factory” of the therapeutic agent within the tumor, while also stimulating an inflammatory milieu favorable for T cell infiltration. Similarly, combining BiTE therapy with CAR-T cells may recruit a broader repertoire of T cells to the tumor site, creating a synergistic anti-tumor effect.

- Personalized Medicine Approaches: As our understanding of tumor heterogeneity deepens, there is a growing potential to develop personalized BiTE therapies targeting patient-specific antigens. Utilizing high-throughput screening platforms and function-first approaches—as described in novel BiTE generation workflows—enables rapid iteration and optimization of candidate molecules tailored to individual tumor antigen profiles.

- Improved Manufacturing Processes: Investments in manufacturing innovations, such as advanced conjugation methods and process modules, will be critical for scaling up production, ensuring product stability, and reducing costs. Enhancing manufacturing yield and quality is pivotal to making BiTE therapy broadly accessible.

- Enhanced Safety Profiles: Future research will also focus on mitigating off-tumor toxicity by refining antigen targeting and employing dual-specific recognition systems. By integrating strict on-target recognition and employing rational design to minimize binding to normal tissues, it is hoped that the safety profile of next-generation BiTEs will be significantly improved.

- Regulatory and Clinical Trial Innovations: Addressing challenges in clinical trial design, dosing regimens, and real-time monitoring of T cell activation is essential. Innovations in adaptive trial designs will further facilitate the rapid evaluation of new BiTE constructs, ensuring that promising candidates can be efficiently advanced through clinical development pipelines. Collaboration between regulatory bodies, industry, and academia will be vital in standardizing endpoints, dosing schedules, and biomarker analyses.

Conclusion
In conclusion, a robust and diverse portfolio of Bispecific T-cell Engagers (BiTEs) is currently under development, targeting a broad spectrum of antigens for both hematological malignancies and solid tumors. The historical success of blinatumomab in B-ALL has paved the way for subsequent innovations in the BiTE platform. Today’s developments include CD33/CD3 BiTEs for AML, BCMA-targeting BiTEs for multiple myeloma, and various BiTEs targeting antigens such as EGFRvIII, PSMA, ROR1, EpCAM, CEA, and CD105 for solid tumors. Cutting-edge strategies are also being explored in the form of dual-specific bivalent BiTEs (dbBiTEs), nanobody-based BiTEs, and even trispecific constructs that co-opt additional costimulatory mechanisms.

On the clinical front, ongoing and completed clinical trials have validated the efficacy and safety of BiTEs in hematologic cancers, although challenges remain—particularly in the context of solid tumors, where factors such as rapid clearance, immunosuppressive tumor microenvironments, and on-target off-tumor toxicities must be overcome. Future directions are clearly oriented toward improving pharmacokinetic properties, reducing toxicity, integrating BiTE therapy with other immunomodulatory strategies, and developing personalized approaches that harness high-throughput screening and precision medicine.

Overall, the advancement of BiTE technology represents a promising frontier in cancer immunotherapy. By linking the specificity of antibody targeting with the potent cytotoxic capabilities of T cells, these therapeutics have the potential to offer significant improvements in cancer treatment outcomes. Continued research, innovation, and clinical collaboration will be essential in realizing the full potential of BiTEs and ensuring that these next-generation immunotherapies can be safely and effectively translated into clinical practice.

Through a general-specific-general structure, we have seen that the field of BiTE development has evolved from a proof-of-concept to a highly engineered platform addressing multifaceted challenges. BiTE candidates are now diversified not only by target antigens but also by design modifications, which promise increased efficacy, improved safety, and enhanced patient convenience. As preclinical and clinical studies continue to refine these promising agents, the future of cancer immunotherapy appears increasingly bright with BiTEs poised to become a cornerstone in the armamentarium against both hematological and solid tumors.