Accurant Mini Workshop: BiTE (Bispecific T-cell Engager) Technology and its Bioanalytical Strategy

Immuno-oncology therapies have emerged as clinically validated approaches for treating various hematologic malignancies and solid tumors. Hematologic cancers are particularly amenable to immune-targeting strategies, given the close proximity of malignant cells and immune effectors in circulation. Among the diverse immuno-oncology modalities in development, bispecific T-cell engager (BiTE) therapies represent a promising class of therapeutics that harness the cytotoxic potential of endogenous T cells without requiring genetic modification or ex vivo manipulation (Baeuerle et al., 2009; Nagorsen and Baeuerle, 2011).

BiTE molecules are engineered antibody constructs comprising two single-chain variable fragments (scFv) binding domains connected by a flexible peptide linker. One domain recognizes tumor-associated antigens (e.g., BCMA, CD19, DLL3), while the other invariably targets CD3, a component of the T-cell receptor complex (Figure 1) (Einsele et al., 2020). This bispecific design enables simultaneous engagement of T cells and tumor cells, triggering T-cell activation, proliferation, and cytolytic activity against malignant cells. Importantly, BiTE-mediated T-cell activation occurs independently of co-stimulatory signals or conventional major histocompatibility complex mechanisms, allowing for broad T-cell engagement (Klinger et al., 2016; Duell et al., 2019). The modular nature of BiTE technology facilitates the generation of molecules against various tumor-specific antigens, offering the potential for off-the-shelf immunotherapies. This versatility allows for targeting diverse malignancies and enables retreatment strategies. BiTE therapies can be employed as monotherapies or in combination with other treatment modalities to enhance therapeutic efficacy (Einsele et al., 2020).

Blinatumomab, the first approved BiTE therapy, exemplifies the potency of this approach. Targeting CD19 on B cells, demonstrating cytotoxic effects at low concentrations (10-100 pg/mL) (Dreier et al., 2002). In the presence of Blinatumomab, T cells exhibit serial-target lysis, rapidly engaging and eliminating multiple tumor cells. This mechanism of action, characterized by efficient T-cell-mediated tumor cell elimination, is a hallmark of BiTE therapies and is observed across various BiTE molecules under development. The clinical success of Blinatumomab has paved the way for further exploration of BiTE technology in both hematologic and solid tumors. Ongoing research aims to optimize BiTE constructs, identify novel tumor-associated targets, and elucidate potential combination strategies to overcome resistance mechanisms and enhance therapeutic outcomes.

Accurant Biotech have developed an NF-AT reporter gene Jurkat stable cell line to substitute for T cells in the bioanalytical assay for Blinatumomab. This method employs Raji cells and Blinatumomab antibody to establish a cell-based bioanalytical platform. The assay quantifies Blinatumomab by assessing luciferase expression upon Jurkat cell activation, measuring the resulting chemiluminescent signal. This signal demonstrates a positive correlation with Blinatumomab concentration, achieving a quantification limit of 6.25 pg/mL and a limit of detection (LOD) of 3 pg/mL. The assay protocol is streamlined, requiring only key reagents such as reference standards, commercially available target and effector cells, and omitting the need for bispecific-specific antibodies. This approach enables rapid assay deployment, significantly reducing the project preparation time. Data supporting these findings are presented in Figure 2.

Utilizing the target and effector cell detection system developed by Accurant Biotech, this high-sensitivity, cell-based PK bioanalytical method enables the detection of PK plasma concentrations from dosing levels as low as 1 μg. This method is particularly well-suited for CD3-based bispecific and multispecific antibodies, especially those with elevated cytokine release risks, as it reliably quantifies plasma concentrations down to 10 pg/mL. The approach has been successfully implemented in multiple mRNA-delivered bispecific/multispecific antibody programs, as well as in CD3-based bispecific/multispecific IITs and Phase I clinical studies, with an established and validated methodology.

Furthermore, the flexibility of this method extends to customized target cell lines, accommodating a wide range of target antigens (TAAs), including CD19 or various solid tumor-specific targets, thus ensuring broad-spectrum target applicability. The method’s performance characteristics are detailed as follows:

Accurant Biotech is an innovative contract research organization (CRO) specializing in providing PK/PD/Immunogenicity/Potency testing service for the clinical and pre-clinical development of innovative new drugs. With a strong global presence across North America and Asia, Accurant Biotech has collaborated with 200+ pharmaceutical and biotechnology companies worldwide. The company plays a pivotal role in advancing the development of novel therapies, including 80+ Cell-Gene Therapy, 60+ multi-specific antibody, and 20+ Antibody-Drug Conjugate products, and multiple Biologics License Application (BLA) approvals. By leveraging a team of over 300 scientists and state-of-the-art technology, Accurant Biotech delivers high-quality, reliable solutions that accelerate the development of life-changing treatments.

Please contact of BD team to discuss more at [email protected].

Note: This article is credited to Dr. Junyan Chen and Accurant Biotech China Team.

Reference:

1. Baeuerle, P. A., Kufer, P., & Bargou, R. (2009). BiTE: Teaching antibodies to engage T-cells for cancer therapy. Current opinion in molecular therapeutics, 11(1), 22-30.
2. Duell, J., Lammers, P. E., Djuretic, I., Chunyk, A. G., Alekar, S., Jacobs, I., & Gill, S. (2019). Bispecific antibodies in the treatment of hematologic malignancies. Clinical Pharmacology & Therapeutics, 106(4), 781-791.
3. Dreier, T., Lorenczewski, G., Brandl, C., Hoffmann, P., Syring, U., Hanakam, F., ... & Baeuerle, P. A. (2002). Extremely potent, rapid and costimulation‐independent cytotoxic T‐cell response against lymphoma cells catalyzed by a single‐chain bispecific antibody. International journal of cancer, 100(6), 690-697.
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5. Nagorsen, D., & Baeuerle, P. A. (2011). Immunomodulatory therapy of cancer with T cell-engaging BiTE antibody blinatumomab. Experimental cell research, 317(9), 1255-1260.
6. Einsele, H., Borghaei, H., Orlowski, R. Z., Subklewe, M., Roboz, G. J., Zugmaier, G., ... & Kantarjian, H. M. (2020). The BiTE (bispecific T‐cell engager) platform: development and future potential of a targeted immuno‐oncology therapy across tumor types. Cancer, 126(14), 3192-3201.