T-cell engagers (TCEs) are immunotherapeutic molecules that simultaneously recognize T cells and specific target cells, thereby inducing directed T-cell cytotoxicity. In cancer research, a classical TCE binds a tumor-cell surface antigen through one arm and T-cell CD3 through the other. This intercellular bridge triggers T-cell activation, degranulation, and target-cell death.

Keywords: T-cell engager; TCE; bispecific antibody; BiTE; CD3; T-cell redirection; cytotoxicity; CRS; tumor immunity

1 Basic Concepts of T-Cell Engagers

1.1 Basic Structure and Mode of Action

The core function of a TCE is to complete two recognition events simultaneously: one side binds a specific antigen on the target-cell surface, whereas the other binds CD3 or another T-cell-associated molecule. Dual binding brings the T cell and target cell into close proximity, promotes local clustering of CD3/TCR signaling complexes, and creates an intercellular contact interface capable of supporting cytotoxic responses.

Classical CD3-directed TCEs redirect T cells with different TCR clonotypes toward cells expressing a specific target antigen. Unlike natural TCR recognition, this redirection generally does not require a tumor antigen to be presented as a specific peptide–MHC complex recognized by a particular TCR clone. CD3-mediated T-cell effector programs can be initiated when the target cell expresses sufficient surface antigen to support stable TCE-mediated bridging.

1.2 TCEs, Bispecific Antibodies, and BiTEs

Classical BiTE molecules generally consist of two connected single-chain variable fragments (scFvs) that recognize CD3 and a target antigen, respectively. Modern TCEs have expanded into IgG-like, 2+1, multivalent, and multispecific formats; BiTEs represent only one of these molecular architectures.

1.3 Differences Between TCEs and Conventional Antibody Therapy

Conventional targeted antibodies may act through receptor blockade, ligand neutralization, Fc-mediated antibody-dependent cellular cytotoxicity, or complement activation. The defining feature of a TCE is direct T-cell recruitment. CD3-mediated intercellular bridging redirects the T-cell cytotoxic program toward antigen-positive target cells.

TCE function depends not only on binding to the target antigen but also on antigen density, CD3 binding, intercellular-bridging efficiency, contact-interface architecture, and the functional state of the T cell. Simply increasing binding affinity does not necessarily produce stronger or safer T-cell redirection.

2 Mechanisms of TCE-Mediated Directed T-Cell Cytotoxicity

2.1 Bridging T Cells and Target Cells

After a TCE binds both the target-cell antigen and T-cell CD3, it stabilizes otherwise transient and random intercellular contacts. Accumulation of the TCE at the contact site increases the local effective concentrations of CD3 and the target antigen, providing a spatial foundation for signal initiation and the formation of a cytotoxic cell–cell interface.

TCE activity is jointly regulated by binding affinity, antigen density, epitope position, intercellular distance, and molecular geometry.

Target-antigen binding + CD3 binding → T-cell–target-cell bridging → CD3 signaling → T-cell activation → Target-cell killing

2.2 CD3/TCR Signaling Initiates T-Cell Activation

CD3 is an essential signaling component of the T-cell receptor (TCR) complex. Stable TCE-mediated bridging promotes clustering of CD3/TCR complexes at the contact site, initiates proximal TCR signaling, and subsequently induces intracellular calcium signaling, cytoskeletal rearrangement, and polarization of cytotoxic granules.

TCE-mediated T-cell activation is strongly spatially dependent. CD3 binding is only one prerequisite; stable binding at the target-antigen arm and the quality of intercellular contact jointly determine the final signaling strength.

2.3 Perforin–Granzyme-Mediated Target-Cell Death

After T-cell activation, cytotoxic granules polarize toward the intercellular contact site and undergo exocytosis. Perforin 1 (PRF1) facilitates the delivery and activity of granzymes within target cells, whereas granzyme B (GZMB) initiates proteolytic cascades and apoptotic programs. Activated T cells can also produce cytokines such as IFN-γ, TNF, and IL-2, thereby altering the local immune environment.

TCE bridging → T-cell activation → Cytotoxic-granule polarization → PRF1/GZMB release → Apoptotic-program initiation → Target-cell clearance

2.4 Degranulation and Serial Killing

During cytotoxic-granule release, the granule-membrane protein LAMP1/CD107a transiently appears on the T-cell surface. CD107a is therefore commonly used to evaluate cytotoxic T-cell degranulation.

After completing one killing event, some T cells can detach from the original target, locate another antigen-positive cell, and establish a new TCE-dependent contact. This serial-killing mechanism allows a single effector T cell to eliminate multiple target cells sequentially. However, persistent and high-intensity CD3 stimulation may also be accompanied by impaired T-cell function and increased inflammatory cytokine release.

3 TCE Molecular Design and Functional Optimization

3.1 Target Antigens Determine TCE Selectivity

(1) Tumor Selectivity and Normal-Tissue Expression

An ideal TCE target should be expressed at relatively high and stable levels on tumor-cell surfaces while showing minimal expression in essential normal tissues. Because TCEs directly recruit highly cytotoxic T cells, even low antigen expression in normal tissues may support effective bridging and produce on-target/off-tumor effects.

A sufficiently large difference in antigen expression between the tumor and essential normal tissues is therefore fundamental to establishing a therapeutic window.

(2) Antigen Density, Heterogeneity, and Expression Stability

TCE target selection must also consider surface-antigen density, intratumoral expression heterogeneity, and antigen stability under therapeutic pressure. Higher antigen density generally favors stable bridging, whereas low-expressing or antigen-negative cell populations may reduce overall cytotoxic efficacy and contribute to antigen escape during sustained treatment.

The presence of an antigen in tumor tissue alone is insufficient to establish its suitability as a TCE target. Normal-tissue distribution and expression patterns across the tumor-cell population must also be considered.

3.2 CD3-Binding Strength and the T-Cell Activation Threshold

CD3 binding that is too weak may fail to recruit T cells stably, whereas excessively strong binding may increase undesirable T-cell activation and cytokine release. Modern TCE design focuses on identifying an appropriate CD3-binding window instead of simply maximizing affinity.

Some molecules use reduced or carefully tuned CD3 affinity so that effective activation depends more strongly on stable bridging through the target-antigen arm. This approach seeks to balance T-cell recruitment, target-cell selectivity, cytotoxic activity, and cytokine release.

3.3 Epitope Position and Intercellular Contact Geometry

The distance of the target antigen from the cell membrane, the location of the CD3-binding epitope, antibody-arm length, and molecular flexibility collectively influence the spatial relationship between the T cell and target cell.

Different epitopes on the same antigen can produce different levels of T-cell activation and cytotoxicity even when their binding affinities are similar. TCE engineering must therefore consider both molecular binding and intercellular geometry. Binding parameters measured using recombinant proteins alone are insufficient to predict final cellular activity.

3.4 1+1, 2+1, and Multispecific Formats

(1) 1+1 and 2+1 Formats

A classical 1+1 TCE contains one tumor-antigen-binding site and one CD3-binding site. A 2+1 format introduces a second tumor-antigen-binding site, enabling stronger avidity on cells with high antigen density while retaining monovalent CD3 binding.

Glofitamab, which contains two CD20-binding sites and one CD3-binding site, represents this design strategy. The 2+1 format can exploit differences in antigen density to increase binding stability on high-expressing target cells, while low-antigen normal cells are less likely to reach equivalent T-cell activation conditions.

(2) Multispecific and Costimulatory Designs

Higher-order multispecific TCEs can recognize two tumor antigens simultaneously, reducing escape caused by downregulation of a single antigen. Other designs incorporate costimulatory modules involving CD28, 4-1BB, or related pathways in addition to CD3, providing additional functional support to appropriately redirected T cells.

The central objective of multispecific formats is to increase the number of activation requirements. Multiple tumor-associated signals jointly determine cell bridging or costimulation, thereby concentrating strong T-cell activation on target cells with a defined antigen combination.

3.5 Molecular Size, Fc Regions, and Half-Life

(1) Molecular Size and Half-Life Extension

Early scFv-based TCEs were relatively small and displayed rapid tissue diffusion but could also undergo rapid systemic clearance. IgG-like scaffolds, Fc fusion, and other half-life-extension strategies can increase molecular stability and prolong in vivo exposure, reducing the need for continuous administration associated with short-lived molecules.

Prolonged exposure can also extend potential immune effects. Half-life extension must therefore be coordinated with CD3-binding strength, dose, and the overall safety window.

(2) Fc Effector Functions

The early TCE Catumaxomab contains a functional Fc region capable of recruiting Fcγ receptor-positive accessory immune cells. Subsequent TCE development has placed greater emphasis on Fc-function engineering, with Fcγ receptor- and complement-associated activities retained, reduced, or silenced according to the intended mechanism.

In modern TCEs, the Fc region may support half-life extension and molecular stability but may also produce additional immune effects. Its function must therefore be aligned with the overall mechanism of action.

4 Major TCE Targets and Applications

4.1 CD19 and CD20 in B-Cell Malignancies

CD19 and CD20 have well-defined B-cell-lineage expression and represent relatively mature TCE targets. CD19×CD3 and CD20×CD3 TCEs use CD3-positive T cells to recognize and eliminate B cells expressing the corresponding antigen.

Blinatumomab represents the classical CD19×CD3 BiTE format. CD20×CD3 molecules such as Mosunetuzumab, Epcoritamab, Glofitamab, and Odronextamab use different bispecific-antibody engineering strategies, creating multiple technical approaches involving molecular scaffolds, binding valency, and pharmacokinetics.

4.2 BCMA and GPRC5D in Multiple Myeloma

(1) BCMA-Targeted TCEs

B-cell maturation antigen (BCMA) is primarily associated with the plasma-cell lineage and is an important TCE target in multiple myeloma. BCMA×CD3 TCEs such as Teclistamab, Elranatamab, and Linvoseltamab connect BCMA-positive myeloma cells with T cells and induce directed T-cell cytotoxicity.

Sustained treatment may still be affected by changes in target-antigen expression, disease heterogeneity, and the functional state of the patient’s T cells.

(2) GPRC5D as a Non-BCMA Targeting Axis

G protein-coupled receptor class C group 5 member D (GPRC5D) provides an alternative targeting axis in multiple myeloma. Talquetamab recruits T cells through GPRC5D×CD3 bispecific binding and provides a target-antigen option distinct from BCMA.

The coexistence of BCMA- and GPRC5D-directed strategies also provides a foundation for sequential treatment and investigation of antigen escape across different antigen backgrounds.

4.3 DLL3 and EpCAM as Solid-Tumor-Associated Targets

(1) DLL3 and Small-Cell Lung Cancer

Delta-like ligand 3 (DLL3) is highly expressed in certain neuroendocrine tumors, particularly small-cell lung cancer. DLL3×CD3 has become an important direction in solid-tumor TCE research. Tarlatamab establishes a tumor-cell–T-cell bridge through DLL3 and CD3 and is a representative molecule in this field.

The relatively restricted expression of DLL3 in normal tissues and its accessibility on the tumor-cell surface provide a basis for establishing a therapeutic window in solid-tumor TCE development.

(2) EpCAM and Early Solid-Tumor TCE Development

Epithelial cell adhesion molecule (EpCAM) is expressed in multiple epithelial tumors and was one of the earlier solid-tumor-associated antigens investigated for T-cell engagement. Catumaxomab creates a multicellular immune response through EpCAM, CD3, and Fcγ receptor-positive accessory immune cells and represents an early trifunctional-antibody design.

EpCAM expression in normal epithelial tissues illustrates a typical challenge for solid-tumor TCE development: antigens with broad tumor coverage do not necessarily possess optimal normal-tissue selectivity.

4.4 Specific Barriers to Solid-Tumor TCEs

Solid-tumor TCEs must overcome both target-related and tissue-environmental barriers. Abnormal tumor vasculature, dense extracellular matrix, limited T-cell infiltration, hypoxia, nutrient competition, and immunosuppressive cells can all restrict T-cell entry and sustained cytotoxicity.

Most solid-tumor antigens are not truly tumor-specific. Low-level expression in normal tissues can therefore produce on-target/off-tumor toxicity. Exploiting antigen-density differences, tumor-localized activation, and spatial selectivity has become an important direction in solid-tumor TCE design.

5 Major Limitations of TCE Therapy

5.1 Cytokine Release Syndrome

Cytokine release syndrome (CRS) is a representative immune-related toxicity of TCE therapy. Rapid activation of large numbers of T cells can produce IFN-γ, TNF, IL-2, secondary IL-6, and other inflammatory mediators, resulting in systemic inflammatory responses of varying severity.

CD3-binding strength, TCE dose, target-antigen density, tumor burden, and effector T-cell state can all influence cytokine output. TCE development must establish an appropriate therapeutic window between target-cell killing and systemic inflammation.

5.2 Neurotoxicity

Some T-cell-redirection therapies can be accompanied by immune effector cell-associated neurotoxicity syndrome (ICANS). Its development is associated with systemic inflammation, vascular endothelial changes, and the immune environment of the central nervous system. Risks vary across different TCEs and disease contexts.

TCE safety cannot be evaluated solely through target-cell cytotoxicity. The magnitude of T-cell activation, cytokine release, and duration of systemic exposure must also be controlled.

5.3 On-Target/Off-Tumor Toxicity

When a target antigen is also expressed by essential normal cells, a TCE may mediate effective bridging between those cells and T cells and consequently induce cytotoxicity. This is classified as on-target/off-tumor toxicity because the molecule still recognizes its intended antigen.

Loss of certain normal hematopoietic cell lineages may be clinically manageable, whereas essential solid organs generally provide a much narrower safety margin. Normal-tissue expression profiles are therefore a major constraint in solid-tumor TCE target selection.

5.4 Antigen Heterogeneity and Antigen Escape

Tumors may simultaneously contain cells with high, low, or absent target-antigen expression. After a TCE preferentially eliminates high-antigen cells, low-expressing or antigen-negative subclones may gain a relative survival advantage. Antigen downregulation, altered internalization, and clonal selection can all reduce subsequent TCE activity.

Increasing multivalent binding at the tumor-antigen arm, recognizing two tumor antigens simultaneously, or using combined or sequential strategies against different targets may reduce the effect of single-target antigen escape.

5.5 T-Cell Functional Decline and the Immunosuppressive Microenvironment

Prolonged CD3 stimulation can be accompanied by reduced T-cell effector function and changes in inhibitory receptors such as PD-1, TIM-3, LAG-3, and TIGIT. TGF-β, immunosuppressive myeloid cells, hypoxia, and nutrient competition in solid tumors can further restrict T-cell metabolism and cytotoxicity.

Successful initial bridging represents only one component of therapeutic activity. T-cell abundance, functional state, and serial-killing capacity after entry into the tumor also influence the final outcome.

6 Advances in TCE Research

6.1 Catumaxomab and Early T-Cell Redirection

Catumaxomab is a representative molecule from the early development of TCEs. It recognizes EpCAM on tumor cells and CD3 on T cells, while its intact Fc region interacts with Fcγ receptor-positive accessory immune cells. It can therefore establish multiple immune connections among tumor cells, T cells, and other effector cells.

Catumaxomab was authorized in the European Union in 2009 for the intraperitoneal treatment of malignant ascites associated with EpCAM-positive cancers. Its early development also highlighted challenges involving broad Fc-mediated immune activation and control of systemic toxicity.

6.2 Blinatumomab Establishes the CD19×CD3 BiTE Model

Blinatumomab uses the classical BiTE design and consists of two connected scFvs recognizing CD19 and CD3, respectively. It does not contain the Fc region of a conventional intact IgG. The CD19-binding end recognizes B-lineage target cells, whereas the CD3-binding end recruits T cells and establishes a direct cytotoxic bridge between the two cell types.

Blinatumomab received accelerated FDA approval in 2014 for relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Its clinical development demonstrated that a soluble bispecific molecule can redefine T-cell target-cell selection without ex vivo genetic modification of patient T cells. The relatively short in vivo half-life and associated administration requirements of classical BiTEs also promoted the subsequent development of half-life-extended and IgG-like TCE formats.

6.3 CD20×CD3 Drives IgG-Like and 2+1 Formats

CD20×CD3 TCEs have driven the expansion of TCEs from small BiTE molecules toward complete or engineered IgG-like structures. Mosunetuzumab, Epcoritamab, Glofitamab, and Odronextamab all target CD20-positive B cells but use different antibody-engineering designs.

Glofitamab has a representative 2:1 format with two CD20-binding sites and one CD3-binding site. Bivalent CD20 binding increases avidity on B cells with high antigen density. At this stage, TCE development progressed from determining whether cellular bridging was possible toward coordinated optimization of molecular half-life, binding valency, CD3 activation strength, and route of administration.

6.4 BCMA and GPRC5D Expand TCEs in Multiple Myeloma

Multiple myeloma has expanded TCE targeting from B-cell antigens toward plasma-cell-associated antigens. BCMA×CD3 strategies have produced molecules such as Teclistamab, Elranatamab, and Linvoseltamab, whereas GPRC5D×CD3 has provided an alternative targeting axis represented by Talquetamab.

BCMA and GPRC5D have different expression characteristics. The coexistence of these targeting axes has expanded multiple myeloma research from a single target toward sequential use, combination strategies, and management of antigen escape across different antigen contexts.

6.5 Expansion of T-Cell-Engagement Strategies in Solid Tumors

(1) DLL3×CD3 in Small-Cell Lung Cancer

Tarlatamab represents the clinical development of DLL3×CD3 TCEs in small-cell lung cancer. DLL3 is highly expressed on the surface of some small-cell lung cancer cells, and Tarlatamab directly redirects T cells through dual binding to DLL3 and CD3. Its development demonstrates the feasibility of extending conventional antibody-based TCEs into solid tumors.

(2) Engineered TCR Engagers Expand the Target Space

Tebentafusp represents a different T-cell-engagement strategy. Rather than using an antibody to recognize intact gp100 protein on the tumor-cell surface, it uses a high-affinity engineered TCR to recognize a gp100-derived peptide presented by HLA-A*02:01 and recruits T cells through a CD3-binding module.

Compared with TCEs that directly recognize surface proteins, engineered TCR-based engagers can target peptide–HLA complexes derived from intracellular proteins, thereby expanding the antigen space accessible to T-cell-engagement strategies.

7 Products

7.1 TCE Molecules for Research

7.2 Products for TCE Target Validation and Binding Research

7.3 Products for T-Cell Activation and Cytotoxic Function Research

TCEs have evolved from early trifunctional antibodies and classical BiTEs toward IgG-like, multivalent, and multispecific architectures. Multiple mature targets have been established in hematologic malignancies, while DLL3 and engineered TCR-based engagers have expanded the scope of T-cell redirection in solid tumors. Current TCE optimization increasingly focuses on coordinated control of target-cell selectivity, sustained cytotoxicity, and the therapeutic safety window.

Major Abbreviations

References

[1] van de Donk NWCJ, Zweegman S. T-cell-engaging bispecific antibodies in cancer. Lancet. 2023;402(10396):142–158. doi:10.1016/S0140-6736(23)00521-4.

[2] Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585–608. doi:10.1038/s41573-019-0028-1.

[3] Goebeler ME, Bargou RC. T cell-engaging therapies—BiTEs and beyond. Nat Rev Clin Oncol. 2020;17(7):418–434. doi:10.1038/s41571-020-0347-5.

[4] Heiss MM, Murawa P, Koralewski P, et al. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: results of a prospective randomized phase II/III trial. Int J Cancer. 2010;127(9):2209–2221. doi:10.1002/ijc.25423.

[5] Ahn MJ, Cho BC, Felip E, et al. Tarlatamab for patients with previously treated small-cell lung cancer. N Engl J Med. 2023;389(22):2063–2075. doi:10.1056/NEJMoa2307980.

[6] Nathan P, Hassel JC, Rutkowski P, et al. Overall survival benefit with tebentafusp in metastatic uveal melanoma. N Engl J Med. 2021;385(13):1196–1206. doi:10.1056/NEJMoa2103485.

[7] Kantarjian H, Stein A, Gökbuget N, et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med. 2017;376(9):836–847. doi:10.1056/NEJMoa1609783.

[8] Dickinson MJ, Carlo-Stella C, Morschhauser F, et al. Glofitamab for relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med. 2022;387(24):2220–2231. doi:10.1056/NEJMoa2206913.