Bispecific and multispecific T-cell engagers: advancing the future of immunotherapy.

Introduction

The therapeutic antibody landscape has evolved dramatically over recent decades, progressing from traditional monoclonal antibodies (mAbs) to highly engineered constructs capable of eliciting precise immune responses. Initially celebrated for their antigen-specific targeting, mAbs provided a foundation for immunotherapy with improved specificity over small-molecule drugs. However, their dependence on single-target binding and limited efficacy in complex diseases, such as cancer, necessitated the development of advanced antibody formats [ 1 ]. Bispecific antibodies (bsAbs), which engage two distinct antigens or epitopes, and multispecific antibodies (msAbs), targeting three or more, have emerged as versatile solutions to unmet clinical needs.

Among these innovations, T-cell engager (TCE) antibodies represent a significant advancement. Engineered as bsAbs and msAbs, TCEs link T cells—the immune system’s key cytotoxic effectors—to pathological targets, such as tumor-associated antigens (TAAs) or autoreactive antigens, thereby redirecting T-cell activity to combat tumors and autoimmune disorders [ 2 ]. By binding CD3 receptors on T cells and a disease-related antigen, TCEs achieve potent and specific cell elimination, surpassing the indirect mechanisms (e.g. antibody-dependent cellular cytotoxicity) of traditional mAbs [ 3 ]. This approach has shown particular promise in oncology, where TCEs enhance immune responses against resistant malignancies, and in autoimmune diseases, where they enable targeted immune modulation.

This review provides a comprehensive analysis of TCE bsAbs and msAbs, covering their mechanisms, critical challenges, design strategies, and clinical applications. Additionally, we discuss our efforts in this field, focusing on refining TCE specificity and T-cell activation, with several projects advancing through preclinical and clinical evaluation. While TCEs hold significant potential to transform immunotherapy, their development faces hurdles such as on-target off-tumor effects and immune-related toxicities. This review aims to elucidate these dynamics and underscore the importance of ongoing innovation to expand TCE applications in diverse disease areas.

Mechanisms of T-cell engager antibodies

At the core of TCE functionality is their ability to bridge T cells and diseased cells, redirecting cytotoxic activity against target cells. The primary mechanism involves the simultaneous binding of the CD3ε subunit of the T-cell receptor (TCR) complex and a TAA, such as CD19 on leukemic cells or GPRC5D on myeloma cells. This physical tethering forms immunological synapses, mimicking natural T-cell activation while bypassing the need for antigen presentation via major histocompatibility complex (MHC) molecules. Upon synapse formation, T cells release perforins and granzymes, inducing necrosis in target cells, while also secreting cytokines (e.g. Interferon γ (IFN-γ), Tumor Necrosis Factor α (TNF-α)) that amplify the immune response [ 4 ]. For multi-specific TCEs, additional binding domains can modulate this process. Co-stimulatory targets, such as CD28, CD2 and 4-1BB, enhance T-cell proliferation and activation, while binding to checkpoint inhibitors (e.g. anti-PD-1/PD-L1) can counteract T-cell exhaustion, further optimizing the therapeutic effect.

The efficiency of this mechanism hinges on the interplay of several factors: the affinity of the CD3-binding arm (where excessive affinity may trigger off-target activation and cytokine release syndrome (CRS), while insufficient affinity limits efficacy), the epitope specificity of the antigen-binding arm, and the spatial orientation of the immunological synapse [ 5 ]. Our institution has focused on tuning the CD3-binding affinity, developing TCE constructs with adjustable affinities to optimize efficacy while minimizing safety risks. This approach is crucial given the potential for CRS in clinical settings.

Challenges in T-cell engager antibody development

The clinical success of TCE bsAbs and msAbs underscores their transformative potential, yet their development is fraught with challenges that must be addressed to fully realize their therapeutic promise. These hurdles span on-target off-tumor toxicities, CRS, as well as TAA loss, and T cell dysfunction, each requiring innovative solutions to ensure efficacy, scalability, and patient tolerability [ 6 ].

On-target off-tumor toxicity

A primary safety concern for TCE bsAbs and msAbs is on-target off-tumor toxicity, where TCEs bind to antigens on healthy cells, causing unintended tissue damage [ 7 ]. For instance, antigens like CD19 or B-Cell Maturation Antigen (BCMA), expressed at low levels on normal tissues—such as CD19 on healthy B-cell precursors or BCMA on non-malignant plasma cells—can trigger T-cell-mediated cytotoxicity against these cells, leading to dose-limiting toxicities (DLTs). In B-cell acute lymphoblastic leukemia (B-ALL), blinatumomab (a CD3 × CD19 bsAb) induces transient B-cell aplasia, increasing infection risk [ 8 ], while BCMA-targeted TCEs for multiple myeloma (MM) cause hypogammaglobulinemia, impairing immune function [ 9 ].

This issue is particularly pronounced in solid tumors, where antigens are often present on vital organs or normal tissues. For example, in a study of solitomab (a CD3 × Epithelial Cell Adhesion Molecule (EpCAM) bsAb) involving 65 patients with EpCAM-positive solid tumors (e.g. ovarian, lung, gastric cancers) treated with 1–96 μg/day for ≥28 days, 15 patients experienced DLTs, including 8 with transient liver abnormalities, 1 with Grade 3 supraventricular tachycardia, and 6 with diarrhea (Grade 3, n = 3; Grade 4, n = 1; one progressing to Grade 5 post-discontinuation) [ 10 ]. These effects, linked to EpCAM expression on normal epithelial tissues like the gastrointestinal tract and liver, limited the maximum tolerated dose to 24 μg/day [ 10 ]. Similarly, CEA TCB (Carcinoembryonic antigen T cell bispecific antibody) for CEA-positive solid tumors (e.g. colorectal cancer) caused severe gastrointestinal toxicity, including diarrhea and colitis, due to CEA expression on normal intestinal epithelium, restricting dose escalation [ 11 ]. HER2-targeted TCEs also risk cardiotoxicity due to HER2 expression on cardiac tissue, underscoring the challenge of balancing efficacy and safety when antigens lack tumor-specific expression [ 12 ]. These examples underscore a key challenge in TCE development: balancing efficacy and toxicity when antigens lack tumor-specific expression. In solid tumors, this issue is exacerbated by the broader distribution of target antigens across healthy tissues, making on-target off-tumor toxicity a significant barrier to success.

To mitigate these risks, several strategies are under investigation, including the critical selection of TAAs with high expression in tumors and low or no expression in normal tissues to enhance specificity [ 7 ]. Taking CLDN18.2 and the emerging target CDH17 as examples, both are highly expressed in tumors while being expressed at tight junction sites in normal tissues, thus demonstrating their specific expression patterns [ 13 , 14 ]. CLDN18.2/CD3 bsAbs, such as IBI389 and QLS31905 [ 15 , 16 ], have shown excellent efficacy and safety in clinical trials. Additional approaches include adjusting CD3-binding affinity to reduce excessive T-cell activation, conditional activation using protease-cleavable linkers to activate TCEs in the tumor microenvironment, dual-antigen targeting requiring co-expression of two TAAs, and stepwise dosing to allow immune adaptation. Our group has made significant progress in this field with two innovative TCEs: LBL-034 and LBL-043.

LBL-034 is a bispecific TCE targeting GPRC5D and CD3. GPRC5D is highly expressed on MM cells and only in hair follicles at low levels, making it an ideal target to minimize off-tumor effects [ 17 ]. LBL-043 is a bispecific TCE targeting LILRB4 and CD3. LILRB4 is highly expressed on MM cells, M4-M5 Acute Myeloid Leukemia (AML) cells (per French-American-British (FAB) classification), and myeloid-derived suppressor cells (MDSCs), but minimally expressed on hematopoietic stem and progenitor cells (HSPCs) [ 18 ]. This profile makes LBL-043 a promising candidate for MM and AML, addressing unmet medical needs. It reduces on-target off-tumor toxicity and immunotoxicity risks through enhanced tumor specificity and optimized T-cell engagement.

Cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome

TCE therapies, while promising, face significant safety challenges due to T-cell overstimulation, leading to severe adverse events like CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) [ 19 ]. CRS, driven by excessive release of pro-inflammatory cytokines such as Interleukin 6 (IL-6) and TNF-α, often manifests as high fever, hypotension, and respiratory distress, and has been a DLT in CD3-targeted TCE trials for hematological malignancies [ 19 ]. Management typically involves tocilizumab (an IL-6 receptor antagonist) or corticosteroids to prevent life-threatening outcomes. ICANS, linked to immune activation in the central nervous system, presents with symptoms ranging from confusion and tremors to severe seizures and encephalopathy, posing further risks to patient safety and quality of life. What’s more, Tarlatamab, a DLL3/CD3 bispecific antibody developed by Amgen, has been approved for small cell lung cancer (SCLC), but it carries a black box warning for potentially causing severe or life-threatening CRS and ICANS [ 20 ]. This indicates that there is still room for improvement in TCE therapies with respect to CRS and ICANS.

To address these issues, innovative strategies are being explored, including fine-tuning CD3-binding affinity to temper T-cell activation, selecting highly tumor-specific TAAs to reduce off-target effects, and implementing stepwise dosing to allow immune adaptation [ 21 ]. We have made significant strides in this area with two novel TCEs: LBL-034 and LBL-043. LBL-034, with its 2:1 molecular design, features two high-affinity arms targeting GPRC5D and one low-affinity CD3 arm (~100-fold lower), optimizing T-cell cytotoxicity while minimizing CRS and off-target effects [ 22 ]. Phase 1 data from the 2024 ASH meeting confirmed its safety, reporting no Grade ≥ 3 CRS or ICANS [ 23 ]. Similarly, LBL-043, developed via the proprietary LeadsBody™ platform in this area with two high-affinity variable domain of heavy chain antibody (VHH) arms targeting LILRB4 and a single low-affinity scFv arm for CD3, is designed to enhance efficacy while reducing immunotoxicity risks [ 24 ]. These advancements reflect a balanced approach to improving the safety and efficacy of TCE therapies.

Tumor-associated antigen loss

TAA loss presents a significant obstacle to the sustained efficacy of TCE bsAbs and msAbs, compromising their ability to maintain therapeutic responses. This occurs when cancer cells downregulate or lose expression of targeted antigens—such as CD19, BCMA, or GPRC5D—as a response to TCE-mediated T-cell cytotoxicity, leading to antigen escape and disease relapse [ 25 ]. Clinical evidence underscores this challenge. For instance, in B-ALL patients treated with blinatumomab, CD19-negative relapses have been observed in 5% to 15% of cases across various studies, with rates reaching up to 20% in specific CAR-T therapy contexts, though less commonly with blinatumomab alone [ 26 , 27 ]. Similarly, in MM patients receiving teclistamab, BCMA loss has been identified as a key resistance mechanism, with emerging data linking relapse to reduced or absent BCMA expression [ 28 ]. To address TAA loss, strategies such as simultaneously targeting multiple TAAs with multispecific TCEs are being explored to minimize the likelihood of antigen escape and enhance treatment durability.

T cell dysfunction and exhaustion

T cell dysfunction and exhaustion pose significant challenges to the development of TCE bsAbs and msAbs. In the tumor microenvironment, T cells often become exhausted due to chronic antigen exposure, leading to upregulation of inhibitory receptors such as PD-1, TIM-3, and LAG-3. This exhaustion reduces T cell activation, function, and responsiveness to antibody-based therapies [ 29 ].

T cell heterogeneity further complicates antibody targeting. Tumor-infiltrating T cells are diverse, with only a small portion being tumor-specific. It is challenging to ensure that bispecific or trispecific antibodies target and activate tumor-specific T cell subsets [ 30 ]. Moreover, the tumor microenvironment disrupts T cell metabolism. Tumor cells consume nutrients, depriving T cells of essential resources [ 31 ]. Inhibitory metabolites in the tumor microenvironment further suppress T cell activity. To overcome these challenges, multimodal strategies are employed. Trispecific antibodies engineered with co-stimulatory signals (e.g. targeting CD28 or 4-1BB) selectively enhance proliferation and effector functions of tumor-specific T cells while minimizing off-target effects [ 32–34 ]. Concomitant checkpoint blockade (e.g. anti-PD-1/PD-L1) counteracts T cell exhaustion induced by chronic antigen exposure and tumor microenvironment (TME) suppression, restoring robust T cell activity. Additionally, γδ T-cell engagers (e.g. Epidermal Growth Factor Receptor (EGFR）-Vδ2 and Prostate-Specific Membrane Antigen (PSMA)-Vδ2 bsTCEs) bypass αβ T-cell heterogeneity and TME immunosuppression [ 35 , 36 ]. EGFR-Vδ2 bsTCEs demonstrated preferential cytotoxicity against EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and NK cells, without the concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs [ 37 ].

Design and engineering of T-cell engager bsAbs or msAbs

The therapeutic potential of TCE bsAbs or msAbs stems from their advanced design and engineering. Unlike traditional mAbs, which rely on single-target binding and indirect effector functions, TCEs engage multiple targets—CD3 on T cells and TAA on tumor cells to redirect T-cell cytotoxicity with precision. Their efficacy depends on diverse structural formats, optimized mechanisms of action, and innovations in antibody engineering technology.

The molecular design of TCEs has evolved significantly. Early constructs, such as blinatumomab (Amgen) and mosunetuzumab (Roche), adopted a 1:1 binding format [ 38 ]. Blinatumomab comprises two scFvs linked by a flexible peptide, targeting CD19 and CD3, whereas mosunetuzumab employs two Fab arms—one for CD20 and the other for CD3 [ 39 , 40 ]. To improve tumor selectivity, subsequent TCEs transitioned to a 2:1 format (two TAA-binding arms, one CD3-binding arm) [ 41 ]. A prominent example is glofitamab (Roche), which targets CD20 and CD3 in a 2:1 configuration. It has been approved for use in combination with gemcitabine and oxaliplatin (GemOx) for the treatment of adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) [ 42 ].

Some TCEs have also been engineered in a 2:2 format to further enhance selectivity. For instance, givastomig is a tetravalent bispecific antibody targeting CLDN18.2 on tumor cells and 4-1BB on immune cells, utilizing an IgG scaffold fused with two scFvs. This design ensures 4-1BB cross-linking and activation occur only upon simultaneous binding to CLDN18.2 + tumor cells and 4-1BB + immune cells, thereby minimizing nonspecific 4-1BB activation in peripheral tissues (e.g. liver) [ 43 ]. In a Phase 1 monotherapy trial, givastomig demonstrated promising activity in 45 patients with advanced gastric and esophageal cancer: the objective response rate (ORR) was 18% (8/45), the disease control rate (DCR) was 49%, and Grade ≥ 3 treatment-related adverse events occurred in 33% of patients. Notably, the incidences of elevated Alanine Aminotransferase (ALT), elevated Aspartate Aminotransferase (AST), and CRS were low (2%, 4%, and 0%, respectively). Overall, 2:1 and 2:2 TCE formats remain the preferred designs for exploiting avidity effects, as they bolster tumor selectivity without increasing the risk of nonspecific T-cell cross-linking.

Tri-specific TCEs represent an advanced evolution of TCE technology, designed to overcome limitations of bispecific constructs by targeting three distinct molecular entities. These molecules are classified into two functional categories: enhancing tumor specificity and amplifying T-cell activation. The first category improves tumor selectivity and sensitivity by targeting two TAAs or dual epitopes of a single TAA alongside CD3. For instance, an IgG-like tri-specific TCE targeting CD3, Ly6E, and B7-H4 leverages their frequent tumor co-expression (78% Ly6E, 71% B7-H4, 54% both), contrasting with weak to moderate expression in normal tissues, optimizing specificity [ 44 ]. Similarly, a BiTE molecule targeting CD3, P-cadherin (CDH3), and mesothelin (MSLN) addresses the on-target off-tumor toxicity risks, capitalizing on their co-expression in solid tumors and reduced overlap in normal tissues, as confirmed by immunohistochemistry [ 45 ]. These designs enhance tumor recognition precision and minimize off-target effects on single-antigen-expressing healthy tissues.

In contrast, the second category focuses on amplifying T-cell effector functions and countering immunosuppressive tumor microenvironments by targeting CD3, a single TAA (e.g. Her2, CD38, or CLDN6), and a co-stimulatory molecule (e.g. CD28, CD2 or 4-1BB) [ 34 , 46 , 47 ]. Co-stimulatory signaling boosts T-cell proliferation, cytokine production (e.g. IL-2, IFN-γ), and survival [ 48 ]. For example, SAIL66 (CLDN6/CD3/4-1BB) enhances antitumor activity with high specificity and superior efficacy over conventional TCEs via CD3 activation and 4-1BB co-stimulation in preclinical studies [ 46 ]. Likewise, SAR443216 (CD28/CD3/HER2) is able to activate primary human CD4 and CD8 T cells, resulting in T cell proliferation and secretion of cytokines and granzyme B [ 47 ]. However, its clinical trials were halted due to lack of efficacy (ORR = 0%) and safety concerns, with three DLTs: one heart failure case at 180 μg and two transaminase elevations at 720 μg [ 12 ]. Another emerging immune costimulatory signal is CD2. Notably, CD2 exhibits preferential expression in CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), with ratios significantly exceeding those of CD28 and 4-1BB [ 49 ], suggesting its potential as a more effective immunotherapeutic target in tumors. PIT565, the most advanced therapeutic program leveraging this biology, targets CD19/CD2/CD3 simultaneously and has already entered Phase 1 clinical trials [ 50 ]. These outcomes highlight the critical role of TAA selection and tri-specific design in balancing efficacy and safety.

Early TCEs had a short plasma half-life, and TCE platforms designed to extend the half-life are continuously being improved. BiTEs, such as blinatumomab, are compact and non-Fc-containing, enabling efficient tissue penetration, and rapid T-cell activation. However, their short serum half-life (≈2 h) necessitates continuous intravenous infusion to maintain active exposure in clinical settings. One of the strategies to extend the half-life is to incorporate an Fc domain into the TCE construct. For example, tarlatamab is a half-life extended (HLE) BiTE molecule that binds to DLL3 and CD3. It has demonstrated impressive clinical efficacy in the treatment of extensive-stage SCLC, with an ORR of 40% in the pivotal study, and is administered every 2 weeks [ 25 ]. In addition to HLE-BiTE, other strategies for extending half-life include enhancing binding to albumin, such as the TriTAC™ platform developed by Harpoon Therapeutics [ 51 ]. TriTAC antibodies contain a domain that binds to human serum albumin to extend the antibody’s half-life. Cyno studies have shown that the half-life of TriTAC is ~80–90 h, while the half-life of the control BiTE antibody is only about 2 h [ 52 ]. Enhancing FcRn binding extends antibody half-life by improving pH-dependent recycling [ 53 ]. For example, the YTE mutation (M252Y/S254T/T256E) modifies the Fc region to increase FcRn binding affinity at low pH (~6.0), significantly prolonging half-life [ 54 ]. Similarly, Xencor’s Xtend platform uses Fc mutations [e.g. M428L/N434S, LS(Xtend) mutation] to boost low-pH FcRn binding and extend therapeutic antibody half-life; these two amino acid changes increase affinity for human/cynomolgus monkey FcRn at pH 6 by ~11-fold, and IV administration of an anti-VEGF mAb (Xtend-based) to cynomolgus monkeys (4 mg/kg) extended its serum half-life from 9.7 to 31.1 days [ 55 ].

Clinical applications of T-cell engager antibodies

TCE bsAbs/msAbs have evolved from experimental constructs into clinically validated therapeutics, demonstrating significant efficacy across diverse diseases, including hematological malignancies and solid tumors. Beyond cancer, TCEs are emerging as tools for autoimmune disorders through targeted immune modulation. As of 2025, more than 60% of the bsAbs globally approved for cancer treatment are TCEs, reflecting their dominance among bispecific therapeutics. In addition to the 12 approved TCE programs worldwide, 5 TCE projects have advanced to Phase 3 clinical trials ( Table 1 ). Notably, STEAP1, an emerging TAA in prostate cancer, demonstrates higher homogeneity and more abundant expression in metastatic prostate cancer patients compared to traditional targets like PSMA [ 56 , 57 ]. AMG 509, anti-STEAP1/CD3 developed by Amgen, has shown a 28.6% ORR at the Phase 3 dosage of 1.5 mg Q2W and is currently in pivotal Phase 3 trials [ 58 ]. Meanwhile, a robust pipeline persists, with many multi-specific TCEs under investigation in clinical trials worldwide, targeting a broad range of conditions, including cancer, infectious diseases, and autoimmune disorders [ 59 , 60 ]. This dual- and multi-specific progression underscores TCEs’ transformative potential in precision medicine.

T-cell engager antibodies in oncology

TCE antibodies have emerged as a pivotal therapeutic strategy in oncology, achieving notable success in hematological malignancies and, increasingly, solid tumors. As of May 2025, 12 TCEs have gained approval from U.S. Food and Drug Administration (FDA) and European Medicines Agency since 2014, with 9 targeting hematological malignancies such as MM, follicular lymphoma, and DLBCL. These agents exemplify the ability of TCEs to redirect T-cell cytotoxicity with clinical precision.

In hematological malignancies, blinatumomab, a CD19/CD3 BiTE, became the first TCE bsAb approved by the FDA in 2014 for R/R B-ALL. By linking T cells to CD19-expressing leukemic blasts, it induces rapid tumor cell lysis, yielding complete remission rates of 40%–50% in heavily pretreated patients [ 61 ]. Its success catalyzed further TCE development, leading to approvals targeting CD19, CD20, BCMA, and GPRC5D. For instance, elranatamab, a BCMA/CD3 bsAb approved in 2023 for R/R MM, achieves overall response rates exceeding 60%, with sustained responses in a disease resistant to prior therapies [ 62 ]. These outcomes highlight TCE potency in liquid tumors, where accessible antigens and a supportive immune microenvironment enhance efficacy.

Solid tumors pose greater challenges, including physical stromal barriers, immunosuppressive microenvironments, and antigen heterogeneity, yet TCEs are advancing [ 60 ]. Tebentafusp, a TCR/CD3 bispecific fusion protein targeting gp100 in HLA-A*02:01-positive uveal melanoma (UM), became the first TCE approved for a solid tumor in 2022, improving 1-year survival to 73% versus 59% with checkpoint inhibitors [ 63 ]. Likewise, tarlatamab, a DLL3/CD3 bsAb, received accelerated approval in 2024 for SCLC, with a 40% ORR in a disease with limited options [ 64 ]. These advances demonstrate TCE adaptability, though penetration and immunosuppression remain key hurdles.

Building on these successes, the clinical pipeline for TCEs remains robust, with dozens of candidates in Phases 1-3 as of May 2025. Next-generation TCEs address limitations like short half-life, off-target effects, CRS, and TAA loss. For example, MK-6070 (DLL3/CD3/HSA) is in Phase 2 for SCLC and neuroendocrine tumors, incorporating an Fc domain for extended pharmacokinetics [ 65 ]. Similarly, SIM0500, a tri-specific TCE targeting GPRC5D and BCMA to counter antigen loss and tumor heterogeneity, is in Phase 1 for R/R MM, reflecting ongoing efforts to refine TCE technology [ 66 ].

T-cell engager antibodies in autoimmune diseases

Beyond oncology, TCE antibodies are gaining attention for their potential in autoimmune diseases, where precise immune modulation could address unmet needs [ 67 ]. Traditional therapies, such as corticosteroids or biologics targeting broad immune pathways (e.g. TNF-α), often lack specificity, leading to systemic immunosuppression and side effects [ 68 ]. TCEs offer a targeted alternative by either depleting pathogenic immune cells or redirecting T-cell activity to restore immune homeostasis.

B-cell depletion therapy using TCEs is a highly promising approach for treating autoantibody-driven autoimmune diseases. By targeting antigens such as CD19, CD20, BAFF-R, and BCMA on B cells and plasma cells, TCEs effectively deplete these cells, offering a robust treatment for autoantibody-mediated autoimmune disorders [ 69 , 70 ]. Blinatumomab, a CD19/CD3 TCE, successfully treated patients with refractory rheumatoid arthritis (RA) and gMG, reducing disease activity and autoantibody levels by depleting CD19-positive B cells via T-cell cytotoxicity [ 71 , 72 ]. Teclistamab, a CD3/BCMA TCE—has shown promising clinical results in treating systemic sclerosis, idiopathic inflammatory myositis, and IgG4-related disease, confirming that TCEs targeting plasma cells are a valuable approach for autoimmune diseases [ 73 ]. These findings confirm eTCEs as a promising option for B cell-mediated autoimmune diseases, expanding their scope beyond oncology. Industry reports indicate that many TCE bsAbs are under investigation globally for autoimmune indications, primarily targeting CD3 × CD19, CD3 × CD20, and CD3 × BCMA combinations. Additionally, tri-specific TCEs, such as those targeting CD3 × CD19 × BCMA, CD3 × CD19 × CD20, and CD3 × CD28 × CD19, are being developed to effectively deplete pathogenic B cells across a broader range of conditions [ 69 , 74 , 75 ]. What’s more, some research teams have designed novel bispecific molecules, Bispecific AutoAntigen-T cell Engagers (BiAATEs): one arm targets CD3 on T cells, and the other targets disease-specific pathogenic autoantigens (e.g. the CysR domain of PLA₂R in membranous nephropathy, MN) to precisely eliminate autoreactive B cells. Experiments showed BiAATEs deplete PLA₂R-specific B cells from MN patients in vitro and reduce anti-PLA₂R antibodies in human CD3 transgenic mice, holding broad prospects for antibody-mediated autoimmune diseases with known pathogenic autoantigens [ 76 ].

Overview of Leads Biolabs’ efforts in T-cell engager development

Leads Biolabs has been instrumental in advancing TCE therapies, pioneering innovative candidates to meet unmet medical needs in oncology and autoimmune diseases. Leveraging its proprietary bispecific and multispecific antibody platforms, LeadsBody™, the company has developed a robust portfolio of TCE programs, with multiple candidates progressing into clinical stages for both cancer and autoimmune indications. LeadsBody’s TCEs employ a 2:1 asymmetric architecture, integrating low-affinity CD3 binding with high-affinity TAA engagement. This configuration mitigates off-tumor CD3 cross-linking risks, attenuates T-cell exhaustion and apoptosis—a prevalent hurdle in TCE development—and markedly improves the therapeutic safety profile.

LBL-024

LBL-024 is one of the world’s first few PD-L1/4-1BB–targeted therapeutic candidates to advance into pivotal or late clinical trials. It is designed with highly differentiated binding affinities, exhibiting an affinity for PD-L1 over 300-fold higher than for 4-1BB. This design enables LBL-024 to preferentially localize to the PD-L1-rich tumor microenvironment, minimizing systemic 4-1BB activation—particularly in peripheral circulation and the liver—thus reducing potential toxicities while optimizing T-cell-mediated efficacy. The costimulatory activity of LBL-024 on 4-1BB depends on simultaneous PD-L1 binding, as evidenced in a human 4-1BB reporter gene system ( Fig. 1A ). In this system, LBL-024 activates 4-1BB signaling in the presence of PD-L1-expressing target cells, outperforming a urelumab analog ( Fig. 1B ), with activation levels strongly correlating with PD-L1 expression [ 77 ]. Specifically, LBL-024 effectively activates the 4-1BB reporter gene in HCC827 and MDA-MB-231 cells, which exhibit high PD-L1 expression, whereas it demonstrates efficacy in HT29 cells, with lower PD-L1 expression, only at high concentrations, highlighting its PD-L1-dependent specificity [ 77 ].

In preclinical studies, LBL-024 (at 5 mg/kg) exhibited superior anti-tumor efficacy compared to PD-L1 or 4-1BB monotherapies in the MC38-PD-L1 syngeneic mouse model ( Fig. 1C ), while also increasing the proportion of CD8+ T cells within TILs, highlighting its immunomodulatory potential [ 77 ]. Clinically, LBL-024 has shown significant efficacy in treating extrapulmonary neuroendocrine carcinoma (EP-NEC). In a Phase 1/2 study involving 45 patients with previously treated EP-NEC, it achieved an ORR of 33.3% [ 78 ]. In a clinical trial evaluating its chemotherapy combination as first-line (1L) therapy for EP-NEC and SCLC, the ORR was 75% and 86.5%, respectively. Regarding safety, Grade ≥ 3 elevations in AST and ALT each occurred in <4% of patients, indicating a favorable safety profile ( Table 2 ).

LBL-034

LBL-034, a humanized, IgG1-like bispecific antibody targeting GPRC5D and CD3, is currently being evaluated in a Phase 1/2 clinical trial for the treatment of R/R MM. In a Phase 1 clinical study for the treatment of R/R MM, at doses of 400–800 μg/kg, the ORR reached 82.1% (23/28). At the dose of 1200 μg/kg, the ORR was 83.3% (5/6). In terms of safety, CRS, ICANS, and hematological toxicities were all safe and manageable ( Table 2 ) [ 23 ]. In non-head-to-head clinical studies, LBL-034 outperforms talquetamab in efficacy and safety. At 400–800 μg/kg, LBL-034 has an 82.1% ORR; talquetamab—the only approved GPRC5D bispecific antibody—only reaches a 64% ORR at the same 800 μg/kg dose in MM patients [ 79 ]. In terms of safety, LBL-034 induces lower levels of CRS and hematological toxicity than talquetamab. This indicates a potentially superior safety profile.

In preclinical studies, LBL-034 demonstrated potent T-cell-dependent cellular cytotoxicity (TDCC) activity against various GPRC5D-expressing tumor cells, outperforming benchmark treatments. Notably, LBL-034 conditionally activates CD3+ T cells only in the presence of GPRC5D-expressing target cells ( Fig. 2A ), enhancing specificity [ 22 ]. It also exhibits a significant bystander killing effect, as evidenced by its activity against Raji cells (GPRC5D-negative) and RPMI-8226 cells (low GPRC5D expression) when co-cultured with MM.1R cells (high GPRC5D expression) [ 22 ]. In preclinical studies, LBL-034 showed strong antitumor efficacy in mouse tumor models expressing GPRC5D ( Fig. 2B and C ) [ 22 ].

LBL-043

LBL-043 is a TCE engineered to target both CD3 and LILRB4 using LeadsBody™ platform and is designed as a 2:1 bispecific antibody. It incorporates two VHH arms that bind LILRB4 with high affinity and a single scFv arm that targets CD3 with carefully calibrated low affinity [ 24 ]. Specific mutations were introduced to eliminate FcγR binding and to abolish ADCC and CDC effects, while preserving FcRn binding capacity. LBL-043 activates Jurkat-CD3/NFAT reporter cells in a manner dependent on LILRB4 binding ( Fig. 3A ) [ 80 ]. Notably, it significantly enhances the activation of Jurkat-CD3/NFAT reporter cells exclusively in the presence of LILRB4+ cells, exhibiting negligible activity in their absence, even at the highest concentrations tested [ 24 , 80 ]. LBL-043 demonstrates potent cytotoxic effects across various MM and AML cell lines [ 24 , 80 ]. Furthermore, it exhibits no toxicity toward CD34+ hematopoietic stem cells (HSCs) or other LILRB4-negative cells ( Fig. 3B ) [ 80 ]. In preclinical studies, LBL-043 showcased robust antitumor efficacy in the NCI-H929 xenograft PBMC-humanized mouse model (MM) ( Fig. 3C ) and Molm-13 xenograft PBMC-humanized mouse model (AML) [ 24 , 80 ]. Additionally, LBL-043 was well tolerated in cynomolgus monkeys, displaying a favorable PK profile.

LBL-051

LBL-051 is a first-in-class CD19/BCMA/CD3 tri-specific TCE antibody designed for B cell-driven autoimmune diseases developed using the LeadsBody™ Platform. This design enhances safety while optimizing anti-tumor potency through the fine-tuning of the relative potency of each targeting domain. By simultaneously targeting CD19 and BCMA, LBL-051 has the potential to elicit more potent and sustained responses. This is achieved through the depletion of a broader spectrum of pathogenic B-cell populations, making it applicable to a wide range of B-cell-driven autoimmune disorders.

Future directions and perspectives

As TCE bispecific and msAbs solidify their position in immunotherapy, their future depends on broadening their therapeutic scope, enhancing targeted cell elimination, and integrating them into comprehensive treatment strategies. Advances in target selection, combination approaches, and delivery technologies, hold the potential to unlock new applications, particularly for complex diseases such as solid tumors and autoimmune disorders.

Novel targets and targets combinations

Expanding the antigen repertoire remains a critical frontier. While current TCEs primarily target CD19, CD20, or BCMA, emerging neoantigens and TAAs (e.g. CDH17, LY6G6D, ENPP3) offer opportunities for improved specificity in oncology [ 45 ]. Multispecific TCEs that engage multiple TAAs or co-stimulatory molecules (e.g. 4-1BB, CD28, CD2) could overcome resistance and enhance T-cell persistence, as exemplified by JNJ-79635322, a trispecific TCE being investigated in clinical trials [ 81 ]. Specifically, its molecular design enables enhanced myeloma cell targeting via a “double lock-down” effect from binding two myeloma antigens, enabling more comprehensive targeting of myeloma cells—including BCMA-/GPRC5D+, BCMA+/GPRC5D-, and dual BCMA+/GPRC5D+ subsets—thereby preventing antigen escape [ 82 ]. At the 2025 American Society of Clinical Oncology (ASCO) meeting, Phase 1 clinical data for JNJ-79635322 were reported: at the RP2D of 100 mg Q4W, the ORR reached 100% (27/27) in triple-class exposed R/R MM patients naive to BCMA/GPRC5D- targeted therapies, with a 12-month progression-free survival rate of 95% [ 83 ]. EVOLVE104 represents another innovative TCE design, a first-in-class trispecific molecule developed for solid tumors that uniquely targets ULBP2/5/6 while simultaneously engaging CD2 (for co-stimulation) and CD3 via the EVOLVE platform [ 84 ]. Using EVOLVE-105 (anti-CD20/CD2/CD3) developed on the same platform, in vitro studies demonstrate that EVOLVE-105’s tumor-killing potency is within two-fold of glofitamab’s activity while inducing lower cytokine release [ 85 ]. The co-stimulatory role of CD2 requires further validation in clinical data to confirm its efficacy and safety.

Incorporating immune checkpoint modulation into TCE design has emerged as a transformative strategy. HY05350, a PD-L1/MSLN/CD3 trispecific TCE, simultaneously recruits T cells via CD3, targets tumor cells through MSLN/PD-L1, and relieves PD-L1-mediated immune suppression, triggering stronger T cell activation, which is already in the Phase 1 stage [ 86 ].

Beyond TAA targeting, optimizing T cell engagement specificity via alternative T cell receptors improves safety. SAR444200, the first NANOBODY®-based TCE clinical candidate, binds to TCRαβ and GPC3 to co-engage T cells and GPC3+ tumor cells, inducing TDCC while reducing CRS and expanding the therapeutic window [ 87 ]. However, in Phase 1 clinical studies, the ORR was 0%, suggesting that TCR binding did not yield clear clinical benefits. Furthermore, AZD9793, a novel CD8-guided TCE, is an asymmetric trispecific IgG1 monoclonal antibody with two Fab domains binding to human GPC3, one VHH domain binding to the human TCR, and one VHH domain targeting CD8. Unlike conventional TCEs that broadly activate CD3+ T cells, AZD9793 preferentially engages CD8+ T cells via CD8/TCR binding, driving potent tumor killing with minimal CD4+ T cell activation and reduced cytokine secretion [ 88 ].

In autoimmune diseases, a key direction for future exploration is to identify specific antigens on pathogenic cells—beyond those on B cells and plasma cells—and to develop targeted TCEs for their treatment. Additionally, targeting dual pathogenic cell types (such as B cells and plasma cells) or incorporating co-stimulatory signals like CD28 in systemic lupus erythematosus (SLE) may enable more comprehensive immune modulation. For example, CC312—a trispecific TCE targeting CD19/CD28/CD3 for autoimmune disease therapy—incorporates the CD28 co-stimulatory signal to prevent T-cell exhaustion and enhance T-cell engagement efficacy. In a Phase 1 clinical trial with five SLE patients, CC312 administration led to significant reductions in SLEDAI-2K scores and notable depletion of peripheral blood CD19+ B cells [ 89 ]. Further efficacy validation requires additional clinical data.

Currently, TCE technology has evolved from the initial “single-target, broad activation” model to a new phase characterized by “multi-target synergy, precise regulation, and cross-field application”: In the oncology field, through designs such as antigen combinations, T-cell subset-preferential activation, and immune checkpoint integration, core challenges including antigen escape, CRS toxicity, and T-cell exhaustion are being gradually addressed. However, it is important to note that the translation of TCE technology into clinical practice still requires overcoming three key hurdles: “accuracy of target validation,” “control of clinical dose window,” and “assessment of long-term safety.”

T-cell receptor-based T-cell engager antibodies

TCR-mimic antibodies based TCEs (TCRm-TCEs) represent a distinct evolution, leveraging TCR-like specificity to target intracellular antigens presented by MHC molecules [ 90 ]. Unlike traditional TCEs that bind surface antigens, TCRm-TCEs, such as tebentafusp (gp100/TCR/CD3), engage peptide–MHC complexes, enabling access to a broader range of tumor-specific targets [ 90 ]. This approach has shown success in solid tumors like UM, where tebentafusp improved survival rates.

Developing TCRm antibodies faces significant hurdles, including the difficulty of selecting highly tumor-specific pMHC complexes to minimize off-tumor toxicity, achieving high affinity and peptide selectivity to avoid cross-reactivity, addressing the vast diversity of over 20 000 HLA alleles that restrict patient applicability, countering tumor escape mechanisms like MHC-I downregulation, and overcoming technical platform limitations that struggle to reconcile antibody-TCR structural-energy differences [ 91 , 92 ]. Looking ahead, advancements in AI-driven tools (e.g. RFdiffusion, AlphaFold) for neoantigen-pMHC structure prediction and high-throughput screening will enhance target identification [ 93 , 94 ]. Emerging trispecific formats (e.g. the WT1/CD3/4-1BB trispecific antibody, which sustains T cell function even with low MHC expression) promise to broaden patient coverage, improve therapeutic precision, and counter tumor escape [ 95 ]. Integrating these technologies with combination therapies, such as pairing TCRm-TCEs or TCRm-based trispecific antibodies with checkpoint inhibitors, could significantly boost efficacy against solid tumors, paving the way for scalable, off-the-shelf immunotherapies that transform cancer treatment outcomes.

Conditionally activated T-cell engager

Conditionally active antibodies are engineered to exploit tumor microenvironment features—such as elevated metalloproteinase levels, low pH, and high Adenosine triphosphate (ATP) concentrations—to enhance specificity and minimize off-target effects [ 96 , 97 ]. For example, antibodies can be masked with metalloproteinase-cleavable polypeptides, blocking epitope binding in normal tissues until activated by tumor-specific proteases. Similarly, pH-sensitive designs exhibit high affinity in the acidic tumor milieu but low or no affinity in neutral conditions, restricting activity to tumors. Chugai’s ATP-mediated Switch antibodies, which require ATP binding for antigen engagement, leverage high ATP levels in tumors for selective activation [ 96 , 98 ].

Companies like Janux Therapeutics [with Tumor-Activated T Cell Engager (TRACTr) platform, e.g. JANX007 for PSMA and JANX008 for EGFR], Takeda (COBRA platform, e.g. TAK-186 targeting EGFR and TAK-280 for B7-H3), Vir (PRO-XTEN platform, e.g. Vir-5818 for HER2 and Vir-5500 for PSMA), Harpoon Therapeutics (ProTriTAC for TROP2 and Integrin-β6), Revitope Oncology (TwoGATE, e.g. REV-403 for EGFRxPD-L1), and Zymeworks (PROTECT for multispecifics) are leading the charge [ 7 ]. Additionally, candidates like KGX105—a prodrug of EGFRxCD3 bispecific antibody—incorporate dual masking and an albumin-binding domain to extend in vivo half-life [ 99 ].

Conditionally active TCEs face major clinical bottlenecks, with most candidates unvalidated clinically. The core issue is uncontrollable TME activation: TME heterogeneity (e.g. variable metalloproteinase expression) causes insufficient activation in some patients, while low TME-specific molecules in non-tumor tissues trigger premature systemic activation (e.g. CRS). CytomX’s masked EGFRxCD3 TCE CX-904 was discontinued for poor efficacy, highlighting the challenge of balancing activation efficiency and specificity. A notable example is JANX007, a TCE developed on Janux’s TRACTr platform, targeting PSMA and CD3 for metastatic castration-resistant prostate cancer (mCRPC). In a Phase 1a study, JANX007 demonstrated robust efficacy in 16 mCRPC patients, achieving 100% PSA50, 63% PSA90, and 31% PSA99 declines; durable responses (75% PSA50 and 50% PSA90 at ≥12 weeks); 50% ORR; and 63% DCR. The safety profile was tolerable. The efficacy at high doses and in larger patient populations require further attention.

Overall, conditionally active TCEs, leveraging their TME-targeted activation property, have emerged as a critical direction in the field of tumor immunotherapy. However, they still need to overcome two major hurdles: activation precision and clinical translation. With the continuous optimization and maturation of development platforms, as well as the accumulation of more clinical data, these drugs are expected to become a core tool for solid tumor treatment, bringing benefits to patients with refractory tumors.

Conclusion

TCE bispecific and msAbs have emerged as a transformative class of therapeutics, demonstrating significant potential in treating cancers and autoimmune diseases. By precisely bridging T cells with target cells, these antibodies have driven significant advancements in immunotherapy, with clinical breakthroughs notably observed in hematological malignancies such as lymphoma and R/R MM. In autoimmune diseases, despite being in early development stages, TCEs have yielded promising preclinical and clinical results, indicating a future role in managing chronic, refractory conditions, like SLE.

Nevertheless, critical challenges persist across disease areas. In solid tumors, obstacles such as CRS, off-target antigen effects, and limited T-cell infiltration into the tumor microenvironment remain significant hurdles. In autoimmune settings, issues like deep tissue clearance, sustained efficacy, and long-term safety require further exploration. To address these limitations, ongoing efforts to prioritize innovative strategies, including novel target combinations, optimizing molecular design, and developing combination therapies—such as pairing TCEs with checkpoint inhibitors or cytokine modulators—are poised to propel this field forward. As these innovations advance, TCE antibodies are positioned to become a cornerstone of modern immunotherapy. By enabling highly targeted immune modulation, TCEs hold promise for delivering personalized, effective therapies that improve patient outcomes and quality of life across diverse disease landscapes, from cancer to autoimmune diseases, and beyond.

Contributor Information

Xiaoqiang Kang, Nanjing Leads Biolabs, Nanjing, 210031, China.

Yue Zhao, Strategic & Intelligence Department, Nanjing Leads Biolabs, Nanjing, 210031, China.

Hong Ling, Nanjing Leads Biolabs, Nanjing, 210031, China.

Xiao Huang, R&D Department, Nanjing Leads Biolabs, Nanjing, 210031, China.

Author contributions

Xiaoqiang Kang (Conceptualization [equal], Supervision [equal], Writing—review & editing [equal]), Yue Zhao (Conceptualization [equal], Formal analysis [equal], Methodology [equal], Resources [equal], Visualization [equal], Writing—original draft [equal]), Hong Ling (Conceptualization [equal], Supervision [equal], Writing—review & editing [equal]), and Xiao Huang (Conceptualization [equal], Supervision [equal], Writing—review & editing [equal])

Conflict of interest

All the authors are the employees of Nanjing Leads Biolabs Co., Ltd. Nanjing Leads Biolabs Co., Ltd. is a public company. All authors are shareholders of the company.

Funding

This work was supported by Nanjing Leads Biolabs Co., Ltd.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

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Associated Data

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.