Stepping forward: T-cell redirecting bispecific antibodies in cancer therapy.

Introduction

Cancer has an extremely high incidence and mortality rate worldwide, making it one of the significant public health issues currently, posing a severe threat to human health 1 . Immunotherapy, one of the most promising strategies for tumor treatment that harnesses the body's immune system to combat tumor cells selectively, has received significant attention internationally 2 , 3 , 4 , 5 . T-cell-based therapies hold a prominent position in the scope of immunotherapy, which mainly involves three primary categories: immune checkpoint inhibitors (ICIs), chimeric antigen receptor (CAR) T-cell therapy, and bispecific antibodies, each addressing the disease through distinct mechanisms. ICIs could block T cell activation negative regulators, such as PD-1 and CTLA-4. By inhibiting the interaction between these checkpoint molecules and their ligands, these therapies rejuvenate the immune system's ability to combat cancer. However, ICIs induce long-term remission only in patients with specific cancer types and only in a minority of patients. CAR T therapy is to couple the potency of a T cell with the specificity of an antibody to kill diseased cells precisely. However, the production of CAR T cells necessitates ex vivo genetic modification and proliferation, typically of autologous T cells, which is a time-consuming, relatively inefficient, and costly process. Bispecific antibodies (BsAbs) are composed of two distinct antibodies, enabling them to bind to either two epitopes of the same antigen or two distinct antigens 6 , 7 . Due to the complexity of the tumor's immunosuppressive microenvironment and the immune escape mechanisms, single immune-modulating strategies still have significant limitations, such as their capacity only to solely target individual molecules, insufficient treatment, or side effects 8 , 9 . BsAbs, especially T cell-redirecting BsAbs, have emerged as a promising and progressive avenue in T-cell immunotherapy 10 .

So far, over 100 BsAbs are undergoing clinical research. Among these, thirteen BsAbs have been granted market approval, with nine targeting the CD3 receptor ( Figure 1 , Figure 2 , and Table 1 ) 11 , 12 , 13 , 14 , 15 . In the architecture of a T-cell redirecting BsAb, one arm is designed to bind to the tumor cell surface antigen tumor-associated antigens (TAA), while the other arm is connected to the CD3 ε subunit of the T cell receptor complex (TCR) 16 , 17 , 18 , 19 . These antibodies can remarkably bridge the tumor and T cells, facilitate interactions with T cells, and trigger cytotoxic responses 20 , 21 . In a concise overview, the T-cell redirecting BsAb establishes an immune synapse upon binding the target and T cells 21 . This synapse enables T cells to transport cytolytic proteins such as perforin and apoptosis-inducing proteins like granzymes) to the target cell membrane, effectively leading to tumor cell killing. The cytolytic activity of CD3 + T cells is redirected towards tumor cells, promoting their elimination independent of major histocompatibility complex (MHC) molecules, thus bypassing the constraints of MHC limitation 6 . The synapse formation also triggers the cross-linking of TCR, subsequent activation of T cells, releasing pro-inflammatory cytokines, and induction of T cell proliferation. This review provides a broad overview of the current challenges and future perspectives for developing T-cell redirecting BsAbs for cancer treatment, which provides insights into the research and development of novel cancer therapeutics using BsAbs. Figure 1 Timeline of some major advances in bispecific antibodies. Figure 1 Figure 2 Schematic structures of thirteen bispecific antibodies that have been approved. Bispecific antibodies recognize antigens (blue and purple) by two different antigen-binding arms, and most bispecific antibodies are established on IgG1/4 scaffolds. IgG: immunoglobulin G; CD3: cluster of differentiation 3; EpCAM: epithelial cell adhesion molecule; CD19: cluster of differentiation 19; EGFR: estimated glomerular filtration rate; c-Met: cellular-mesenchymal epithelial transition factor; VEGF: vascular endothelial growth factor; Ang-2: angiopoietin-2; PD-1: programmed cell death protein 1; CTLA-4: cytotoxic T lymphocyte associate protein-4; BCMA: B cell maturation antigen; CD20: cluster of differentiation 20. Figure 2 Table 1 Overview of clinical advances in T-cell redirecting bispecific antibodies in the treatment of tumors. Table 1 Drug Target Indication Phase Clinical trial Status Last update posted Research institution Cibisatamab CD3 × CEA Metastatic colorectal cancer II NCT04826003 Recruiting 2023/8/1 Roche Holding AG Nivatrotamab CD3 × GD2 Neuroblastoma, small cell carcinoma, osteosarcoma II NCT03033303 Active, not recruiting 2023/7/5 Y-mAbs Therapeutics, Inc. Memorial Sloan Kettering Cancer Center BNT-142 CD3 × CLDN6 Solid tumor I/II NCT05262530 Recruiting 2023/6/22 BioNTech SE GEN-1047 CD3 × B7–H4 Breast cancer, endometria cancer, ovarian cancer I/II NCT05180474 Recruiting 2023/4/21 Genmab A/S GEN1044 CD3 × 5T4 Locally advanced or metastatic solid tumor(s), prostate cancer, esophageal cancer I/II NCT04424641 Terminated 2023/2/1 Genmab A/S CD30 biAb-AATC CD3 × CD30 Pediatric cancer, Hodgkin disease, CD30-positive diffuse large B-cell lymphoma I/II NCT05544968 Not yet recruiting 2022/9/19 Medical College of Wisconsin Research Foundation CM-350 CD3 × GPC3 Advanced solid tumor I/II NCT05263960 Recruiting 2022/6/9 Keymed Biosciences Inc. RO-7444973 CD3 × MAGEA4 Solid tumor I NCT05129280 Terminated 2023/8/4 Roche Holding AG A-337 CD3 × EpCAM EpCAM expressing solid tumours I CTR20232278 Not yet recruiting 2023/8/2 Evive Biotech Runimotamab CD3 × HER2 Solid tumor I NCT03448042 Recruiting 2023/7/13 Genentech, Inc. ERY-974 CD3 × GPC3 Hepatocellular carcinoma (HCC) I NCT05022927 Recruiting 2023/7/10 Chugai Pharmaceutical Co., Ltd. AMG-199 CD3 × MUC17 MUC17-positive solid tumors I NCT04117958 Terminated 2023/6/22 BeiGene Pharmaceuticals (Guangzhou) Co., Ltd. BA-1202 CD3 × CEA Advanced solid tumor I NCT05909241 Not yet recruiting 2023/6/22 Luye Pharma AG Vixtimotamab CD3 × CD33 AML, childhood I NCT05077423 Terminated 2023/5/30 Amphivena Therapeutics, Inc. BA-3182 CD3 × EpCAM Advanced adenocarcinoma I NCT05808634 Not yet recruiting 2023/4/11 BioAtla, Inc. EMB-07 CD3 × ROR1 Advanced/metastatic solid tumors I NCT05607498 Recruiting 2023/3/17 EpimAb Biotherapeutics, Inc. CX-904 CD3 × EGFR Solid tumor I NCT05387265 Recruiting 2023/2/28 Amgen, Inc. JNJ-63898081 CD3 × PSMA Neoplasms I NCT03926013 Completed 2022/11/7 Janssen Research & Development LLC GBR-1342 CD3 × CD38 HER2 expressing solid tumors I NCT02829372 Terminated 2020/10/9 Ichnos Sciences, Inc. PF-6671008 CD3 × CDH3 Neoplasms I NCT02659631 Terminated 2020/5/6 MacroGenics, Inc. CEA: carcinoembryonic antigen; GD2: disialoganglioside GD2; CLDN6: claudin-6; GPC3: glypican-3; MAGEA4: melanoma-associated antigen A4; HER2: human epidermal growth factor receptor 2; ROR1: inactive tyrosine-protein kinase transmembrane receptor; EGFR: epidermal growth factor receptor; PSMA: prostate-specific membrane antigen; CDH3: cadherin-3.

Challenges of T-cell redirecting BsAbs

T-cell redirecting BsAbs face numerous challenges in cancer immunotherapy, mainly toxicity, loss of TAA, the tumor microenvironment, infections, and tumor-lysis syndrome. Toxicity includes on-target off-tumor toxicities (target expression in healthy tissues), increased cytokine release leads to cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). The release of tumor cells after lysis also causes metabolic abnormalities. Furthermore, tumor cells can suppress T cell activation and evade immune surveillance through various mechanisms, such as antigen expression loss, antigen endocytosis, or shedding. Immunosuppression of the TME and infection also enhances the side effects of BsAbs ( Fig. 3 ). The clinical presentation ranges from mild symptoms, such as fever, to severe symptoms, such as hypoxia and hypotension. Figure 3 Challenges of T-cell redirecting bispecific antibodies in cancer immunotherapy. ① On-target, off-tumor toxicity. A subset of solid tumor antigens is expressed in tissues of healthy organs, resulting in varying degrees of off-tumor toxicity. ② Cytokine release syndrome (CRS). A significant surge of proinflammatory cytokines due to overactivation of immune cells leads to a cytokine storm. ③ Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). ④ TAA loss. Tumor cells can suppress T cell activation and evade immune surveillance through various mechanisms, such as antigen expression loss, antigen endocytosis, or shedding. ⑤ Tumors microenvironment (TME). It includes insufficient T cell infiltration and reverse recruitment of T cell subsets. A significant feature of solid tumors is the presence of an immunosuppressive TME. Secondly, in addition to recruiting effector T cells, T-cell redirecting bispecific antibodies also recruit other immune cells, which may lead to drug resistance. ⑥ Infection. Infections are common in patients receiving bispecific antibody therapy, and combination therapy increases the risk of infection. ⑦ Tumor-lysis syndrome (TLS) is attributed to the breakdown of tumor cells, which release their contents into the systemic circulation, resulting in metabolic abnormalities. Figure 3

Toxicity

On-target, off-tumor toxicity

The first challenge in T-cell redirecting BsAbs immunotherapy is identifying and differentiating therapeutic targets in normal cells from those in tumor cells. Most membrane proteins are conserved between tumor and healthy cells 22 . Ideal TAAs are limited to a small fraction of tumors, and solid tumor TAAs are usually expressed on tissues of healthy organs 23 , 24 , resulting in varying degrees of off-tumor toxicity 25 . Some proteins with significant expression differences in solid tumors are intracellular proteins, but traditional antibodies cannot penetrate the cells 26 , 27 . These intracellular proteins require recognition through HLA class I molecules, such as ImmTACs or TCR-like antibodies 28 , 29 , 30 . TCR-like CD3 BsAbs still exhibit on-target-off-tumor side effects in clinical trials. For example, dose-limiting toxicity was observed in clinical trials of tebentafusp. Tebentafusp is a bispecific protein composed of a soluble T-cell receptor incorporating single-chain variable fragments targeting the CD3 domain 31 , 32 , 33 . This high-affinity, high-specificity T-cell receptor targets a 9-amino acid peptide derived from intracellular gp100 protein through proteolytic degradation, presented by HLA molecules on the surface of target cells (including melanocytes and melanoma cells). By targeting tumor-associated antigens specifically shared by tumors, these T-cell receptor bispecific molecules can recruit T cells and direct them toward tumors. Patients treated with tebentafusp experienced skin-related adverse events, possibly due to the recognition of gp100-expressing melanocytes. In order to improve the selectivity and specificity of tumor antibodies and reduce off-tumor toxicity, many researchers have designed novel T-cell redirecting BsAbs. For example, they enhance the binding to tumor cells by adding a second tumor-associated antigen (TAA) binding domain. If the affinity to the TAA is too high, it may lead to binding to low-expressing TAAs on healthy cells. On the other hand, if the affinity to the TAA is too low, it may affect the efficiency of T-cell redirecting BsAbs. Besides, the design of antibodies can be improved to develop BsAbs with a modulatory activity that is only activated in the acidic tumor microenvironment. The acidic pH is due to the lactic acid produced by the glycolytic pathway used by tumor cells. The acidic environment creates an immunosuppressive microenvironment that helps tumor cells evade the immune system while also affecting the bioavailability of classical antibodies. Researchers have developed selective antibodies and CAR-T cell therapy technologies based on acidic pH, which can increase the TME targeting of antibodies and CAR-T cells and reduce on-target, off-tumor toxicity. BA3182, a conditionally active biologic, possesses two binding domains for EpCAM and CD3 ε . In vitro studies reveal that BA3182 can specifically and reversibly bind to the target proteins EpCAM and CD3 under the acidic conditions characteristic of the tumor microenvironment. Conversely, at the physiological alkaline pH of 7.4 found in normal cells, its binding affinity is considerably diminished. In vivo research indicates that BA3182 offers a therapeutic index surpassing non-CAB variants by over a hundredfold, underscoring the immense potential of CAB BsAbs compared to their traditional counterparts ( NCT05808634 ).

Furthermore, by combining bispecific antibodies with other forms of treatment, such as immunomodulators and small molecule drugs, comprehensive strategies can significantly enhance the selectivity and specificity of bispecific antibodies, maximize their therapeutic effects, and simultaneously reduce off-target toxicity to normal tissues.

CRS

Cytokine release syndrome (CRS) emerges due to excessive activation of immune cells, marking a severe immune-related adverse reaction 33 . CRS is characterized by a substantial surge in pro-inflammatory cytokines within the bloodstream, including IL-6, IL-10, TNF- α , and IFN- γ , leading to a cytokine storm. Clinical manifestations of CRS include fever, fatigue, muscle pain, respiratory distress, capillary leakage, vasodilatory shock, and systemic organ failure 34 .

The pioneering BsAb drug, Catumaxomab, showed promising efficacy in treating malignant ascites. However, its strong immunogenicity gave rise to pronounced hepatotoxicity and triggered a high-intensity cytokine storm when it went off-target, leading to its withdrawal from the market in 2017 35 . CRS is the predominant toxicity concern in therapies involving BsAbs that bridge T cells 36 . Existing pharmacological interventions, such as the immunosuppressant agent dexamethasone and the IL-6 receptor inhibitor tocilizumab, offer promising avenues for mitigating CRS. In 2017, due to insufficient endogenous immune response to clear patients’ cancer, Ishiguro and colleagues designed a BsAb targeting CD3/GPC3 for solid tumors. In a monkey model, the main adverse event was cytokine storm, mitigated by pre-treatment with dexamethasone without compromising anti-tumor efficacy. In the dose-escalation phase I trial, 29 subjects were enrolled, with a dose range of 0.003–0.81 μg/kg 37 , 38 , 39 . The results showed that more than 20% of the patients experienced CRS reactions and fever. The dose of 0.81 μg/kg was found to be intolerable; however, doses lower than 0.81 μg/kg were generally well-tolerated, and adverse events could be managed with corticosteroids 40 and anti-IL-6 receptor inhibitors ( NCT05022927 ).

TNB-383B, co-developed by Tenebio and AbbVie, is a novel BsAb featuring a unique structural configuration: two α BCMA molecules on one arm, a singular α CD3 arm, and a silent IgG4 Fc segment 41 . This design endows it with a potent T-cell activating capacity and a low-affinity anti-CD3 activity, which balances robust cytotoxic effects with minimized cytokine release. In phase I clinical trials, TNB-383B is evaluated for safety, tolerability, and pharmacokinetics, including its maximum concentration and half-life in the circulatory system. Preliminary data suggest a dosage-dependent therapeutic response, with an impressive 80% overall response rate (ORR) at doses equal to or exceeding 40 mg 42 . Common side effects encountered in the trials include CRS, fatigue, headaches, reduced blood cell counts, infections, and nausea.

In November 2023, the BsAb targeting CD3/CD20, Glofitamab, was approved by the NMPA for marketing in China. Before that, in March 2023, Glofitamab was approved for marketing in Canada; in June, it was approved by the US Food and Drug Administration (FDA) for marketing. Glofitamab, a 2 + 1 CD3/CD20 BsAb, targets T cells to malignant B cells to exert its anti-tumor effects 43 , 44 . In a Phase II clinical trial for the treatment of aggressive lymphoma, CRS was the most common adverse reaction, occurring in the majority (approximately 63%) of patients, mainly at Grade 1 (47.4%) and Grade 2 (11.7%), and one patient discontinued the trial due to CRS.

BsAbs currently demonstrate potential effectiveness in treating certain malignant tumors; however, CRS, as a significant adverse effect issue, remains to be addressed. As a result, future research may focus more on the design and improvement of BsAbs. These antibodies aim to maximize the targeted effects on tumor cells while minimizing the impact on healthy tissues and controlling the host's inflammatory response. Additionally, drugs such as dexamethasone used in pre-treatment have shown potential in reducing CRS induced by BsAbs treatment. Future therapies may adopt more of this kind of strategy, which includes using more advanced immunosuppressants or inflammation pathway-specific inhibitors to decrease the toxic side effects associated with treatment.

Neurotoxicity

Neurotoxicity of BsAbs in tumor immunotherapy is often an indirect consequence, transpiring concurrently with CRS or shortly after CRS 45 , 46 , 47 , 48 , 49 . Nevertheless, it can also occur independently of CRS 41 , 42 . This form of neurotoxicity is referred to as ICANS and is characterized by a range of symptoms, including headaches, confusion, hallucinations, seizures, tremors, ataxia, and aphasia 39 . The risk of ICANS can be mitigated through dose escalation and premedication 45 . Supportive care interventions for ICANS-affected patients involve the administration of tocilizumab (when CRS is concurrent), corticosteroids, or antiepileptic drugs.

Neurotoxicity has been observed in a subset of patients with B-cell hematological malignancies who underwent T-cell engager therapy 50 , 51 , 52 , 53 , 54 , 55 , 56 . Blinatumomab, a T-cell redirecting BsAb, has been developed to target CD19, an antigen expressed on B-cell acute lymphoblastic leukemia cells. In a multicenter, single-arm, phase 2 clinical trial, blinatumomab rarely induced adverse events in tumor immunotherapy; they were largely transient and resolved effectively. However, a majority of these adverse events exhibited a notable association with a high rate of neurotoxicity (all grades at 47%–53%; grade 3 or higher at 7%–13%) 57 , 58 , 59 , 60 . Compared to treatments targeting other antigens, CD19-directed BsAbs demonstrated a heightened frequency of neurotoxicity, a phenomenon potentially attributed to the presence of CD19 on cells in the blood–brain barrier 61 .

Clinicians can formulate precision therapeutic strategies by deepening our understanding of the risk factors and mechanisms underlying neurotoxicity. These strategies involve adjusting dosages and selecting appropriate antibody treatments contingent on a patient's clinical symptoms, genetic predispositions, and immune system status. Enriched knowledge of ICANS paves the way for innovative support treatments. The future holds promise for BsAbs and related immunotherapies to reduce side effects and enhance patient safety while effectively combatting cancer.

TAA loss

The development of T-cell redirecting BsAbs is predicated on identifying and TAAs. Nevertheless, tumor cells can inhibit T cell activation and circumvent immune detection through several strategies, including losing antigen expression, antigen endocytosis, or shedding. The immune system's elimination of cells exhibiting tumor-specific antigens can give rise to variations of the tumor that lack these antigens. Additionally, analogous to viral “antigenic drift”, tumor cells can evade T cell-mediated destruction by modifying their antigenic profile through epitope mutations. The CD19 antigen has emerged as an effective target for the immunotherapeutic treatment of acute lymphoblastic leukemia, yet resistance to the BsAb blinatumomab, targeting CD3/CD19, has been observed during treatment 62 . Flow cytometry analysis of tumor tissues from resistant patients revealed mutations in the CD19 antigen, indicative of TAA loss. Further analysis showed no structural anomalies in the CD19 gene, suggesting the presence of complete antigen loss escape variants, as opposed to single-epitope loss or splice variants.

BsAb research faces obstacles in treating mature solid tumors such as gastrointestinal cancer, melanoma, and glioma due to the scarcity of identifiable tumor-specific antigens and the underlying heterogeneity of solid tumor cells. Therefore, selecting optimal targets predominantly found on tumor cells and displaying minimal expression on healthy cells to reduce collateral damage to normal tissue while enhancing treatment specificity is crucial. Given tumors’ diverse nature and variability, therapies concentrating on a single antigen may prove insufficient to eradicate cancer cells. Future research should pivot towards combination therapies that concurrently target multiple antigens, thereby increasing the likelihood of therapeutic success and mitigating the risk of resistance emergence.

Tumors microenvironment

Insufficient T cell infiltration

Compared to hematologic malignancies, one distinguishing feature of solid tumors is the presence of an immunosuppressive TME 63 , 64 . The TME encompasses a complex interplay of elements, including immune suppressor cells, stromal cells, immune suppressor cytokines, and soluble molecules, all inhibiting anti-tumor immunity 29 . Both tumor cells and immune suppressor cells express immune checkpoint molecules, which upregulation often leads to tumor cell immune evasion 64 , 65 , 66 . Moreover, tumor cells employ other tactics to evade immune system surveillance. Under normal circumstances, there exists a dynamic balance between molecules of immune activation and immune suppression 67 , collectively maintaining the stability of the body's immune system 68 . However, excessive expression of immune suppressor factors on tumor or T cells disrupts T cell activity, leading to exhaustion and restraining the infiltration of CD8 + T cells into the tumor microenvironment 69 , 70 , 71 . Inadequate tumor-infiltrating lymphocytes into tumors may also be attributed to prolonged antigen stimulation in the TME 71 , causing a decline in the frequency of tumor-specific T cell clones and a reduction in effector T cells, culminating in T cell exhaustion 72 , 73 .

Reverse recruitment of T-cell subsets

T-cell redirecting BsAbs are designed to interact with tumor target cells and effector T cells, as brief contact can promote the formation of an immune synapse between T cells and tumor cells 74 . Afterward, the presence of perforin and granzyme in the immune synapse is observed 75 , subsequently facilitating the elimination of tumor cells by T cells. Most cytotoxic T cells (CTL), such as CD8 + T cells, CD4 + T cells, γδ T cells, and NKT cells, can participate in tumor cell lysis through this mechanism 76 . CD8 + T cells within CTL are the primary effector cells 77 . Intravenous administration of T cell redirecting bispecific antibodies has shown the ability to recruit or activate CTL cells directly, alongside recruiting other immune cells 78 . However, this recruitment is not exclusive and may encompass various T cell subgroups, including those with suppressive functions such as naïve or exhausted T cells, regulatory T cells, and different helper T cell subgroups (Th). This indiscriminate recruitment could lead to drug resistance, weakening the therapeutic potential of tumor cell killing by CD8 + T cells. Notably, a clinical study has indicated that resistance to Binatumomab arises from CD3 + CD4 + CD25 hi FoxP3 + Treg cells 20 , which hinder the dissolution of CD8 + T cells and suppress their tumor-killing capabilities. Additionally, the recruited CD4 + T cells, predominantly Th1 and Th17, often release cytokines and spark a cytokine storm. Therefore, enhancing the specificity of BsAbs can lead to better cell killing and reduce cytotoxic effects. Furthermore, exploring alternative targets for T cell recruitment or activation, such as CD28, emerges as a strategic avenue to address the non-selective recruitment of T cells by BsAbs in immune therapy.

Infection

Infections are frequent in patients treated with BsAbs, although their frequency varies considerably across diverse clinical studies 79 , 80 . Apart from tumor type, this could be associated with the extent of prior immunosuppressive therapy. The frequency of neutropenia caused by BsAbs may also fluctuate among hematological malignancies 81 , 82 , 83 . Furthermore, sustained T cell activation mediated by BsAbs may lead to T cell exhaustion, thereby increasing the risk of infection. Additionally, the combinations of T-cell redirecting BsAbs with other treatment strategies may also increase the incidence and severity of infections.

Tumor-lysis syndrome (TLS)

TLS represents a metabolic emergency attributed to the breakdown of a substantial number of tumor cells, which release their contents into the systemic circulation. This phenomenon results in metabolic anomalies like hyperkalemia, hyperuricemia, hyperphosphatemia, and hypocalcemia 84 . These metabolic abnormalities can lead to acute renal failure, seizures, cardiac arrhythmias, acidosis, azotemia, and even sudden death. Although BsAbs can potentially reduce tumor burden, the development of TLS may also occasionally occur. Patients at risk of TLS, particularly those with elevated tumor burden or impaired renal function, should receive prophylaxis measures such as uric acid-lowering medications and adequate hydration 85 , 86 , 87 , 88 .

Building on our understanding of TLS, future research and clinical practices should prioritize the identification of reliable biomarkers for predicting TLS risk, thereby enabling prophylactic interventions before its onset. Given that acute renal failure is one of the principal complications associated with TLS, developing strategies and pharmaceuticals to preserve renal function represents a critical area of investigation.

Implications for treatment strategies

Development of T-cell redirecting BsAbs in different formats

Antibody potency

The design of BsAbs configurations can impact the target binding site 89 . A bivalent BsAb featuring one binding site for each target is called 1 + 1; introducing additional binding sites can result in tri-specific antibodies (2 + 1) or quadrivalent BsAb (2 + 2 or 1 + 3). Although traditional BsAbs maintain a 1:1 ratio of CD3 and TAA 90 , enhancing the binding capacity through a 1 + 2 design may bolster the recognition of tumor cells while avoiding activation of CD3 on T cells in the absence of TAA. IGM2323 is a 10+1 type targeting CD20/CD3 91 . IGM-2323 is structured by the fusion of anti-CD3 scFv to the J chain, forming a 10:1 antibody form of anti-CD20: anti-CD3. IGM-2323 superimposed the TDCC effect on the action of CDC and played a significantly enhanced B-cell clearance effect, demonstrating the synergistic effect of BsAb ( NCT04082936 ). Compared to rituximab, IGM-2323 has a 1000-fold increase in killing activity.

Among the BsAbs that have been marketed, Roche has two BsAbs targeting CD20/CD3-Mosunetuzumab and Glofitamab 92 , 93 , 94 , 95 , 96 . Although the two new drugs are dual antibodies with the same target, their structure and indications differ. Mosunetuzumab has a 1 + 1 structure, while Glofitamab has a 2 + 1 structure. The former is approved for follicular lymphoma and the latter for treating DLBCL. This 2 + 1 structure design makes it more binding to CD20 on the surface of B cells, induces rapid T cell activation and cytokine release, and leads to target cell lysis. The activity is 10–1000 times higher than traditional 1 + 1 BsAbs. In the Phase Ib/II GO40516 study, Mosunetuzumab and Polivy achieved an ORR of 65.0% in severely treated R/R NHL patients, with a CR of 48.3%. CRS occurred in 18%, and all adverse events occurred in the first treatment cycle and were grade 1 to 2. In the Phase I/Ib dose-escalation NP30179 study, Glofitamab alone achieved an ORR of 81.0% for patients with R/R NHL who received severe treatment and 100% ORR in the glofitamab plus Gazyva group. In the Phase Ib/II NP39488 study, Glofitamab and Polivy had an ORR of 73% over a 3.2-month follow-up period, including a CR of 51.5%, and no CRS above grade 3 was observed.

Tri-specific antibody

Among the successful cancer immunotherapies, approved BsAbs such as Blinatumomab have stimulated researchers' interest in other multispecific antibody therapies 97 . T-cell redirected tri-specific antibodies represent a promising multi-targeting immune intervention; some are currently in the clinical research stage (see Table 2 ). Tri-specific antibodies extend from BsAbs by introducing an extra antigen binding site, totaling three sites. This enables simultaneous interaction with target and effector cells (typically T cells or NK cells), enhancing the killing of target cells 97 , 98 . Compared to monoclonal and BsAbs, some tri-specific antibodies have two additional specific antigen binding sites, which are advantageous for increasing binding specificity, improving the accuracy of drug targeting to tumor cells, and reducing off-target toxicity 99 , 100 . Furthermore, some tri-specific antibodies bind to tumor antigens through one binding site; the second binding site on the other arm can specifically bind to the TCR on T cells, thereby activating the first signal pathway and guiding T cells to the vicinity of target cells to achieve the aggregation/localization of immune cells 101 . The third binding site on the arm triggers the second signal pathway, further activating immune cells. Those tri-specific antibodies have a strong cytotoxic effect on target cells through bridging and activating immune cells simultaneously or bridging immune cells and blocking dual signal pathways. Table 2 Representative clinical trials of the combination strategy. Table 2 Combination strategy Antibody Target Phase Clinical trial Adaptive symptom Source of information Antibody potency IGM2323, 1 + 10 CD3/CD20 I/II NCT04082936 Non-Hodgkin lymphoma, follicular lymphoma, DLBCL https://www.clinicaltrials.gov/ Glofitamab, 2 + 1 CD3/CD20 Marketing – DLBCL 2023, FDA Trispecific antibody HPN-424 CD3/PSMA/albumin I/II NCT03577028 Prostatic cancer https://www.clinicaltrials.gov/ CMG1A46 CD3/CD19/CD20 I/II NCT05348889 Acute lymphoblastic leukemia, non-Hodgkin lymphoma, hematologic neoplasms PIT-565 CD2/CD3/CD19 I NCT05397496 B-cell lymphoma, adult lymphoblastic lymphoma, non-hodgkin lymphoma SAR-442257 CD3/CD28+/CD38 I NCT04401020 Neoplasms, non-Hodgkin lymphoma, multiple myeloma JNJ-80948543 CD3/CD20/CD79B I NCT05424822 B-cell chronic lymphocytic leukemia, non-Hodgkin lymphoma Pro-drug AMX-818 CD3+HER2/EGFR I NCT05356741 Locally advanced or metastatic HER2-expressing cancers Ref [ 180 ] ADG138 CD3+HER2 IND-enabling – Solid tumors AACR poster, Adagene's official website CX-904 CD3+EGFR I NCT05387265 Solid tumours AACR poster, Adagene's official website Mobilization of other immune cells AFM13 CD30/CD16 I/II NCT04074746 Anaplastic large cell lymphoma, B-cell non-Hodgkin lymphoma, classic Hodgkin lymphoma https://www.clinicaltrials.gov/ Reducing affinity of CD3 arm Elranatamab CD3/BCMA IV NCT06057402 Multiple myeloma Ref [ 138 , 139 , 140 , 141 , 142 ] TNB-585 CD3/PSMA I NCT04104607 Castration resistant metastatic prostate cancer Ref [ 126 ] MCLA-117 CD3/CLL I NCT03038230 Acute myeloid leukemia Ref [ 130 ] NILK-2301 CD3/CEACAM5 Drug discovery – CEACAM5-expressing cancers Ref [ 181 ] Combining with chemotherapy drugs Chemotherapy+ Mosunetuzumab CD3+CD20 I NCT05464329 Diffuse large B cell lymphoma, high-grade B-cell lymphoma, transformed B-cell lymphoma https://www.clinicaltrials.gov/ Combining with radiation therapy p53-SADA-BsAb GD2 I – Non-small cell lung cancer, melanoma, Sarcoma Ref [ 137 , 155 , 156 , 157 , 158 ] Combining with oncolytic viruses EphA2 TEA VV EphA2 Drug discovery – Solid tumors Ref [ 140 ] OAd-MUC16-BiTE MUC16 Drug discovery – Ovarian cancer Ref [ 165 ] Combining with targeted therapy BHKis + CD3/CD19 BsAb CD3+CD19 Drug discovery – Chronic lymphocytic leukemia Ref [ 170 ] Combining immune checkpoint inhibitors M7824 PD-L1+TGF β II NCT03840902 Non-small cell lung cancer https://www.clinicaltrials.gov/ SHR1701 PD-L1+TGF β I/II NCT04407741 Solid tumors, lymphomas

For example, Wu et al. 100 and Sun et al. 102 developed a T-cell redirected tri-specific antibody, SAR-442257, that interacts with CD38, CD3 and CD28. The engagement of CD3 and low-affinity CD28 affords efficient T cell activation ability, whereas the anti-CD38 antibody directs T cells to certain lymphomas, leukemias, and myeloma cells. In vitro , SAR-442257 showed 1000–10,000-fold higher cytotoxic activity against myeloma cells than CD38 monoclonal antibody. This promising approach is being explored for relapsed/refractory multiple myeloma (MM) and non-classical Hodgkin's lymphoma patients within phase I clinical trials ( NCT04401020 ). Sun et al. 102 and Seung et al. 103 developed a HER2/CD3/CD28 tri-specific antibody based on the HER2/CD3 BsAb. Coupling an anti-CD28 variable domain to the anti-CD3 variable region in a humanized mouse model, regression of breast cancer cells mediated by CD4 + and CD8 + T cells was observed. The single-valent anti-CD28 utilized in this tri-specific antibody did not induce the severe cytokine release observed previously with the bivalent superagonist anti-CD28 agents. This antibody is currently undergoing phase I clinical trials ( NCT05013554 ). T-cell redirecting BsAbs emerge as an ideal anti-tumor therapy, representing a multitargeted immunotherapeutic intervention with promising prospects for immunotherapy against human cancers.

Pro-drug

T-cell redirected bispecific antibodies, exemplified by Blinatumomab (CD19/CD3), have succeeded in hematologic malignancies 104 , 105 , 106 , 107 . However, in solid tumors, the development of such BsAbs faces challenges, primarily due to T-cell overstimulation or on-target off-tumor effects causing damage to normal tissues 20 , 108 . Researchers are addressing these issues from multiple angles, one approach being tumor-specific activation of bispecific antibodies. The pro-drug design is used to reduce the specific killing of normal tissue cells by BsAb drugs 109 , significantly improving the effectiveness and safety of BsAb drugs 110 . In normal tissues, BsAb drugs exist in the form of pro-body without activity 111 . The TME harbors abundant matrix metalloproteinases 112 . Upon reaching specific sites in the tumor area, they undergo enzymatic cleavage by certain enzymes, removing a small peptide segment that masks the binding site, thereby enabling the BsAb to exert its function 111 , 112 .

Tianyan Pharmaceuticals' SAFEbody BsAb platform technology effectively employs this concept in design. ADG138 is a BsAb targeting HER2 × CD3, where the epitopes of HER2 and CD3 are masked 113 . Upon reaching the target site, the masking peptide is removed, exposing the Fab region of the antibody and revealing the binding sites for anti-HER2 and anti-CD3. This allows for the normal binding of the antibody to the antigens on tumor cells, recruiting and activating T cells to kill tumor cells selectively. CI107, a BsAb, consists of the cetuximab antibody targeting EGFR and the humanized SP34 antibody targeting CD3, fused in scFv form at the N-terminus of the cetuximab antibody heavy chain 114 . CI107 utilizes Tianyan Pharmaceuticals’ SAFEbody platform to mask the epitopes of EGFR and CD3. Short masking peptides that can be specifically cleaved are fused at the N-termini of both the cetuximab antibody light chain and the scFv. In vivo , CI107 exhibited a potent, dose-dependent induction of tumor regression in established colon cancer xenografts mice. CI107 significantly reduced cytokine release and showed no elevation in AST levels. In contrast, administering the activated T cell bispecific antibody (TCB) increased IL-6, IFN- γ , and AST levels. These discernible differences underscore the compelling effect of dual masking on the EGFR and CD3 binding domains within CI107. This innovative approach substantially enhances the tolerability and safety profile of the probody TCB. Besides, the maximum tolerated dose of CI107 was determined in cynomolgus monkeys and was over 60 times higher than that of the unmasked TCB. Animals administered with CI107 exhibited significantly lower toxicity levels, further reinforcing the notion of an expanded safety margin for this novel therapeutic. This design prevents damage to normal tissues and side effects. CI107 has demonstrated good safety and efficacy in preclinical stages as well.

Mobilization of other immune cells to precisely eradicate tumors

Cell engagers could bridge tumor and immune cells to form an immune synapse-mediated cytotoxic effect 21 , 115 . In addition to bispecific T cell engager (BiTE), it can also mobilize other immune cells, such as NK cells 116 . The bispecific NK cell engager antibody redirects CD16A + NK cells to tumor cells and activates NK cells. Compared to BiTE, a noteworthy attribute of the bispecific NK cell engager lies in its ability to reduce CRS and neurotoxicity 117 , 118 . AFM13 is the pioneering tetravalent BsAb to target CD30 and CD16 119 . This dual targeting empowersAFM13 to bind CD16A on NK cells and CD30 on lymphoma cells, thereby mediating NK cell killing and clearance of cancer cells 120 . Interventions utilizing modified immune cells AFM13-NK and BsAbs AFM13 for the treatment of relapsed or refractory CD30 + Hodgkin's or non-Hodgkin's lymphoma patients are currently in Phase I/II ( NCT04074746 ) 121 , 122 , 123 . The application of AFM13 immunotherapy may benefit patients by enabling their immune system to attack tumor cells and potentially inhibit tumor growth and metastasis. Combining AFM13 with NK cells may potentiate tumor cell demise and suppression of tumor growth beyond the scope of AFM13 monotherapy.

Reducing affinity of CD3 arm

Studies have highlighted a significant trend in T-cell redirecting BsAbs associating affinity for CD3 with pharmacokinetic properties 124 , 125 . Intriguingly, the antibodies with a low-affinity CD3 arm exhibited slower clearance and higher drug exposure than a higher-affinity CD3 arm 126 , 127 . However, this correlation presents considerable challenges 21 , such as the affinity for CD3 exerts a multifaceted influence on various properties crucial for therapeutic efficacy, including biodistribution, potency, and toxicity 128 , 129 . For instance, high CD3 affinity correlates with CRS and might result in a lack of potency and proper biodistribution. Ideally, optimizing the performance of a T-cell redirecting BsAb, such as the appropriate affinity of the CD3 and TAA arms, could result in the desired balance between potency, pharmacokinetics, and safety for meeting therapeutic objectives.

CD3/CLL1 TDB, a BsAb binding CD3 and CLL-1, is engineered to treat acute myeloid leukemia 130 . Notably, results in monkeys revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells, compared to the high-affinity TDBs 130 . They provide evidence that using a low-affinity CD3 arm could circumvent toxicities related to CRS and provide acceptable safety while maintaining robust activity. Consequently, one of the most promising strategies to generate a promising BsAb involves pairing low-affinity CD3 antibodies with high-affinity antibodies targeting the TAA 130 , 131 , 132 , 133 . Besides, the target selection of antibodies depends on the different expression levels of TAAs, while varying structural formats yield different effects. For instance, compared to tandem scFv-based BsAbs, those based on Fab exhibit superior biophysical properties. Furthermore, Fab-based BsAbs retain the activity of their tandem scFv counterparts and possess unique biological activity 134 . Sebag et al. 135 developed a novel BsAb, Elranatamab, which optimizes affinity for binding to CD3 and the B cell maturation antigen BCMA for treating multiple myeloma 135 , 136 , 137 , 138 , 139 . Decreasing antibody affinity promotes T cell activation and enhances their anti-tumor activity against multiple myeloma, reducing off-target cell toxicity to some extent.

Combining T-cell redirecting BsAb with other antitumor therapies

T-cell redirecting BsAbs has demonstrated remarkable efficacy in tumor treatment 140 . However, the effectiveness achieved through a single treatment approach is limited, and it is challenging to eliminate tumors solely relying on this treatment approach. Building upon the antitumor mechanisms of T-cell redirecting BsAbs, combining them with other treatment modalities, including systemic chemotherapies, radiotherapy, targeted therapies, and immunotherapies, creates a synergistic effect, further enhancing tumor therapy's effectiveness ( Fig. 4 ). Figure 4 T cell-redirecting BsAbs combination therapy strategies. These include systemic chemotherapies, radiotherapy, targeted therapies, oncolytic viruses and immune-checkpoint inhibitors, which can create a synergistic effect, further enhancing the efficacy of tumor therapy. Figure 4

Combining with chemotherapy drugs

One of the challenges in utilizing T-cell redirecting BsAbs for treating solid tumors lies in the limited infiltration of T-cells within the TME 20 , 141 . Traditional cytotoxic agents, like DNA intercalators 142 , 143 , nucleotide analogs 144 , or alkylating agents 145 , are also being considered for combination with T-cell redirecting BsAbs. Chemotherapies are designed to target rapidly dividing cells directly 146 ; thus, combining chemotherapy and T-cell redirecting BsAbs might improve therapeutic responses. Preliminary data substantiating this hypothesis have been reported. For example, ERY974, a T cell-redirecting antibody that targets glypican-3 and CD3, demonstrates modest antitumor effectiveness when used as a monotherapy in non-inflamed NCI–H446 xenograft tumors. This is due to the limited infiltration of ERY974-redirected T cells, primarily at the tumor-stromal boundary. Meanwhile, the cytotoxic agent paclitaxel monotherapy initially showed strong efficacy but could not prevent eventual tumor regrowth. However, ERY974 with chemotherapy synergistically and reciprocally increases antitumor efficacy in non-inflamed tumors by promoting T-cell infiltration into the tumor center and increasing ERY974 distribution in the tumor. This combination therapy may be effective for patients with non-inflamed tumors and those resistant to existing therapies. Chemotherapeutic cyclophosphamide is a potent antitumor drug as an alkylating agent that can alkylate the DNA of tumor cells within their cells 147 , 148 , 149 . At the same time, cyclophosphamide alone can inhibit tumor growth during the initial treatment phase; tumors often resurge within 2–3 weeks 150 . BCMA/CD3 BsAb monotherapy causes an increase in the number of CD8 + T-cells. However, this excessive activation of T-cells can result in T-cell depletion, leading to temporary tumor regression followed by tumor recurrence after 3–4 weeks of treatment. Combining of BsAbs and cyclophosphamide can remarkably eradicate the tumor 150 . Experimental data proved that cyclophosphamide can inhibit the excessive activation of T cells and prevent T cell depletion. It can also inhibit the up-regulation of PD-L1 on the tumor surface, promote the renewal of memory T cells, and ensure that the T cells exhibit prolonged and highly effective antitumor activity. Therefore, the therapeutic strategy of combining T-cell redirecting BsAbs with chemotherapeutic drugs holds significant promise for further development 151 , 152 , 153 .

Combining with radiation therapy

T-cell redirecting BsAbs and radiotherapy are two distinct domains in cancer treatment, each possessing non-overlapping cytotoxic effects 154 , 155 . A synergistic approach materializes through the combination of targeted immunotherapy, exemplified by tumor-targeting agents (BsAbs) and potent payloads (radioisotopes). This multifaceted strategy employs multiple stages, with BsAbs administered over 24 h, followed by a free radical scavenger, and a radioactive metal injection 4 h later. This orchestrated sequence seeks to overcome the limitations of conventional radiation therapy, including inadequate radioisotope uptake and elevated kidney retention. An article has reported a self-assembling and disassembling (SADA) BsAbs that fuses with p53 protein to form a p53–SADA–BsAbs tetramer. After administration, it forms dimers or monomers in the bloodstream, which can be cleared and reduce immunogenicity in rats after kidney filtration, with no short-term or long-term toxicity to the bone marrow, kidneys, or liver 137 . Notably, using anti-GD2 BsAbs in pretargeted radioimmunotherapy for neuroblastoma has showcased improved therapeutic effects in terms of tumor shrinkage, elimination of micrometastases, and prevention of relapse 156 , 157 , 158 .

Radiation therapy operates by stimulating the immune system, thereby bolstering immune-stimulating cell infiltration within tumors, amplifying the immunogenicity of tumor cells, and augmenting antitumor immune responses. Nonetheless, it also engenders local or systemic immune suppression, occasionally even at low doses, culminating in temporary bone marrow inhibition and diminished lymphocyte counts. Radiation therapy can inadvertently impair CD8 + T cells 159 , pivotal for effective immune therapy response. Hence, in some instances, radiation therapy might not accentuate the efficacy of combined immunotherapy and may attenuate its potency.

Combining with oncolytic viruses

Oncolytic viruses can infect, replicate, and lyse tumor cells while enhancing immune therapy and inducing increased permeability of the immune-tumor microenvironment, allowing for sufficient infiltration of CD8 + T cells in the tumor microenvironment 160 . However, due to their non-targeted impact on normal cells, oncolytic viruses are often used in combination therapy strategies in clinical trials, resulting in promising advancements 161 , 162 , 163 , 164 . Yu et al. 140 engineered an oncolytic vaccinia virus (VV) that carries a BiTE antibody targeting the tumor cell surface antigen EphA2 and named it EphA2 TEA VV. In a mouse model of lung cancer xenografts, EphA2 TEA VV triggered CTL activation and redirection, outperforming conventional oncolytic vaccinia virus in achieving more potent anti-tumor outcomes. In another study, Wang et al. 165 designed an oncolytic adenovirus (OAd-MUC16-BiTE) carrying a BiTE antibody targeting MUC16 (mucin 16). Stable BiTE expression within tumor tissues was achieved post-local administration, mitigating systemic toxicity from systemic application. OAd-MUC16-BiTE mediated antigen-specific T cell activation and target cell lysis. Furthermore, it demonstrated the ability to “reverse” the tumor microenvironment, fostering CTL infiltration, amplifying T cell-mediated tumor cell elimination, and significantly enhancing anti-tumor efficacy.

The functional mechanism of oncolytic measles viruses harboring bispecific T-cell engagers (MV-BiTEs) hinges on the production and release of BiTE antibodies from tumor cells infected with BiTEs 166 . This triggers the assembly of multinucleated syncytia, culminating in the destruction of the tumor cells. Concurrently, BiTEs mobilize T cells to target and eliminate non-infected tumor cells, manifesting additional bystander effects. The therapeutic efficacy of MV-BiTEs was assessed in immunocompetent mouse models. Specifically, in the MC38-CEA model, MV-BiTE treatment extended the survival rates and effectively curbed tumor growth compared to the control group. Within the B16-CD20-CD46 model, MV-BiTE therapy augmented tumor-infiltrating T cells’ number and activation levels, thereby bolstering anti-tumor immunity. These findings affirm that MV-BiTEs offer a potent, relapse-free treatment for solid tumors while inducing protective immunity.

Merging BsAbs with oncolytic viruses presents a promising strategy that supersedes the current limitations of individual BiTE treatments, offering a compelling avenue for solid tumor therapy. These studies underscore that combining oncolytic viruses and BiTE achieves a synergistic effect that overcomes the limitations of single-agent therapy.

Combining with targeted therapy

Targeted therapy is a precision treatment that designs corresponding targeted therapeutic drugs at the cellular and molecular levels to target specific cancer sites that have been identified. These drugs typically exert their effects predominantly on tumor cells, minimizing collateral damage to normal cells.

In small molecule targeted inhibitors, the primary targets are often vital kinases involved in cell signaling pathways, such as the mammalian target of rapamycin (mTOR), mitogen-activated protein kinase, and anaplastic lymphoma kinase. An exhaustive examination of 52 FDA-approved small molecule tyrosine kinase inhibitors revealed that select drugs aimed at mTOR, JAK, and Src kinases effectively suppress T cell proliferation 167 . When co-administered with T-cell redirecting BsAbs in vitro , mTOR and JAK kinase inhibitors diminished cytokine release related to CRS without undermining the tumor-killing potential of T-cell bispecific antibodies. In contrast, Src kinase inhibitor dasatinib successfully suppressed CRS-related cytokines but at the cost of impaired T cell cytotoxicity. Therefore, mTOR and JAK kinase inhibitors are preferable for mitigating CRS reactions in clinical settings without sacrificing anti-tumor effectiveness.

Bruton tyrosine kinase inhibitors are the preferred therapeutic agents for patients with chronic lymphocytic leukemia (CLL) 168 , 169 . The efficacy of BHK in CLL can be enhanced by combining it with CD19/CD3 BsAbs 170 . Notably, BTKis actively disrupts CLL cells’ immunosuppressive mechanisms, irrespective of ITK inhibition, thereby bolstering the cytotoxic activity of autologous T cells 171 , 172 , 173 , 174 . Elevated CTLA-4 expression in peripheral blood CLL cells has been documented, and its subsequent downregulation upon BTKi treatment suggests a mechanistic link that may enhance T-cell cytotoxic responses. Thus, combining BTKis with BsAbs is a robust strategy for achieving sustained CLL remission and counteracting drug resistance, which may have broader applications in treating other mature B-cell malignancies.

Targeted therapy has made substantial strides in cancer care, offering personalized treatments by engaging tumor-associated molecules. This not only heightens therapeutic efficacy but also minimizes adverse side effects. The combination of BsAbs can markedly elevate treatment success rates, underscoring the vast potential of this approach.

Combining immune checkpoint inhibitors

Even after BsAbs and T cells navigate multiple barriers to penetrate tumor sites, a host of elements can dampen their therapeutic efficacy. Exhaustion of T cells owing to upregulation of immune-checkpoint proteins during therapy has been described with various BiTEs, in vitro and patients. It is a potential immune escape mechanism underlying both intrinsic and acquired resistance. Combinatorial immunotherapy approaches have been designed to counteract T-cell dysfunction/exhaustion mechanisms using inhibitory checkpoint-blocking antibodies to improve the clinical outcomes of individuals receiving CD3 + bispecific T-cell redirection therapies. Interestingly, resistance to blinatumomab treatment has been attributed to increased PD-L1 expression in CD19 + leukemic cells 175 . In order to antagonize PD-L1 expressed by the leukemic cells, blinatumomab in combination with pembrolizumab (anti-PD-1 therapy) resulted in enhanced T cell-mediated cytotoxicity relative to that observed with the parental BiTE alone 176 . A large number of clinical trials are currently evaluating the safety and efficacy of combinatorial approaches for the treatment of relapsed/refractory acute leukemia or lymphoma patients ( NCT03160079 , NCT03605589 , NCT03340766 , NCT03512405 ).

Belmontes et al. 177 have found that combining PD-1 antibodies, CTLA-4 antibodies, and 4-1BB agonist antibodies with CD3 BsAbs can enhance the efficacy of CD3 BsAbs. Among them, 4-1BB has demonstrated the most substantial impact, particularly in amplifying CD8 + T cells within tumor tissues and overcoming treatment resistance in solid tumors. One of the most rapidly advancing BsAbs, SMET12 (targeting EGFR/CD3), combined with PD-1 antibodies, is now in Phase II clinical trials.

Cibisatamab, TCB targeting CEA/CD3, is a novel T-cell redirecting BsAbs targeting CEA on tumor cells and CD3 on T cells 178 . Cibisatamab displays potent anti-tumor activity in preclinical models, leading to increased intra-tumoral T-cell infiltration and activation and upregulating PD-1/PD-L1. In the ongoing dose-escalation phase I studies ( NCT02324257 and NCT02650713 ), activity appeared to be enhanced with doses in combination with atezolizumab, with a manageable safety profile.

An EGFR/CD3 molecule, SMET12, is currently in preclinical development for treating EGFR-positive advanced/metastatic solid tumors. Pharmacological studies demonstrate that SMET12 combined with Treprinumab (anti-PD-1 therapy) can effectively eliminate the immune suppressive environment caused by the expression of inhibitory receptors such as PD-1 after T cell activation 179 , further exerting the anti-tumor function of SMET12 to achieve synergistic enhancement. It has significant advantages and potential good prospects for treating solid tumors.

Conclusions and prospects

T-cell immunotherapy constitutes a dynamic area in cancer treatment, with three main approaches currently in the spotlight: ICIs, T-cell redirecting BsAbs, and CAR-T cell therapy. Notably, T-cell redirecting BsAbs have achieved a revolutionary breakthrough in cancer therapy. Unlike ICIs, which release the general suppression of the cellular immune system and may trigger adverse reactions to non-tumor cells, T-cell redirecting BsAbs precisely engage T cells with tumor cells, enhancing T cell activation and proliferation. Their mechanism of action enables potentially quicker responses than ICIs by inducing direct T cell-mediated cytotoxicity. In contrast to personalized therapies like CAR-T, which are time-consuming and expensive due to the need for individualized collection, modification, and expansion of a patient's T cells, bispecific antibodies offer a more straightforward and cost-effective “off-the-shelf” solution 182 . They can be manufactured and stockpiled on a large scale, reducing treatment costs. Nevertheless, the progression of T-cell redirecting BsAbs in solid tumors is relatively slower, and there are still many challenges and areas for optimization, including CRS, targeted and non-targeted cytotoxicity, TAA loss, limited T-cell infiltration and suppression of T-cell activity in the tumor microenvironment.

CRS can be observed in patients treated with T-cell redirecting BsAbs such as Binatumomab, and the severity depends on the treatment type and target 89 , 150 . Preclinical studies using humanized mouse models have found that TNF- α produced by activated T cells is the primary mediator of CRS induced by T-cell redirecting BsAbs. This leads to massive secretion of inflammatory cytokines by monocytes 183 . To overcome this challenge, T-cell redirecting BsAbs could be designed using a low-affinity anti-CD3 to reduce the potential for dose-limiting toxicities associated with CRS. Besides, blocking upstream TNF- α and downstream IL-1 β or IL-6 can induce tumor killing with reduced cytokine release 184 , 185 .

The challenges posed by targeted and non-targeted cytotoxicity, the limited number of effector cells, and the suppressed T-cell activity within tumors are deeply intertwined 186 , 187 . These three obstacles are indispensable in tackling the limitations associated with T-cell redirecting BsAbs 188 . Most antibodies currently undergoing development are centered around differentiation markers 189 . Many investigational solid tumor targets, like CEA, EGFR, EpCAM, and HER2, fall within this differentiation marker category 190 . Noteworthy among these can be expressed in normal cells, albeit at limited levels, thereby narrowing the therapeutic window and circumscribing the potential for T-cell redirecting BsAbs design. To enhance the targeting of antibodies to tumor cells and reduce drug resistance, simultaneously targeting two different tumor antigens and tri-specific antibody development can be employed to address these challenges.

Many strategies have been devised to tackle the challenge of on-target off-tumor effects; one of the innovative approaches is utilizing a “mask” to shield the antibody epitope until it reaches the tumor site for conditional activation. The stimuli involved in this strategy include light, temperature, enzymes, pH, ions, small effect molecules, or rare antigens. In addition to the pro-drugs and cleavage enzymes mentioned earlier, structural modification of the antibody Fc domain has achieved significant breakthroughs. The TME caused by changes in tumor cell metabolism is characterized by acidic pH, a widely used parameter in targeted TME research. Low pH significantly impacts the TME, leading to immune suppression, immune escape, and disease progression. This acidic extracellular environment significantly affects the bioavailability of therapeutic antibodies. Harnessing this characteristic as a trigger for therapeutic antibody activation not only affords precise control but also mitigates drug toxicity linked to off-target effects. For instance, Sulea et al. 191 developed a pH-dependent HER2 antibody (bH1-P5P8) that exhibits heightened antigen binding in acidic pH compared to neutral pH, bolstering tissue-specific distribution and diminishing distribution-related side effects in normal tissues.

The objective of achieving a durable cure with limited toxicity is also a driving force in designing and developing new tumor immunotherapies. Efficacy in tumor treatment relies mainly on immune activation strategies, especially the cytotoxic activity of immune T cells within the tumor. However, the TME can affect the infiltrating T cells, curbing their proliferation and function, curtailing effector T-cell quantity and activity. Which may result in reduced or lost activity such as impaired proliferation and immune function. The emergence of T cell exhaustion, characterized by the expression of inhibitory receptors, represents a pivotal mechanism in tumor immune evasion. By countering these signals, interventions like ICIs have demonstrated considerable antitumor potency by revitalizing T-cell functionality within the tumor microenvironment. With the ongoing advancement of science and technology, researchers have proposed innovative approaches combining T-cell redirection bispecific antibodies with other strategies, including ICIs, immune activators, and oncolytic viruses. Current and upcoming clinical trials must prove the value of these combinatory approaches. As the number of BsAbs undergoing clinical trials increases, our understanding of the optimal structures and constructs for targeting particular tumor types is expanding. Such knowledge will improve the antitumor efficacy of T cell-engaging therapies, prevent disease relapse, and drive future advancements in this field.

Author contributions

Xiaojing Qin, Wenjing Ning reviewed the literature and drafted the manuscript. Wenxin Luo and Xue Liu conceived and supervised the project. Other authors participated in the search and collation of literature. All of the authors have read and approved the final manuscript.

Conflicts of interest

The authors declare no conflicts of interest.

References

1 Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA A Cancer J Clin 71 2021 209 249 10.3322/caac.21660 33538338

2 Zhang Y. Zhang Z. The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications Cell Mol Immunol 17 2020 807 821 32612154 10.1038/s41423-020-0488-6 PMC7395159

3 Pilard C. Ancion M. Delvenne P. Jerusalem G. Hubert P. Herfs M. Cancer immunotherapy: it's time to better predict patients' response Br J Cancer 125 2021 927 938 34112949 10.1038/s41416-021-01413-x PMC8476530

4 Wang D.R. Wu X.L. Sun Y.L. Therapeutic targets and biomarkers of tumor immunotherapy: response versus non-response Signal Transduct Targeted Ther 7 2022 331 10.1038/s41392-022-01136-2 PMC9485144 36123348

5 Chehelgerdi M. Chehelgerdi M. Allela O.Q.B. Pecho R.D.C. Jayasankar N. Rao D.P. Progressing nanotechnology to improve targeted cancer treatment: overcoming hurdles in its clinical implementation Mol Cancer Mol Cancer 22 2023 169 37814270 10.1186/s12943-023-01865-0 PMC10561438

6 Ma J. Mo Y. Tang M. Shen J. Qi Y. Zhao W. Bispecific antibodies: from research to clinical application Front Immunol 12 2021 626616 10.3389/fimmu.2021.626616 PMC8131538 34025638

7 Szijj P. Chudasama V. The renaissance of chemically generated bispecific antibodies Nat Rev Chem 5 2021 78 92 37117612 10.1038/s41570-020-00241-6

8 Zhu Y. Yu X. Thamphiwatana S.D. Zheng Y. Pang Z. Nanomedicines modulating tumor immunosuppressive cells to enhance cancer immunotherapy Acta Pharm Sin B 10 2020 2054 2074 33304779 10.1016/j.apsb.2020.08.010 PMC7714985

9 Wu S.Y. Wu F.G. Chen X.Y. Antibody-incorporated nanomedicines for cancer therapy Adv Mater 34 2022 2109210 10.1002/adma.202109210 35142395

10 Wei J. Yang Y. Wang G. Liu M. Current landscape and future directions of bispecific antibodies in cancer immunotherapy Front Immunol 13 2022 1035276 10.3389/fimmu.2022.1035276 PMC9650279 36389699

11 Nisonoff A. Wissler F.C. Lipman L.N. Properties of the major component of a peptic digest of rabbit antibody Science 132 1960 1770 1771 13729245 10.1126/science.132.3441.1770

12 Milstein C. Cuello A.C. Hybrid hybridomas and their use in immunohistochemistry Nature 305 1983 537 540 6137772 10.1038/305537a0

13 Perez P. Hoffman R.W. Shaw S. Bluestone J.A. Segal D.M. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody Nature 316 1985 354 356 3160953 10.1038/316354a0

14 Holliger P. Prospero T. Winter G. “Diabodies”: small bivalent and bispecific antibody fragments Proc Natl Acad Sci U S A 90 1993 6444 6448 8341653 10.1073/pnas.90.14.6444 PMC46948

15 Lindhofer H. Mocikat R. Steipe B. Thierfelder S. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies J Immunol 155 1995 219 225 7602098

16 Bargou R.C. The expanding success of T cell-engaging bispecific antibodies Nat Cancer 4 2023 1054 1055 37316727 10.1038/s43018-023-00586-z

17 Haber L. Olson K. Kelly M.P. Crawford A. DiLillo D.J. Tavaré R. Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning Sci Rep 11 2021 14397 10.1038/s41598-021-93842-0 PMC8277787 34257348

18 van de Donk N.W.C.J. Zweegman S. T-cell-engaging bispecific antibodies in cancer Lancet 402 2023 142 158 37271153 10.1016/S0140-6736(23)00521-4

19 Kamakura D. Asano R. Yasunaga M. T cell bispecific antibodies: an antibody-based delivery system for inducing antitumor immunity Pharmaceuticals 14 2021 1172 34832954 10.3390/ph14111172 PMC8619951

20 Singh A. Dees S. Grewal I.S. Overcoming the challenges associated with CD3 + T-cell redirection in cancer Br J Cancer 124 2021 1037 1048 33469153 10.1038/s41416-020-01225-5 PMC7960983

21 Xiong W. Chen Y. Kang X. Chen Z. Zheng P. Hsu Y.H. Immunological synapse predicts effectiveness of chimeric antigen receptor Cells Mol Ther 29 2021 1349 1351 33592166 10.1016/j.ymthe.2021.01.025 PMC7934788

22 Lin C.Y. Lee C.H. Chuang Y.H. Lee J.Y. Chiu Y.Y. Wu Lee Y.H. Membrane protein-regulated networks across human cancers Nat Commun 10 2019 3131 31311925 10.1038/s41467-019-10920-8 PMC6635409

23 Jhunjhunwala S. Hammer C. Delamarre L. Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion Nat Rev Cancer 21 2021 298 312 33750922 10.1038/s41568-021-00339-z

24 Andersen M.H. Tumor microenvironment antigens Semin Immunopathol 45 2023 253 264 36175673 10.1007/s00281-022-00966-0 PMC10335965

25 Peri A. Salomon N. Wolf Y. Kreiter S. Diken M. Samuels Y. The landscape of T cell antigens for cancer immunotherapy Nat Cancer 4 2023 937 954 37415076 10.1038/s43018-023-00588-x

26 Raman V. Van Dessel N. Hall C.L. Wetherby V.E. Whitney S.A. Kolewe E.L. Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases Nat Commun 12 2021 6116 34675204 10.1038/s41467-021-26367-9 PMC8531320

27 Pishesha N. Harmand T.J. Ploegh H.L. A guide to antigen processing and presentation Nat Rev Immunol 22 2022 751 764 35418563 10.1038/s41577-022-00707-2

28 Xu Y. Salazar G.T. Zhang N. An Z. T-cell receptor mimic (TCRm) antibody therapeutics against intracellular proteins Antib Ther 2 2019 22 32 33928218 10.1093/abt/tbz001 PMC7990144

29 Tian Z. Liu M. Zhang Y. Wang X. Bispecific T cell engagers: an emerging therapy for management of hematologic malignancies J Hematol Oncol 14 2021 75 33941237 10.1186/s13045-021-01084-4 PMC8091790

30 Passariello M. Yoshioka A. Takahashi K. Hashimoto S.I. Inoue T. Nakamura K. Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo J Exp Clin Cancer Res 41 2022 269 36071464 10.1186/s13046-022-02474-3 PMC9450414

31 Damato B.E. Dukes J. Goodall H. Carvajal R.D. Tebentafusp: T cell redirection for the treatment of metastatic uveal melanoma Cancers 11 2019 971 31336704 10.3390/cancers11070971 PMC6679206

32 Middleton M.R. McAlpine C. Woodcock V.K. Corrie P. Infante J.R. Steven N.M. Tebentafusp, a TCR/anti-CD3 bispecific fusion protein targeting gp100, potently activated antitumor immune responses in patients with metastatic melanoma Clin Cancer Res 26 2020 5869 5878 32816891 10.1158/1078-0432.CCR-20-1247 PMC9210997

33 Morris E.C. Neelapu S.S. Giavridis T. Sadelain M. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy Nat Rev Immunol 22 2022 85 96 34002066 10.1038/s41577-021-00547-6 PMC8127450

34 Frey N. Cytokine release syndrome: who is at risk and how to treat Best Pract Res Clin Haematol 30 2017 336 340 29156206 10.1016/j.beha.2017.09.002

35 Frampton J.E. Catumaxomab: in malignant ascites Drugs 72 2012 1399 1410 22676343 10.2165/11209040-000000000-00000

36 Cao Y. Wang J. Jiang S. Lyu M. Zhao F. Liu J. JAK1/2 inhibitor ruxolitinib promotes the expansion and suppressive action of polymorphonuclear myeloid-derived suppressor cells via the JAK/STAT and ROS–MAPK/NF- κ B signalling pathways in acute graft- versus -host disease Clin Transl Immunology 12 2023 e1441 10.1002/cti2.1441 PMC9968240 36855558

37 Sano Y. Azuma Y. Tsunenari T. Kayukawa Y. Shinozuka J. Fujii E. Combination of T cell-redirecting bispecific antibody ERY974 and chemotherapy reciprocally enhances efficacy against non-inflamed tumours Nat Commun 13 2022 5265 36071036 10.1038/s41467-022-32952-3 PMC9452528

38 Safran H. Druta M. Morse M. Lynce F. Pintova S. Almhanna K. Abstract CT111: results of a phase 1 dose escalation study of ERY974, an anti-glypican 3 (GPC3)/CD3 bispecific antibody, in patients with advanced solid tumors Cancer Res 81 2021 CT111

39 Moreau P. Garfall A.L. van de Donk N.W.C.J. Nahi H. San-Miguel J.F. Oriol A. Teclistamab in relapsed or refractory multiple myeloma N Engl J Med 387 2022 495 505 35661166 10.1056/NEJMoa2203478 PMC10587778

40 Ishiguro T. Sano Y. Komatsu S.I. Kamata-Sakurai M. Kaneko A. Kinoshita Y. An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors Sci Transl Med 9 2017 eaal4291 10.1126/scitranslmed.aal4291 28978751

41 Moreau P. Kumar S.K. San Miguel J. Davies F. Zamagni E. Bahlis N. Treatment of relapsed and refractory multiple myeloma: recommendations from the International Myeloma Working Group Lancet Oncol 22 2021 e105 e118 33662288 10.1016/S1470-2045(20)30756-7

42 Shah N. Chari A. Scott E. Mezzi K. Usmani S.Z. B-cell maturation antigen (BCMA) in multiple myeloma: rationale for targeting and current therapeutic approaches Leukemia 34 2020 985 1005 32055000 10.1038/s41375-020-0734-z PMC7214244

43 Minson A. Dickinson M. Glofitamab CD20-TCB bispecific antibody Leuk Lymphoma 62 2021 3098 3108 34263696 10.1080/10428194.2021.1953016

44 Dickinson M.J. Carlo-Stella C. Morschhauser F. Bachy E. Corradini P. Iacoboni G. Glofitamab for relapsed or refractory diffuse large B-cell lymphoma N Engl J Med 387 2022 2220 2231 36507690 10.1056/NEJMoa2206913

45 Lee D.W. Gardner R. Porter D.L. Louis C.U. Ahmed N. Jensen M. Current concepts in the diagnosis and management of cytokine release syndrome Blood 124 2014 188 195 24876563 10.1182/blood-2014-05-552729 PMC4093680

46 Ludwig H. Terpos E. van de Donk N. Mateos M.V. Moreau P. Dimopoulos M.A. Prevention and management of adverse events during treatment with bispecific antibodies and CAR T cells in multiple myeloma: a consensus report of the European Myeloma Network Lancet Oncol 24 2023 e255 e269 37269857 10.1016/S1470-2045(23)00159-6

47 Cohen A.D. Mateos M.V. Cohen Y.C. Rodriguez-Otero P. Paiva B. van de Donk N.W.C.J. Efficacy and safety of cilta-cel in patients with progressive multiple myeloma after exposure to other BCMA-targeting agents Blood 141 2023 219 230 36095849 10.1182/blood.2022015526 PMC10562529

48 Gaballa M.R. Banerjee P. Milton D.R. Jiang X. Ganesh C. Khazal S. Blinatumomab maintenance after allogeneic hematopoietic cell transplantation for B-lineage acute lymphoblastic leukemia Blood 139 2022 1908 1919 34914826 10.1182/blood.2021013290 PMC8952188

49 Roth P. Winklhofer S. Müller A.M.S. Dummer R. Mair M.J. Gramatzki D. Neurological complications of cancer immunotherapy Cancer Treat Rev 97 2021 102189 10.1016/j.ctrv.2021.102189 33872978

50 San-Miguel J. Dhakal B. Yong K. Spencer A. Anguille S. Mateos M.V. Cilta-cel or standard care in lenalidomide-refractory multiple myeloma N Engl J Med 389 2023 335 347 37272512 10.1056/NEJMoa2303379

51 Foà R. Bassan R. Vitale A. Elia L. Piciocchi A. Puzzolo M.C. Dasatinib-blinatumomab for pH-positive acute lymphoblastic leukemia in adults N Engl J Med 383 2020 1613 1623 33085860 10.1056/NEJMoa2016272

52 Trudel S. Cohen A.D. Krishnan A.Y. Fonseca R. Spencer A. Berdeja J.G. Cevostamab monotherapy continues to show clinically meaningful activity and manageable safety in patients with heavily pre-treated relapsed/refractory multiple myeloma (RRMM): updated results from an ongoing phase I study Blood 138 2021 157

53 van der Sluis I.M. de Lorenzo P. Kotecha R.S. Attarbaschi A. Escherich G. Nysom K. Blinatumomab added to chemotherapy in infant lymphoblastic leukemia N Engl J Med 388 2023 1572 1581 37099340 10.1056/NEJMoa2214171

54 Brown P.A. Ji L. Xu X. Devidas M. Hogan L.E. Borowitz M.J. Effect of postreinduction therapy consolidation with blinatumomab vs chemotherapy on disease-free survival in children, adolescents, and young adults with first relapse of B-cell acute lymphoblastic leukemia: a randomized clinical trial JAMA 325 2021 833 842 33651090 10.1001/jama.2021.0669 PMC7926290

55 Locatelli F. Zugmaier G. Rizzari C. Morris J.D. Gruhn B. Klingebiel T. Effect of blinatumomab vs chemotherapy on event-free survival among children with high-risk first-relapse B-cell acute lymphoblastic leukemia: a randomized clinical trial JAMA 325 2021 843 854 33651091 10.1001/jama.2021.0987 PMC7926287

56 Jabbour E. Short N.J. Jain N. Huang X. Montalban-Bravo G. Banerjee P. Ponatinib and blinatumomab for Philadelphia chromosome-positive acute lymphoblastic leukaemia: a US, single-centre, single-arm, phase 2 trial Lancet Haematol 10 2023 e24 e34 36402146 10.1016/S2352-3026(22)00319-2

57 Topp M.S. Gökbuget N. Stein A.S. Zugmaier G. O'Brien S. Bargou R.C. Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study Lancet Oncol 16 2015 57 66 25524800 10.1016/S1470-2045(14)71170-2

58 Kantarjian H. Stein A. Gökbuget N. Fielding A.K. Schuh A.C. Ribera J.M. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia N Engl J Med 376 2017 836 847 28249141 10.1056/NEJMoa1609783 PMC5881572

59 Gökbuget N. Dombret H. Bonifacio M. Reichle A. Graux C. Faul C. Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia Blood 131 2018 1522 1531 29358182 10.1182/blood-2017-08-798322 PMC6027091

60 Stein A.S. Schiller G. Benjamin R. Jia C. Zhang A. Zhu M. Neurologic adverse events in patients with relapsed/refractory acute lymphoblastic leukemia treated with blinatumomab: management and mitigating factors Ann Hematol 98 2019 159 167 30238148 10.1007/s00277-018-3497-0 PMC6334725

61 Parker K.R. Migliorini D. Perkey E. Yost K.E. Bhaduri A. Bagga P. Single-cell analyses identify brain mural cells expressing CD19 as potential off-tumor targets for CAR-T immunotherapies Cell 183 2020 126 142 32961131 10.1016/j.cell.2020.08.022 PMC7640763

62 Braig F. Brandt A. Goebeler M. Tony H.P. Kurze A.K. Nollau P. Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking Blood 129 2017 100 104 27784674 10.1182/blood-2016-05-718395

63 Mancini S.J.C. Balabanian K. Corre I. Gavard J. Lazennec G. Le Bousse-Kerdilès M.C. Deciphering tumor niches: lessons from solid and hematological malignancies Front Immunol 12 2021 766275 10.3389/fimmu.2021.766275 PMC8631445 34858421

64 Montironi C. Muñoz-Pinedo C. Eldering E. Hematopoietic versus solid cancers and T cell dysfunction: looking for similarities and distinctions Cancers 13 2021 284 33466674 10.3390/cancers13020284 PMC7828769

65 Nakamura K. Smyth M.J. Myeloid immunosuppression and immune checkpoints in the tumor microenvironment Cell Mol Immunol 17 2020 1 12 31611651 10.1038/s41423-019-0306-1 PMC6952382

66 Tie Y. Tang F. Wei Y.Q. Wei X.W. Immunosuppressive cells in cancer: mechanisms and potential therapeutic targets J Hematol Oncol 15 2022 61 35585567 10.1186/s13045-022-01282-8 PMC9118588

67 Galli F. Aguilera J.V. Palermo B. Markovic S.N. Nisticò P. Signore A. Relevance of immune cell and tumor microenvironment imaging in the new era of immunotherapy J Exp Clin Cancer Res 39 2020 89 32423420 10.1186/s13046-020-01586-y PMC7236372

68 Kalaora S. Nagler A. Wargo J.A. Samuels Y. Mechanisms of immune activation and regulation: lessons from melanoma Nat Rev Cancer 22 2022 195 207 35105962 10.1038/s41568-022-00442-9

69 Jiang W. He Y. He W. Wu G. Zhou X. Sheng Q. Exhausted CD8 + T cells in the tumor immune microenvironment: new pathways to therapy Front Immunol 11 2021 622509 10.3389/fimmu.2020.622509 PMC7902023 33633741

70 Lopez de Rodas M. Schalper K.A. Tumour antigen-induced T cell exhaustion—the archenemy of immune-hot malignancies Nat Rev Clin Oncol 18 2021 749 750 34556846 10.1038/s41571-021-00562-5 PMC9235859

71 Brummel K. Eerkens A.L. de Bruyn M. Nijman H.W. Tumour-infiltrating lymphocytes: from prognosis to treatment selection Br J Cancer 128 2023 451 458 36564565 10.1038/s41416-022-02119-4 PMC9938191

72 Chow A. Perica K. Klebanoff C.A. Wolchok J.D. Clinical implications of T cell exhaustion for cancer immunotherapy Nat Rev Clin Oncol 19 2022 775 790 36216928 10.1038/s41571-022-00689-z PMC10984554

73 Wherry E.J. Kurachi M. Molecular and cellular insights into T cell exhaustion Nat Rev Immunol 15 2015 486 499 26205583 10.1038/nri3862 PMC4889009

74 Haber L. Olson K. Kelly M.P. Crawford A. DiLillo D.J. Tavaré R. Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning Sci Rep 11 2021 14397 10.1038/s41598-021-93842-0 PMC8277787 34257348

75 Voskoboinik I. Whisstock J.C. Trapani J.A. Perforin and granzymes: function, dysfunction and human pathology Nat Rev Immunol 15 2015 388 400 25998963 10.1038/nri3839

76 Weigelin B. den Boer A.T. Wagena E. Broen K. Dolstra H. de Boer R.J. Cytotoxic T cells are able to efficiently eliminate cancer cells by additive cytotoxicity Nat Commun 12 2021 5217 34471116 10.1038/s41467-021-25282-3 PMC8410835

77 Raskov H. Orhan A. Christensen J.P. Gögenur I. Cytotoxic CD8 + T cells in cancer and cancer immunotherapy Br J Cancer 124 2021 359 367 32929195 10.1038/s41416-020-01048-4 PMC7853123

78 Xie F. Zhang L. Shi S. Zheng A. Di J. Jin S. Liposomal T cell engager and re-director for tumor cell eradication in cancer immunotherapy mAbs 14 2022 2115205 10.1080/19420862.2022.2115205 PMC9450900 36041060

79 Raje N. Anderson K. Einsele H. Efebera Y. Gay F. Hammond S.P. Monitoring, prophylaxis, and treatment of infections in patients with MM receiving bispecific antibody therapy: consensus recommendations from an expert panel Blood Cancer J 13 2016 116 10.1038/s41408-023-00879-7 PMC10394080 37528088

80 Sim B.Z. Longhitano A. Er J. Harrison S.J. Slavin M.A. Teh B.W. Infectious complications of bispecific antibody therapy in patients with multiple myeloma Blood Cancer J 13 2023 34 36894539 10.1038/s41408-023-00808-8 PMC9998258

81 Lamers C.H. Sleijfer S. van Steenbergen S. van Elzakker P. van Krimpen B. Groot C. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity Mol Ther 21 2013 904 912 23423337 10.1038/mt.2013.17 PMC5189272

82 Thistlethwaite F.C. Gilham D.E. Guest R.D. Rothwell D.G. Pillai M. Burt D.J. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity Cancer Immunol Immunother 66 2017 1425 1436 28660319 10.1007/s00262-017-2034-7 PMC5645435

83 Viardot A. Goebeler M.E. Hess G. Neumann S. Pfreundschuh M. Adrian N. Phase 2 study of the bispecific T-cell engager (BiTE) antibody blinatumomab in relapsed/refractory diffuse large B-cell lymphoma Blood 127 2016 1410 1416 26755709 10.1182/blood-2015-06-651380 PMC4797019

84 Budde L.E. Sehn L.H. Matasar M. Schuster S.J. Assouline S. Giri P. Safety and efficacy of mosunetuzumab, a bispecific antibody, in patients with relapsed or refractory follicular lymphoma: a single-arm, multicentre, phase 2 study Lancet Oncol 23 2022 1055 1065 35803286 10.1016/S1470-2045(22)00335-7

85 Philipp N. Kazerani M. Nicholls A. Vick B. Wulf J. Straub T. T-cell exhaustion induced by continuous bispecific molecule exposure is ameliorated by treatment-free intervals Blood 140 2022 1104 1118 35878001 10.1182/blood.2022015956 PMC10652962

86 Howard S.C. Jones D.P. Pui C.H. The tumor lysis syndrome N Engl J Med 364 2011 1844 1854 21561350 10.1056/NEJMra0904569 PMC3437249

87 Bannerji R. Arnason J.E. Advani R.H. Brown J.R. Allan J.N. Ansell S.M. Odronextamab, a human CD20×CD3 bispecific antibody in patients with CD20-positive B-cell malignancies (ELM-1): results from the relapsed or refractory non-Hodgkin lymphoma cohort in a single-arm, multicentre, phase 1 trial Lancet Haematol 9 2022 e327 e339 35366963 10.1016/S2352-3026(22)00072-2 PMC10681157

88 Hutchings M. Mous R. Clausen M.R. Johnson P. Linton K.M. Chamuleau M.E.D. Dose escalation of subcutaneous epcoritamab in patients with relapsed or refractory B-cell non-Hodgkin lymphoma: an open-label, phase 1/2 study Lancet 398 2021 1157 1169 34508654 10.1016/S0140-6736(21)00889-8

89 Oates J. Hassan N.J. Jakobsen B.K. ImmTACs for targeted cancer therapy: why, what, how, and which Mol Immunol 67 2015 67 74 25708206 10.1016/j.molimm.2015.01.024

90 Labrijn A.F. Janmaat M.L. Reichert J.M. Parren P.W.H.I. Bispecific antibodies: a mechanistic review of the pipeline Nat Rev Drug Discov 18 2019 585 608 31175342 10.1038/s41573-019-0028-1

91 Budde E. Gopal A.K. Kim W.S. Flinn I.W. Cheah C.Y. Nastoupil L. A phase 1 dose escalation study of Igm-2323, a novel anti-CD20 × anti-CD3 IgM T cell engager (TCE) in patients with advanced B-cell malignancies Blood 138 2021 132

92 Assouline S.E. Kim W.S. Sehn L.H. Schuster S.J. Cheah C.Y. Nastoupil L.J. Mosunetuzumab shows promising efficacy in patients with multiply relapsed follicular lymphoma: updated clinical experience from a phase I dose-escalation trial Blood 136 2020 42 44

93 Hutchings M. Carlo-Stella C. Bachy E. Offner F.C. Morschhauser F. Crump M. Glofitamab step-up dosing induces high response rates in patients with hard-to-treat refractory or relapsed non-Hodgkin lymphoma Blood 136 2020 46 48

94 Fajgenbaum D.C. June C.H. Cytokine storm N Engl J Med 383 2020 2255 2273 33264547 10.1056/NEJMra2026131 PMC7727315

95 Olszewski A.J. Avigdor A. Babu S. Levi I. Abadi U. Holmes H. Single-agent mosunetuzumab is a promising safe and efficacious chemotherapy-free regimen for elderly/unfit patients with previously untreated diffuse large B-cell lymphoma Blood 136 2020 43 45

96 Phillips T.J. Olszewski A.J. Munoz J. Kim T.M. Yoon D.H. Greil R. Mosunetuzumab, a novel CD20/CD3 bispecific antibody, in combination with CHOP confers high response rates in patients with diffuse large B-cell lymphoma Blood 136 2020 37 38

97 Blanco B. Domínguez-Alonso C. Alvarez-Vallina L. Bispecific immunomodulatory antibodies for cancer immunotherapy Clin Cancer Res 27 2021 5457 5464 34108185 10.1158/1078-0432.CCR-20-3770 PMC9306338

98 Runcie K. Budman D.R. John V. Seetharamu N. Bi-specific and tri-specific antibodies- the next big thing in solid tumor therapeutics Mol Med 24 2018 50 30249178 10.1186/s10020-018-0051-4 PMC6154901

99 Mullard A. Trispecific antibodies take to the clinic Nat Rev Drug Discov 19 2020 657 658 32917978 10.1038/d41573-020-00164-3

100 Wu L. Seung E. Xu L. Rao E. Lord D.M. Wei R.R. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation Nature Cancer 1 2019 86 98 35121834 10.1038/s43018-019-0004-z

101 Garfall A.L. June C.H. Trispecific antibodies offer a third way forward for anticancer immunotherapy Nature 575 2019 450 451 31740852 10.1038/d41586-019-03495-3

102 Sun Y. Yu X. Wang X. Yuan K. Wang G. Hu L. Bispecific antibodies in cancer therapy: target selection and regulatory requirements Acta Pharm Sin B 13 2023 3583 3597 37719370 10.1016/j.apsb.2023.05.023 PMC10501874

103 Seung E. Xing Z. Wu L. Rao E. Cortez-Retamozo V. Ospina B. A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells Nature 603 2022 328 334 35197632 10.1038/s41586-022-04439-0

104 Goebeler M.E. Bargou R. Blinatumomab: a CD19/CD3 bispecific T cell engager (BiTE) with unique anti-tumor efficacy Leuk Lymphoma 57 2016 1021 1032 27050240 10.3109/10428194.2016.1161185

105 van der Sluis I.M. de Lorenzo P. Kotecha R.S. Attarbaschi A. Escherich G. Nysom K. Blinatumomab added to chemotherapy in infant lymphoblastic leukemia N Engl J Med 388 2023 1572 1581 37099340 10.1056/NEJMoa2214171

106 Advani A.S. Moseley A. O'Dwyer K.M. Wood B.L. Fang M. Wieduwilt M.J. Swog 1318: a phase II Trial of blinatumomab followed by POMP maintenance in older patients with newly diagnosed Philadelphia chromosome-negative B-cell acute lymphoblastic leukemia J Clin Oncol 40 2022 1574 1582 35157496 10.1200/JCO.21.01766 PMC9084435

107 Viardot A. Bargou R. Bispecific antibodies in haematological malignancies Cancer Treat Rev 65 2018 87 95 29635163 10.1016/j.ctrv.2018.04.002

108 Yu S. Li A. Liu Q. Yuan X. Xu H. Jiao D. Recent advances of bispecific antibodies in solid tumors J Hematol Oncol 10 2017 155 28931402 10.1186/s13045-017-0522-z PMC5607507

109 Jornada D.H. dos Santos Fernandes G.F. Chiba D.E. de Melo T.R. dos Santos J.L. Chung M.C. The prodrug approach: a successful tool for improving drug solubility Molecules 21 2015 42 26729077 10.3390/molecules21010042 PMC6273601

110 Autio K.A. Boni V. Humphrey R.W. Naing A. Probody therapeutics: an emerging class of therapies designed to enhance on-target effects with reduced off-tumor toxicity for use in immuno-oncology Clin Cancer Res 26 2020 984 989 31601568 10.1158/1078-0432.CCR-19-1457 PMC8436251

111 Lin W.W. Lu Y.C. Chuang C.H. Cheng T.L. Ab locks for improving the selectivity and safety of antibody drugs J Biomed Sci 27 2020 76 32586313 10.1186/s12929-020-00652-z PMC7318374

112 Kessenbrock K. Plaks V. Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment Cell 141 2010 52 67 20371345 10.1016/j.cell.2010.03.015 PMC2862057

113 Liu G. Li Y. Dai Z. Qu J. Shi J. Zhao Q. Abstract 2869: ADG138, a novel HER2×CD3 POWERbody™ integrating bispecific TCE with precision masking to control cytokine release syndrome and on-target off-tumor toxicity for single agent and combination therapies in HER2-expressing solid tumors Cancer Res 82 2022 2869

114 Boustany L.M. LaPorte S.L. Wong L. White C. Vinod V. Shen J. A probody T cell-engaging bispecific antibody targeting EGFR and CD3 inhibits colon cancer growth with limited toxicity Cancer Res 82 2022 4288 4298 36112781 10.1158/0008-5472.CAN-21-2483 PMC9664135

115 Shin H.G. Yang H.R. Yoon A. Lee S. Bispecific antibody-based immune-cell engagers and their emerging therapeutic targets in cancer immunotherapy Int J Mol Sci 23 2022 5686 35628495 10.3390/ijms23105686 PMC9146966

116 Demaria O. Gauthier L. Debroas G. Vivier E. Natural killer cell engagers in cancer immunotherapy: next generation of immuno-oncology treatments Eur J Immunol 51 2021 1934 1942 34145579 10.1002/eji.202048953

117 Sivori S. Pende D. Quatrini L. Pietra G. Della Chiesa M. Vacca P. NK cells and ILCs in tumor immunotherapy Mol Aspect Med 80 2021 100870 10.1016/j.mam.2020.100870 32800530

118 Vacca P. Pietra G. Tumino N. Munari E. Mingari M.C. Moretta L. Exploiting human NK cells in tumor therapy Front Immunol 10 2020 3013 32010130 10.3389/fimmu.2019.03013 PMC6978749

119 Wu J. Fu J. Zhang M. Liu D. AFM13: a first-in-class tetravalent bispecific anti-CD30/CD16A antibody for NK cell-mediated immunotherapy J Hematol Oncol 8 2015 96 26231785 10.1186/s13045-015-0188-3 PMC4522136

120 Kerbauy L.N. Marin N.D. Kaplan M. Banerjee P.P. Berrien-Elliott M.M. Becker-Hapak M. Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated blood and cord blood-derived NK cells facilitates CAR-like responses against CD30 + malignancies Clin Cancer Res 27 2021 3744 3756 33986022 10.1158/1078-0432.CCR-21-0164 PMC8254785

121 Rothe A. Sasse S. Topp M.S. Eichenauer D.A. Hummel H. Reiners K.S. A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma Blood 125 2015 4024 4031 25887777 10.1182/blood-2014-12-614636 PMC4528081

122 Bartlett N.L. Herrera A.F. Domingo-Domenech E. Mehta A. Forero-Torres A. Garcia-Sanz R. A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma Blood 136 2020 2401 2409 32730586 10.1182/blood.2019004701 PMC7685206

123 Sasse S. Bröckelmann P.J. Momotow J. Plütschow A. Hüttmann A. Basara N. AFM13 in patients with relapsed or refractory classical Hodgkin lymphoma: final results of an open-label, randomized, multicenter phase II trial Leuk Lymphoma 63 2022 1871 1878 35848865 10.1080/10428194.2022.2095623

124 Mandikian D. Takahashi N. Lo A.A. Li J. Eastham-Anderson J. Slaga D. Relative target affinities of T cell-dependent bispecific antibodies determine biodistribution in a solid tumor mouse model Mol Cancer Therapeut 17 2018 776 785 10.1158/1535-7163.MCT-17-0657 29339550

125 Liu L. Lam C.K. Long V. Widjaja L. Yang Y. Li H. MGD011, A CD19 × CD3 dual-affinity retargeting Bi-specific molecule incorporating extended circulating half-life for the treatment of B-cell malignancies Clin Cancer Res 23 2017 1506 1518 27663593 10.1158/1078-0432.CCR-16-0666

126 Dang K. Castello G. Clarke S.C. Li Y. Balasubramani A. Boudreau A. Attenuating CD3 affinity in a PSMA×CD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release J Immunother Cancer 19 2021 e002488 10.1136/jitc-2021-002488 PMC8183203 34088740

127 Yu X. Orr C.M. Chan H.T.C. James S. Penfold C.A. Kim J. Reducing affinity as a strategy to boost immunomodulatory antibody agonism Nature 614 2023 539 547 36725933 10.1038/s41586-022-05673-2

128 Wang N. Patel H. Schneider I.C. Kai X. Varshney A.K. Zhou L. An optimal antitumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers Antib Ther 4 2021 90 100 34169228 10.1093/abt/tbab009 PMC8220303

129 Staflin K. Zuch de Zafra C.L. Schutt L.K. Clark V. Zhong F. Hristopoulos M. Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody JCI Insight 5 2020 e133757 10.1172/jci.insight.133757 PMC7205277 32271166

130 Leong S.R. Sukumaran S. Hristopoulos M. Totpal K. Stainton S. Lu E. An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia Blood 129 2017 609 618 27908880 10.1182/blood-2016-08-735365 PMC5290988

131 Betts A. van der Graaf P.H. Mechanistic quantitative pharmacology strategies for the early clinical development of bispecific antibodies in oncology Clin Pharmacol Ther 108 2020 528 541 32579234 10.1002/cpt.1961 PMC7484986

132 Bacac M. Fauti T. Sam J. Colombetti S. Weinzierl T. Ouaret D. A novel carcinoembryonic antigen T-cell bispecific antibody (CEA TCB) for the treatment of solid tumors Clin Cancer Res 22 2016 3286 3297 26861458 10.1158/1078-0432.CCR-15-1696

133 Wu X. Sereno A.J. Huang F. Lewis S.M. Lieu R.L. Weldon C. Fab-based bispecific antibody formats with robust biophysical properties and biological activity mAbs 7 2015 470 482 25774965 10.1080/19420862.2015.1022694 PMC4622572

134 Luo Y. Ye S. Li X. Lu W. Emerging structure–function paradigm of endocrine FGFs in metabolic diseases Trends Pharmacol Sci 40 2019 142 153 30616873 10.1016/j.tips.2018.12.002

135 Sebag M. Raje N.S. Bahlis N.J. Costello C. Dholaria B. Solh M. Elranatamab (PF-06863135), a B-cell maturation antigen (BCMA) targeted CD3-engaging bispecific molecule, for patients with relapsed or refractory multiple myeloma: results from magnetismm-1 Blood 138 2021 895

136 Wu L. Huang Y. Sienkiewicz J. Sun J. Guiang L. Li F. Bispecific BCMA-CD3 antibodies block multiple myeloma tumor growth Cancers 14 2022 2518 35626122 10.3390/cancers14102518 PMC9139578

137 Santich B.H. Cheal S.M. Ahmed M. McDevitt M.R. Ouerfelli O. Yang G. A self-assembling and disassembling (SADA) bispecific antibody (BsAb) platform for curative two-step pretargeted radioimmunotherapy Clin Cancer Res 27 2021 532 541 32958698 10.1158/1078-0432.CCR-20-2150 PMC7855367

138 van de Donk N.W.C.J. O’Neill C. de Ruijter M.E.M. Verkleij C.P.M. Zweegman S. T-cell redirecting bispecific and trispecific antibodies in multiple myeloma beyond BCMA Curr Opin Oncol 35 2023 601 611 37501530 10.1097/CCO.0000000000000983 PMC10566598

139 Ma R. Li Z. Chiocca E.A. Caligiuri M.A. Yu J. The emerging field of oncolytic virus-based cancer immunotherapy Trends Cancer 9 2023 122 139 36402738 10.1016/j.trecan.2022.10.003 PMC9877109

140 Yu F. Wang X. Guo Z.S. Bartlett D.L. Gottschalk S.M. Song X.T. T-cell engager-armed oncolytic vaccinia virus significantly enhances antitumor therapy Mol Ther 22 2014 102 111 24135899 10.1038/mt.2013.240 PMC3978793

141 Crawford A. Chiu D. Targeting solid tumors using CD3 bispecific antibodies Mol Cancer Therapeut 20 2021 1350 1358 10.1158/1535-7163.MCT-21-0073 34045228

142 Venugopal S. Sharma V. Mehra A. Singh I. Singh G. DNA intercalators as anticancer agents Chem Biol Drug Des 100 2022 580 598 35822451 10.1111/cbdd.14116

143 Godzieba M. Ciesielski S. Natural DNA intercalators as promising therapeutics for cancer and infectious diseases Curr Cancer Drug Targets 20 2020 19 32 31589125 10.2174/1568009619666191007112516

144 Liu Z. Zou H. Dang Q. Xu H. Liu L. Zhang Y. Biological and pharmacological roles of m6A modifications in cancer drug resistance Mol Cancer 21 2022 220 36517820 10.1186/s12943-022-01680-z PMC9749187

145 Lajous H. Lelièvre B. Vauléon E. Lecomte P. Garcion E. Rethinking alkylating(-like) agents for solid tumor management Trends Pharmacol Sci 40 2019 342 357 30979523 10.1016/j.tips.2019.03.003

146 Burgess J.T. Rose M. Boucher D. Plowman J. Molloy C. Fisher M. The therapeutic potential of DNA damage repair pathways and genomic stability in lung cancer Front Oncol 10 2020 1256 32850380 10.3389/fonc.2020.01256 PMC7399071

147 Ahlmann M. Hempel G. The effect of cyclophosphamide on the immune system: implications for clinical cancer therapy Cancer Chemother Pharmacol 78 2016 661 671 27646791 10.1007/s00280-016-3152-1

148 Emadi A. Jones R.J. Brodsky R.A. Cyclophosphamide and cancer: golden anniversary Nat Rev Clin Oncol 6 2009 638 647 19786984 10.1038/nrclinonc.2009.146

149 Bracci L. Moschella F. Sestili P. La Sorsa V. Valentini M. Canini I. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration Clin Cancer Res 13 2007 644 653 17255288 10.1158/1078-0432.CCR-06-1209

150 Meermeier E.W. Welsh S.J. Sharik M.E. Du M.T. Garbitt V.M. Riggs D.L. Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy Blood Cancer Discov 2 2021 354 369 34258584 10.1158/2643-3230.BCD-21-0038 PMC8266040

151 Oostindie S.C. Lazar G.A. Schuurman J. Parren P.W.H.I. Avidity in antibody effector functions and biotherapeutic drug design Nat Rev Drug Discov 21 2022 715 735 35790857 10.1038/s41573-022-00501-8 PMC9255845

152 Birrer M.J. Moore K.N. Betella I. Bates R.C. Antibody–drug conjugate-based therapeutics: state of the science J Natl Cancer Inst 111 2019 538 549 30859213 10.1093/jnci/djz035

153 Tsuchikama K. An Z. Antibody–drug conjugates: recent advances in conjugation and linker chemistries Protein Cell 9 2018 33 46 27743348 10.1007/s13238-016-0323-0 PMC5777969

154 Rallis K.S. Lai Yau T.H. Sideris M. Chemoradiotherapy in cancer treatment: rationale and clinical applications Anticancer Res 41 2021 1 7 33419794 10.21873/anticanres.14746

155 Sgouros G. Bodei L. McDevitt M.R. Nedrow J.R. Radiopharmaceutical therapy in cancer: clinical advances and challenges Nat Rev Drug Discov 19 2020 589 608 32728208 10.1038/s41573-020-0073-9 PMC7390460

156 Chan G.C. Chan C.M. Anti-GD2 directed immunotherapy for high-risk and metastatic neuroblastoma Biomolecules 12 2022 358 35327550 10.3390/biom12030358 PMC8945428

157 Zirngibl F. Ivasko S.M. Grunewald L. Klaus A. Schwiebert S. Ruf P. GD2-directed bispecific trifunctional antibody outperforms dinutuximab beta in a murine model for aggressive metastasized neuroblastoma J Immunother Cancer 9 2021 e002923 10.1136/jitc-2021-002923 PMC8292814 34285106

158 Deppisch N. Ruf P. Eissler N. Neff F. Buhmann R. Lindhofer H. Efficacy and tolerability of a GD2-directed trifunctional bispecific antibody in a preclinical model: subcutaneous administration is superior to intravenous delivery Mol Cancer Therapeut 14 2015 1877 1883 10.1158/1535-7163.MCT-15-0156 26063765

159 Zhai D. An D. Wan C. Yang K. Radiotherapy: brightness and darkness in the era of immunotherapy Transl Oncol 19 2022 101366 10.1016/j.tranon.2022.101366 PMC8881489 35219093

160 Harrington K. Freeman D.J. Kelly B. Harper J. Soria J.C. Optimizing oncolytic virotherapy in cancer treatment Nat Rev Drug Discov 18 2019 689 706 31292532 10.1038/s41573-019-0029-0

161 Kaufman H.L. Kohlhapp F.J. Zloza A. Oncolytic viruses: a new class of immunotherapy drugs Nat Rev Drug Discov 14 2015 642 662 26323545 10.1038/nrd4663 PMC7097180

162 Hemminki O. Dos Santos J.M. Hemminki A. Oncolytic viruses for cancer immunotherapy J Hematol Oncol 13 2020 84 32600470 10.1186/s13045-020-00922-1 PMC7325106

163 Tian Y. Xie D. Yang L. Engineering strategies to enhance oncolytic viruses in cancer immunotherapy Signal Transduct Targeted Ther 7 2022 117 10.1038/s41392-022-00951-x PMC8987060 35387984

164 Ma R. Li Z. Chiocca E.A. Caligiuri M.A. Yu J. The emerging field of oncolytic virus-based cancer immunotherapy Trends Cancer 9 2023 122 139 36402738 10.1016/j.trecan.2022.10.003 PMC9877109

165 Wang Q. Ma X. Wu H. Zhao C. Chen J. Li R. Oncolytic adenovirus with MUC16-BiTE shows enhanced antitumor immune response by reversing the tumor microenvironment in PDX model of ovarian cancer OncoImmunology 11 2022 2096362 10.1080/2162402X.2022.2096362 PMC9255048 35800156

166 Speck T. Heidbuechel J.P.W. Veinalde R. Jaeger D. von Kalle C. Ball C.R. Targeted BiTE expression by an oncolytic vector augments therapeutic efficacy against solid tumors Clin Cancer Res 24 2018 2128 2137 29437789 10.1158/1078-0432.CCR-17-2651

167 Leclercq G. Haegel H. Toso A. Zimmermann T. Green L. Steinhoff N. JAK and mTOR inhibitors prevent cytokine release while retaining T cell bispecific antibody in vivo efficacy J Immunother Cancer 10 2022 e003766 10.1136/jitc-2021-003766 PMC8785208 35064010

168 Burger J.A. Bruton tyrosine kinase inhibitors: present and future Cancer J 25 2019 386 393 31764119 10.1097/PPO.0000000000000412 PMC7083517

169 Pal Singh S. Dammeijer F. Hendriks R.W. Role of Bruton's tyrosine kinase in B cells and malignancies Mol Cancer 17 2018 57 29455639 10.1186/s12943-018-0779-z PMC5817726

170 Mhibik M. Gaglione E.M. Eik D. Kendall E.K. Blackburn A. Keyvanfar K. BTK inhibitors, irrespective of ITK inhibition, increase efficacy of a CD19/CD3-bispecific antibody in CLL Blood 138 2021 1843 1854 34046681 10.1182/blood.2020009686 PMC8586964

171 Long M. Beckwith K. Do P. Mundy B.L. Gordon A. Lehman A.M. Ibrutinib treatment improves T cell number and function in CLL patients J Clin Invest 127 2017 3052 3064 28714866 10.1172/JCI89756 PMC5531425

172 Niemann C.U. Herman S.E. Maric I. Gomez-Rodriguez J. Biancotto A. Chang B.Y. Disruption of in vivo chronic lymphocytic leukemia tumor-microenvironment interactions by ibrutinib-findings from an investigator-initiated phase II study Clin Cancer Res 22 2016 1572 1582 26660519 10.1158/1078-0432.CCR-15-1965 PMC4818677

173 Kondo K. Shaim H. Thompson P.A. Burger J.A. Keating M. Estrov Z. Ibrutinib modulates the immunosuppressive CLL microenvironment through STAT3-mediated suppression of regulatory B-cell function and inhibition of the PD-1/PDL1 pathway Leukemia 32 2018 960 970 28972595 10.1038/leu.2017.304 PMC6128536

174 Zou Y.X. Zhu H.Y. Li X.T. Xia Y. Miao K.R. Zhao S.S. The impacts of zanubrutinib on immune cells in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma Hematol Oncol 37 2019 392 400 31420873 10.1002/hon.2667

175 Köhnke T. Krupka C. Tischer J. Knösel T. Subklewe M. Increase of PD-L1 expressing B-precursor ALL cells in a patient resistant to the CD19/CD3-bispecific T cell engager antibody blinatumomab J Hematol Oncol 8 2015 111 26449653 10.1186/s13045-015-0213-6 PMC4599591

176 Kosmaczewska A. Ciszak L. Suwalska K. Wolowiec D. Frydecka I. CTLA-4 overexpression in CD191/CD51 cells correlates with the level of cell cycle regulators and disease progression in B-CLL patients Leukemia 19 2005 301 304 15549146 10.1038/sj.leu.2403588

177 Belmontes B. Sawant D.V. Zhong W. Tan H. Kaul A. Aeffner F. Immunotherapy combinations overcome resistance to bispecific T cell engager treatment in T cell-cold solid tumors Sci Transl Med 13 2021 eabd1524 10.1126/scitranslmed.abd1524 34433637

178 Martinez-Cannon B.A. Castro-Sanchez A. Barragan-Carrillo R. de la Rosa Pacheco S. Platas A. Fonseca A. Adherence to adjuvant tamoxifen in mexican young women with breast cancer Patient Prefer Adherence 15 2021 1039 1049 34040357 10.2147/PPA.S296747 PMC8141391

179 Liu D. Bao L. Zhu H. Yue Y. Tian J. Gao X. Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy J Control Release 354 2023 606 614 36669532 10.1016/j.jconrel.2023.01.041

180 Cattaruzza F. Nazeer A. To M. Hammond M. Koski C. Liu L.Y. Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors Nat Cancer 4 2023 485 501 36997747 10.1038/s43018-023-00536-9 PMC10132983

181 Seckinger A. Majocchi S. Moine V. Nouveau L. Ngoc H. Daubeuf B. Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers J Hematol Oncol 16 2023 117 38087365 10.1186/s13045-023-01516-3 PMC10717981

182 Strohl W.R. Naso M. Bispecific T-cell redirection versus chimeric antigen receptor (CAR)-T cells as approaches to kill cancer cells Antibodies 8 2019 41 31544847 10.3390/antib8030041 PMC6784091

183 Li J. Piskol R. Ybarra R. Chen Y.J. Li J. Slaga D. CD3 bispecific antibody-induced cytokine release is dispensable for cytotoxic T cell activity Sci Transl Med 11 2019 eaax8861 10.1126/scitranslmed.aax8861 31484792

184 Norelli M. Camisa B. Barbiera G. Falcone L. Purevdorj A. Genua M. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells Nat Med 24 2018 739 748 29808007 10.1038/s41591-018-0036-4

185 Schildberger A. Rossmanith E. Eichhorn T. Strassl K. Weber V. Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide Mediat Inflamm 2013 2013 697972 10.1155/2013/697972 PMC3681313 23818743

186 Iwata Y. Sasaki M. Harada A. Taketo J. Hara T. Akai S. Daily ascending dosing in cynomolgus monkeys to mitigate cytokine release syndrome induced by ERY22, surrogate for T-cell redirecting bispecific antibody ERY974 for cancer immunotherapy Toxicol Appl Pharmacol 379 2019 114657 10.1016/j.taap.2019.114657 31326447

187 Trinklein N.D. Pham D. Schellenberger U. Buelow B. Boudreau A. Choudhry P. Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies mAbs 11 2019 639 652 30698484 10.1080/19420862.2019.1574521 PMC6601548

188 Vafa O. Trinklein N.D. Perspective: designing T-cell engagers with better therapeutic windows Front Oncol 10 2020 446 32351885 10.3389/fonc.2020.00446 PMC7174563

189 Wu Z. Cheung N.V. T cell engaging bispecific antibody (T-cell redirecting BsAb): from technology to therapeutics Pharmacol Ther 182 2018 161 175 28834699 10.1016/j.pharmthera.2017.08.005 PMC5785550

190 Peters I.T. Hilders C.G. Sier C.F. Vahrmeijer A.L. Smit V.T. Baptist Trimbos J. Identification of cell-surface markers for detecting breast cancer cells in ovarian tissue Arch Gynecol Obstet 294 2016 385 393 26946151 10.1007/s00404-016-4036-7 PMC4937067

191 Sulea T. Rohani N. Baardsnes J. Corbeil C.R. Deprez C. Cepero-Donates Y. Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment mAbs 12 2020 1682866 10.1080/19420862.2019.1682866 PMC6927761 31777319

Acknowledgments

This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (Nos. 32070940 and 81991491), the 10.13039/501100002858 China Postdoctoral Science Foundation (No. 2021M700115), the Postdoctoral Innovation Talents Support Program (No. BX20220189, China), the Science and Technology Planning Project of Fujian Province (No.2022L3080, China), the CAMS Innovation Fund for Medical Sciences (No. 2019RU022, China). We also thank some materials in the graphical abstract and figures that are produced by BioRender ( https://biorender.com ).

Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.