Bispecific antibodies and nanotechnology: a strategic alliance in cancer immunotherapy.

Introduction

Bispecific antibody (bsAb)-based immunotherapeutics have gained significant attention in cancer immunotherapy for their versatility and ability to achieve therapeutic effects that may surpass those of conventional antibodies, considering their potential to overcome resistance and enhance target specificity. The strength of bsAbs lies in their capacity to simultaneously bind two distinct antigens or epitopes, enabling a multitargeting approach. This feature is especially valuable for addressing the complexity of diseases involving multiple receptors, ligands or signaling pathways. The growing number of approved bsAbs (Table 1 ), along with a robust pipeline of over 200 candidates in clinical development [ 1 ], underscores their great potential in cancer therapy and diagnosis.

Table 1 BsAbs approved by EMA and/or FDA or currently in fast-track designation for cancer immunotherapy Fist approval Trade Name BsAb name Targets Type Indication 2009 (withdrawn 2017) Removab Catumaxomab EpCAM x CD3 TCE Ovarian ascites 2014 Blincyto Blinatumomab CD19 x CD3 TCE Philadelphia chromosome-negative relapsed or refractory B cell precursor acute lymphoblastic leukaemia 2021 Rybrevant Amivantamab EGFR x cMet Signaling inhibitor Locally advanced or metastatic non-small cell lung cancer 2022 Kimmtrak Tebentafusp gp100-HLA-A*02 × CD3 TCE Unresectable or metastatic uveal melanoma 2022 Tecvayli Teclistamab BCMA x CD3 TCE Relapsed or refractory multiple myeloma 2022 (China) Kaitanni Cadonilimab PD-L1 x CTLA-4 Checkpoint inhibitor Hepatocellular carcinoma 2022 Lunsumio Mosunetuzumab CD20 x CD3 TCE Relapsed or refractory follicular lymphoma 2023 Epkinly Epcoritamab CD20 x CD3 TCE Relapsed or refractory diffuse large B-cell lymphoma 2023 Columvi Glofitamab CD20 x CD3 TCE Relapsed or refractory diffuse large B-cell lymphoma or large B-cell lymphoma 2023 Elrexfio Elranatamab BCMA x CD3 TCE Relapsed or refractory multiple myeloma 2023 Talvey Talquelamab GPRC5D x CD3 TCE Relapsed or refractory multiple myeloma 2024 Imdelltra Tarlatamab DLL3 x CD3 TCE Small cell lung cancer 2024 Ziihera Zanidatamab HER2 x HER2 Signaling inhibitor HER2-positive biliary tract cancer 2024 Bizengri Zenocutuzumab HER2 x HER3 Signaling inhibitor Non-small cell lung cancer and pancreatic adenocarcinoma 2024 (EU) Ordspono Odronextamab CD20 x CD3 TCE Relapsed/refractory follicular lymphoma or diffuse large B-cell lymphoma 2024 FDA Fast-track designation - Linvosentamab BCMA x CD3 TCE Relapsed or refractory multiple myeloma 2024 FDA Fast-track designation - Ivonescimab PD-1 x VEGF Signaling/ Checkpoint inhibitor Non-small cell lung cancer 2024 FDA Fast-track designation - PT217 DDL3 x CD47 Signaling inhibitor Small cell lung cancer and neuroendocrine prostate cancer 2024 FDA Fast-track designation - CTX-009 DLL4 x VEGFA Signaling inhibitor Metastatic or locally advanced biliary tract cancer 2024 FDA Fast-track designation - IBI363 PD-1 x IL2 Signaling/ Checkpoint inhibitor Unresectable locally advanced or metastatic melanoma BCMA B-cell maturation antigen, CD cluster of differentiation, cMET tyrosine kinase mesenchymal–epithelial transition, CTLA-4 cytotoxic T-lymphocyte–associated antigen 4, DLL delta-like ligand, EGFR epidermal growth factor receptor, epcam epithelial cell adhesion molecule, gp100 glycoprotein 100, GPRC5D G protein–coupled receptor class C group 5 member D, HER human epidermal growth factor receptor, HLA-A human leukocyte antigen A, IL2 Interleukin 2, PD-1 programmed death 1, PD-L1 programmed death ligand 1, TCE T cell engager, VEGFA vascular endothelial growth factor A

BsAbs are available in a variety of formats [ 2 ] and can mediate anticancer effects through several molecular mechanisms of action (MoA). These MoA, reviewed below, include delivering therapeutic payloads to a specific target (“targeters”), bridging two cell types (“engagers”), or engaging two molecules on the same cell membrane or in the tumor microenvironment (“enhancers”) (Fig. 1 ) [ 1 , 3 ]. Fig. 1 Mechanisms of action of bsAbs. Functional classification of bispecific antibodies (bsAbs) in cancer therapy. “Targeters”: direct therapeutic agents (e.g., drugs, nanoparticles, radiotherapeutics) to tumor-associated antigens (TAA). “Engagers”: recruit immune effector cells to malignant cells. “Enhancers”: modulate the tumor microenvironment by targeting cytokines, angiogenic factors, receptors, or immune checkpoints. Created in https://BioRender.com

“Targeters”

Targeted delivery of payloads, including chemotherapeutics or radiotherapeutics, and nanoparticles (NPs) for cancer treatment or diagnosis can be facilitated by the bsAb technology [ 4 ]. Their multi-binding capability can be leveraged in both targeting or pre-targeting strategies, where one arm can bind the cargo while the other directs it to a tumor-associated antigen (TAA), or both arms can engage distinct TAAs to enhance tumor specificity. In addition, bsAbs can enhance payload internalization and intracellular trafficking by targeting transmembrane proteins, such as the prolactin receptor (PRLR), CD63, or cytokine receptors, thereby increasing uptake into tumor cells [ 5 ]. These approaches are being investigated to improve the efficacy of radiotheranostics, bsAb-drug conjugates, and nanomedicines, particularly in heterogeneous tumor populations.

“Engagers”

Multiple bsAbs have been developed to bring two cell types together, primarily to transiently connect cancer cells with cytotoxic T cells. Bispecific T cell engagers (TCEs) accomplish this dual targeting by simultaneously binding to a selected TAA on the cancer cell surface and to the extracellular CD3 subunit on the T cell surface, thereby inducing specific T cell-mediated tumor cell killing. As shown in Table 1 , most approved TCEs are indicated for hematological malignancies and have shown their ability to target CD19 in B-cell precursor acute lymphoblastic leukemia (B-ALL), CD20 in non-Hodgkin lymphoma, B-cell maturation antigen (BCMA), and G protein–coupled receptor GPRC5D in multiple myeloma. A key factor in their success is that, although these targets are present on regular blood cells, their depletion can be tolerated without causing severe adverse effects. Progress with TCEs in the treatment of solid tumors has been slower, likely due to the heterogeneity of tumor antigens and the immunosuppressive nature of the tumor microenvironment (TME) [ 6 ]. In this context, the TCE Tebentafusp has shown a significant improvement in overall survival in patients with solid tumors. This effect is mediated by the recognition of a peptide fragment of gp100, an intracellular melanoma-associated antigen, presented by HLA-A2:01 molecules on the surface of cancer cells [ 7 ]. While T cells have dominated the field of cell-engagers, significant efforts are underway to develop next-generation bsAbs engaging other immune cells such as natural killer (NK) cells, dendritic cells, neutrophils, or macrophages/monocytes [ 8 ].

“Enhancers”

Another application of bsAbs in cancer therapy is related to the restoration and enhancement of antitumor immunity. Among these approaches, bsAbs can block two interrelated signaling pathways by targeting two epitopes on tumor cells or in the TME. The antigen pairs may include angiogenic factors, tyrosine kinase receptors, cytokines, or cytokine receptors [ 9 ]. An example of the success of this approach is illustrated by Amivantamab (Rybrevant), which targets epidermal growth factor (EGFR) and tyrosine kinase receptors (c-MET) (EGFR x c-MET) and was approved in May 2021 for non-small cell lung cancer treatment [ 10 ]. Another common design principle is the simultaneous targeting of two immune checkpoints, thereby reducing the probability of resistance, and potentially achieving better efficacy compared to monotherapy in solid tumor treatment [ 11 ]. Cadonilimab, a programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) (PD-L1 x CTLA-4) blocker, became the first dual immune checkpoint inhibitor bsAb. It was approved in China in 2022 for the treatment of relapsed or metastatic cervical cancer [ 12 ]. Most of the recent fast-track designations are for this class of bsAbs, illustrating their active investigation and progress toward clinical application. Novel combinations are also arising, including the anti-PD-1/interleukin-2 (IL-2) bsAb fusion protein, IBI363, that achieves simultaneous PD-1 blockade and IL-2 delivery for melanoma treatment.

This review explores how advances in nanotechnology and bsAb technology can synergistically benefit cancer therapy (Fig. 2 ). First, we examine the potential of bsAbs as active targeting agents in nanomedicine to improve tumor specificity. We then discuss how nanotechnology can overcome key limitations of bsAb therapies, including limited half-life and instability, ultimately advancing their clinical translation.

Fig. 2 Synergies between bsAbs and nanotechnology in cancer therapy. Nanomedicine strategies leveraging bsAbs include: bsAbs as active targeting ligands to direct nanocarriers to TAAs (left), therapeutic bsAbs incorporated into or assembled with nanocarriers aimed to increase bsAb therapeutic efficacy (middle), mRNA-loaded nanocarriers encoding bsAbs enabling gene or cell therapy approaches (right). Created in https://BioRender.com

BsAbs as targeting agents for drug-loaded nanocarriers

A major limitation to the antitumor efficacy of drug-loaded NPs is their off-target accumulation in organs such as the liver and spleen [ 13 , 14 ]. While monoclonal antibodies (mAbs) have been widely employed as targeting ligands, antibody-conjugated NPs have yet to reach clinical translation [ 15 ]. In this context, the multi-specificity of bsAbs may offer additional advantages. By simultaneously binding two distinct TAAs, bsAbs can increase tumor specificity and NP retention. Moreover, incorporating a bsAb domain that recognizes a NP moiety can streamline NP functionalization [ 16 ].

Various types of NPs functionalized with bsAbs have been investigated, including liposomes, lipid nanoparticles (LNPs), and polymeric NPs. These formulations are primarily designed for the targeted delivery and protection of therapeutic nucleic acids, or cytotoxic agents like doxorubicin and docetaxel, linked to significant side effects. An overview of these strategies is presented in Table 2 . Approaches for active targeting using bsAbs are discussed in two main categories: bsAb conjugation to the NP surface and pre-targeting delivery.

Table 2 BsAb-based active targeting strategies in nanomedicine Nanocarrier Payload Ab format Targets NP functionalization Tumor model Ref Liposome - bispecific sdAb HER2 (bivalent) Covalent (maleimide-thiol) HER-positive cancer cell lines [ 17 ] Manganese-doped iron oxide NP Fluorescent dye (CyTE777) bsAb HER2 x EGFR Covalent (EDC/NHS) Breast and colorectal cancer [ 18 ] PEGylated liposome Doxorubicin bsAb HER2 x PEG EGFR x PEG Non-covalent Colorectal cancer [ 19 ] PEGylated liposome Doxorubicin bsAb HER2 x PEG Non-covalent Ovarian cancer [ 20 ] PEGylated liposome Doxorubicin bsAb HER2 x PEG Non-covalent Breast cancer [ 21 ] PEGylated liposome Doxorubicin bsAb CD20 x PEG CD22 x PEG CD38 x PEG Non-covalent Leukemia [ 22 ] PEG NP - bsAb EGFR x PEG Non-covalent Breast cancer [ 23 , 24 ] Polymeric NP Doxorubicin bsAb HER2 x PEG Non-covalent Breast cancer brain metastasis [ 25 ] Polymeric NP Doxorubicin bsAb Ephrin A2 x PEG Non-covalent Brain cancer [ 26 ] mPEGylated lecithin micelle Docetaxel bsAb DNS x PEG HER2 x PEG Non-covalent Breast cancer [ 27 ] Micelle Docetaxel bsAb and TrAb EGF3 x PEG FAP x PEG EGF3 x FAP x PEG Non-covalent Pancreatic cancer [ 28 ] PEGylated nano-emulsion Docetaxel and Pictilisib bsAb HER x PEG HER-IV x PEG Non-covalent Breast cancer [ 29 ] Polymeric NP Doxorubicin, camptothecin and imaging tracer (Cy5) bsAb EGFR x PEG Non-covalent Breast cancer [ 30 ] LNP mRNA encoding PE38 bsAb GRP78 x PEG Non-covalent Hepatocellular carcinoma [ 31 ] LNP siRNA encoding PLK1 bsAb EGFR x PEG Non-covalent High-risk neuroblastoma [ 32 ] LNP Model mRNA bsAb PD-L1 x HA CD4 x HA CD5 x HA Non-covalent - [ 33 ] PEGylated liposome Doxorubicin bsAb EGFR x PEG Pre-targeting Breast cancer [ 34 ] PEGylated liposome Doxorubicin bsAb HER2 x PEG Pre-targeting Breast cancer [ 35 ] Polystyrene NP Doxorubicin bsAb HER2 x PEG Pre-targeting Breast cancer [ 36 ] PEGylated liposome Doxorubicin bsAb CD20 x PEG Pre-targeting Leukemia [ 37 ] LNP Model mRNA bsAb EGFR x PEG Non-covalent and pre-targeting Breast cancer [ 38 ] bsab bispecific antibody, bsabs bispecific antibodies, CD cluster of differentiation, DNS Dansyl (negative control), EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EGF epidermal growth factor, FAP fibroblast activation protein, GRP78 glucose-regulated protein 78, HER human epidermal growth factor receptor, LNP lipid nanoparticle, mRNA messenger RNA, NHS N-Hydroxy succinimide, PEG polyethylene glycol, PLK1 polo-like kinase 1, NP nanoparticle, sirna small interfering RNA, sdab single domain antibody, trab trispecific antibody

Functionalization of NPs with BsAbs

Among the different approaches for bsAb functionalization of NPs (Fig. 3 ) covalent functionalization has been scarcely explored. This is likely due to the need of antibody modification, as well as specific pH and media conditions, which may compromise both antibody and nanocarrier stability [ 39 ].

Fig. 3 BsAbs as active targeting agents in nanomedicine. BsAbs used as targeting agents to functionalize drug-loaded nanocarriers either covalently, through non-covalent antigen-antibody interactions or allowing pre-targeted strategies. Created in https://BioRender.com

Despite these technical hurdles, liposomes functionalized with bivalent single-domain (sd) bispecific anti-HER antibodies have shown ~ 10-fold higher binding affinity to human epidermal growth factor receptor 2 (HER2) and up to 3-fold increased cellular uptake in HER-positive cancer cell lines compared to monospecific formats [ 17 ]. Covalent bsAb functionalization has also been applied in molecular imaging. For instance, iron oxide NPs functionalized with HER x EGFR bsAbs using carbodiimide chemistry enabled MRI-based tumor imaging in mouse models [ 18 ]. Although still limited, innovative approaches such as chemo-enzymatic methods are being explored for bispecific sdAb attachment to NPs [ 40 ].

Non-covalent strategies based on bsAbs with a domain specific towards NP surface moieties may facilitate NP functionalization. Typically, the anti-NP domain targets polyethylene glycol (PEG) on NP surfaces. Meanwhile, the second (or third, in case of trispecific Abs) binding domain is used to target cancer cells. This approach has been applied to several PEGylated nanocarriers, including liposomes, micelles, nanoemulsions, polymeric NPs, quantum dots and AuNPs [ 24 – 30 , 41 ] and, also, to approved PEGylated nanomedicines such as liposomal doxorubicin (Doxil, Caelyx) [ 21 , 22 , 42 , 43 ]. Preclinical studies showed that mixing anti-PEG x TAA bsAbs with Doxil enhanced tumor accumulation and therapeutic efficacy across various models, including ovarian, breast, colorectal cancers, and leukemia [ 19 , 21 , 22 ]. For example, intravenous injection of anti-PEG x HER2 bsAbs-targeted Doxil into mice bearing ovarian adenocarcinoma tumors, significantly increased the tumor fluorescence uptake signal by up to 240% at 72 h post treatment compared with isotype control bsAbs-Doxil. Moreover, HER2⁺ tumor growth was significantly inhibited compared with both isotype control bsAbs-Doxil and unmodified Doxil. Based on this conceptual evidence, there is increasing interest in implementing this strategy to improve the targeted delivery of therapeutic nucleic acids through non-covalently functionalized LNPs [ 31 – 33 , 38 ].

Several studies have addressed key technical aspects of bsAb-functionalized NPs, providing insights into how bsAb surface density, PEG architecture, and multi-bsAb conjugation influence NP behaviour and offer strategies to maximize therapeutic outcomes.

The impact of anti-PEG bsAb density on the in vivo NP behavior was studied, for example, using PEGylated mesoporous silica NPs. BsAbs anti-mPEG x EGFR were attached via incubation and indirectly quantified by fluorescence, yielding 45 to 173 bsAbs per particle. Notably, radiolabelled particles with lower bsAb surface density showed a non-significant trend toward improved tumor accumulation, together with a significant reduction in spleen accumulation upon intravenous administration in mice bearing breast cancer tumors [ 24 ]. This may reflect that a high functionalization degree compromises stealth properties. Hence, accurate quantification of bsAbs per particle is crucial for elucidating correlations between bsAb density and tumor accumulation. However, the existing literature lacks rigorous characterization, as most quantification methods are indirect and parameters such as ligand density, orientation, and stability in physiological media are often not reported, leading to a poor correlation between bsAb number and tumor targeting or therapeutic outcome.

Several studies have highlighted the critical role of PEG density and architecture on NP functionalization via PEG recognition. Lecithin micelles prepared with DSPE-PEG2K or DSPE-PEG5K were compared for cellular uptake following bsAb (anti HER or DNS x PEG) binding. Here, longer PEG chains (5 K) improved uptake by ~ 25% compared to shorter chains (2 K), as measured by mean fluorescence intensity in flow cytometry using NPs loaded with a fluorescent dye, likely due to better display of the targeting agent [ 27 ]. Another study that made use of hyperbranched mPEG in PEG-based polymeric NPs, concluded that this structure facilitated the binding of anti-PEG bsAbs and, ultimately, in vivo efficacy in a breast cancer model. However, because no direct comparison between hyperbranched and linear mPEG was performed, no conclusions regarding their relative performance can be drawn [ 23 ]. The effect of different PEG-lipid anchors for bsAbs anti-PEG x anti-EGFR to Polo-Like Kinase 1 (PLK1) siRNA-LNPs was also investigated [ 32 ]. While all LNPs accumulated predominantly in the liver, tumor accumulation at 48 h post-administration showed a 4.3-fold increase for DSPE-PEG LNPs and a 1.9-fold increase for DMG-PEG LNPs, relative to the vehicle control. Despite lower tumor accumulation, DMG-PEG LNPs achieved greater therapeutic efficacy, reducing tumor size by 42.6% ( P < 0.05) after 22 days in a neuroblastoma xenograft model. This might be explained by faster desorption of DMG-PEG, resulting in improved siRNA transfection. This illustrates the importance of balancing target accumulation with delivery efficiency. Collectively, these results underscore the importance of PEGylation parameters, such as chain length, branching and release kinetics, to fine-tune bsAb interaction and optimize therapeutic outcomes.

Another line of research has been the conjugation of multiple anti-TAA x PEG bsAbs to NPs. This strategy is especially promising in cases of loss of TAAs, a common resistance mechanism where tumor cells reduce or internalize specific surface markers and thereby evade recognition. Nanotechnology provides opportunities to mitigate this challenge by incorporating multiplexed targeting strategies into NP design by conjugating multiple anti-TAA×PEG bsAbs to the same platform, thereby broadening antigen recognition and maintaining therapeutic efficacy even in heterogeneous tumor environments. For example, Caelyx ® (a PEGylated liposome) was functionalized with single bsAbs (bsAb-Caelyx) or multiple bsAbs (Trio-Caelyx) to improve doxorubicin delivery in leukemia. In ALL-19 model of chemo-resistant B-cell acute lymphoblastic leukemia, Trio-Caelyx (functionalized with anti-CD19 x PEG, anti-CD22 x PEG, and anti-CD38 x PEG) improved event-free survival by 3.3-fold compared to PBS control. However, bsAb-Caelyx targeting CD22 alone achieved a 4.2-fold increase, outperforming Trio-Caelyx. Notably, a significantly greater accumulation of Trio-Caelyx was observed in the liver, suggesting that the addition of multiple bsAbs may enhance opsonization and clearance by the mononuclear phagocyte system [ 22 ]. However, in some studies it has been shown that the addition of multiple targeting ligands to NPs does not significantly enhance drug accumulation in the tumor [ 29 ], highlighting the need for comprehensive in vivo studies across different tumor models to assess their advantages over single anti-TAA strategies.

While PEGylated NPs have dominated the field of targeted cancer therapeutics, the mounting evidence of PEG-associated immunogenicity and reduced efficacy upon repeated administration has prompted investigation of alternative surface modification strategies [ 44 , 45 ]. PEG alternatives, including zwitterionic polymers [ 46 ] or polysarcosines [ 47 ], or extracellular vesicle (EV)-derived membranes as NP coating material have been used to reduce PEG-associated immunogenicity [ 48 , 49 ]. However, such alternatives are yet to be explored in the context of NP bsAb therapeutics. Recently, biotechnological engineering of dendritic cells enabled the generation of bispecific EVs co-expressing anti-CD19 scFv and PD1, thereby combining tumor antigen targeting with immune checkpoint blockade [ 50 ]. Such pre-functionalized EVs may represent a promising avenue in the development of bsAb-functionalized NPs.

Pre-targeted delivery

Pre-targeted delivery strategies involve a two-step process. First, bsAbs are administered intravenously to bind TAAs on cancer cells; second, drug-loaded PEGylated nanocarriers are administered intravenously, designed to interact with anti-mPEG domains on pre-localized bsAbs [ 36 ]. This strategy has been successfully applied in clinical trials to enhance the delivery of radiolabelled peptides for cancer imaging and therapy [ 51 ]. This concept is also being applied in NP delivery, specifically to enhance the accumulation and payload release of PEGylated liposomal doxorubicin in different cancer cell types (Fig. 3 , right) [ 52 ]. The efficacy of this approach has been shown in triple-negative breast cancer using anti-PEG x EGFR bsAbs [ 34 ], in HER2-positive breast adenocarcinoma with anti-HER2 x anti-mPEG bsAbs [ 35 , 36 ], and in lymphoma with anti-CD20 x anti-mPEG bsAbs [ 37 ]. Therefore, this strategy holds the potential to broaden the application of FDA-approved PEGylated therapeutics to different tumor types without the need for drug re-engineering. This is highly promising; however, the timing and pharmacokinetic coordination of the two-step administration are critical and must be carefully optimized in clinical settings.

A recent study compared non-covalent functionalization and pre-targeting strategies using anti-PEG x HER bsAbs, analyzing the biodistribution of luciferase mRNA-LNPs in mice bearing EGFR + human tumor xenografts [ 38 ]. The pre-targeting strategy, with temporal separation of bsAb and LNP administration, resulted in a comparable increase in mRNA expression in tumor tissue (7-fold) relative to covalent functionalization (8-fold), with pre-targeting maintaining sustained expression over 48 h, exhibiting only a 10% decrease compared to a 60% reduction observed with covalent functionalization. Moreover, pre-targeting achieved a greater reduction in off-target delivery. These differences were linked to altered physicochemical properties of LNPs, including a shift from positive to negative surface charge as well as abundant protein corona formation in pre-functionalized LNPs.

While conjugation provides a one-step approach, bsAb attachment modifies nanocarrier surface properties, alters the resulting protein corona, and complicates manufacturing by requiring highly consistent conjugation chemistry. In contrast, pre-targeting strategies can reduce off-target delivery; however, their two-step administration introduces clinical challenges, including greater procedural complexity. Covalent systems are generally more reproducible across batches, while pre-targeting approaches may display higher variability in pharmacokinetics. Thus, selecting between these strategies involves carefully balancing efficacy, safety, reproducibility, and clinical feasibility.

Enhancing targeting with BsAbs in nanomedicine. Critical considerations and future directions

The functionalization of NPs with bsAbs has been reported exclusively at the preclinical level, where studies consistently demonstrate enhanced antitumor efficacy. This observation is primarily attributed to increased tumor accumulation and prolonged payload retention. However, their impact on overall NP biodistribution and systemic toxicity remains unclear. Biodistribution studies in tumor models following intravenous administration have generally failed to significantly modify the distribution pattern, although they have provided an increased accumulation in the tumor tissue [ 20 , 27 , 29 , 36 ]. This suggests that, while bsAb functionalization improves tumor accumulation, off-target distribution and associated side-effects may persist. A commonly investigated systemic side effect of nanomedicines is chemotherapy-derived cardiotoxicity, a dose-limiting factor that significantly impacts both treatment response and quality of life [ 53 ]. Among the limited studies that have systematically evaluated this side effect, most have not observed a significant improvement upon functionalization with bsAbs [ 21 , 26 , 30 ]. This suggests that functionalization alone may be insufficient to mitigate the principal systemic side-effects of nanomedicines. In contrast, polymeric NPs loaded with doxorubicin and functionalized with bsAbs (anti-HER3 x PEG) showed significantly reduced heart accumulation and cardiotoxicity [ 25 ]. These findings suggest the need for future studies to comprehensively determine biodistribution and systemic toxicity to assess whether bsAb-functionalized NPs can truly overcome these challenges. In fact, some studies suggests that bsAb functionalization does not markedly alter systemic biodistribution, potentially due to rapid bsAb desorption, protein corona masking, or suboptimal ligand orientation and density. Such factors may limit effective target engagement during circulation, underscoring the importance of optimizing surface engineering to enable more selective and safer NP delivery.

BsAbs as therapeutic agents incorporated in nanocarriers

The anticipated widespread use of bsAb therapeutics presents significant challenges for clinical implementation, particularly due to their short plasma half-life and associated adverse effects such as cytokine release syndrome (CRS), neurotoxicity, cytopenia, and infections [ 54 ]. In terms of bsAb design, multiple strategies are being pursued to mitigate bsAb-related toxicities, particularly in TCE therapies. A key approach is the fine-tuning of anti-CD3 affinity to reduce excessive T cell activation and thereby minimize CRS [ 55 , 56 ]. Building on this, recent advances in tri- and tetra-specific antibody (tsAb, ttsAb) engineering seek to overcome different limitations by incorporating: (i) recognition of multiple tumor epitopes to enhance avidity and reduce antigen escape [ 57 ], (ii) agonistic or costimulatory domains (e.g., 4-1BBL, OX40L, CD28) to sustain T-cell function and prevent exhaustion, and (iii) cytokine-blocking modules (e.g., IL-6R or IL-1R antagonists) to dampen inflammatory cascades and reduce CRS risk [ 57 , 58 ]. Another promising avenue is the development of conditionally active TCEs, which remain inactive until activated within the TME, either through protease cleavage, pH-sensitivity, or controlled-release prodrug designs. Such approaches are intended to restrict T cell engagement to tumor sites, thereby improving safety and preventing off-tumor toxicities [ 59 – 62 ]. Collectively, these innovations highlight how rational bsAb engineering is reshaping the therapeutic index of next-generation TCEs.

The short plasma half-life of small bsAbs often requires continuous infusion to maintain therapeutic levels. For example, Blinatumomab is administered via continuous intravenous infusion through an implantable pump for 4 weeks [ 63 ]. Strategies to prolong bsAb half-life include genetic fusion or chemical conjugation to long-circulating proteins like IgG or albumin [ 64 ], as well as the synthesis of bsAb-polymer conjugates with materials like PEG or poly-ADP-ribose [ 65 , 66 ]. Local administration may also improve efficacy in certain clinical contexts. A feasible approach involves solid implants or injectable depots made from biocompatible polymers. For example, subcutaneously injectable PEGylated poly (lactic acid) (PEG-PLA) [ 67 ] or PEGylated poly (lactic acid-co-glycolic acid) (PEG-PLGA) [ 68 ] polymeric depots incorporating bsAbs have shown sustained release in plasma and improved antitumoral efficacy in prostate and breast cancer xenograft models, respectively.

Nanocarriers present an alternative strategy to improve bsAb pharmacokinetics and biodistribution, potentially reducing the required dose and minimizing adverse effects. Beyond delivery optimization, NPs can facilitate combinational therapies by carrying multiple agents within a single platform. This versatility provides opportunities to overcome resistance mechanisms, such as antigen loss and tumor-induced immunosuppression. Through combinatorial or sequential targeting, nanocarriers may help counteract antigen escape, while the localized co-delivery of bsAbs with immunomodulators could remodel the immunosuppressive tumor microenvironment and enhance therapeutic efficacy.

Technical approaches for loading BsAbs into nanocarriers

Most research to date has focused on decorating nanocarriers with multiple classical mAbs to generate bispecific platforms, which have demonstrated favorable pharmacokinetics, and improved antitumor efficacy [ 69 ]. In contrast, the incorporation of bsAbs or trispecific antibodies (trAbs) into NPs remains comparatively underexplored. Several strategies have been used to attach or complex bsAbs to inorganic, hybrid, lipidic, and tumor-responsive nanocarriers (Fig. 4 ). In these studies, therapeutic cell engager bsAbs are incorporated into NPs that simultaneously support combination therapies enhancing their overall antitumor activity.

Fig. 4 BsAbs as therapeutic agents in nanomedicine. BsAbs incorporated into nanocarriers, either by surface attachment or by complexation within the nanoparticle structure, allowing controlled release in response to tumor microenvironment (TME) stimuli. Created in https://BioRender.com

For example, Xu et al. covalently conjugated a novel NK cell-engager (NKCE) bsAb targeting the NK cell marker CD16 and the tumor biomarker carcinoembryonic antigen (CEA) to the surface of PEGylated hollow mesoporous ruthenium NPs. These constructs, intended to function as photothermal therapy agents, were additionally loaded with the fluorescent anti-tumor complex RBT. In vivo studies in a colorectal tumor model showed significantly superior tumor targeting and anticancer efficacy compared to RBT alone, free bsAb, or the combination of RBT-loaded NPs and free bsAb, achieving up to 96% tumor growth inhibition after three injections combined with near-infrared irradiation [ 70 ]. The enhanced therapeutic effect was attributed to the simultaneous delivery of RBT to solid tumors and bsAb-mediated immunotherapy. In contrast, another approach using NPs decorated with trAbs targeting CD3, PD-L1, and PEG did not show added benefits compared to free trAb or drug-loaded NPs alone in a breast cancer model [ 71 ]. The lack of synergy may result from suboptimal trAb exposure, impaired release, or functional inhibition of therapeutic components upon NP integration. These challenges highlight the importance of refining the design and functionalization of NPs to ensure therapeutic benefit.

Another promising approach incorporates bsAbs into tumor-responsive NPs that enable controlled release in response to TME properties, such as pH, hypoxia, or enzyme activity, including metalloproteinases (MMPs) [ 72 ]. Responsive NPs contained bsAbs targeting B7-H3 and CD3 were developed for glioblastoma (GBM) therapy. Their formulation included a multifunctional polymer composed of: (i) hyaluronic acid (HA), which targets CD44 receptors overexpressed in GBM; (ii) PLGLAG (proline-leucine-glycine-leucine-alanine-glycine), the tumor-responsive linker cleavable by MMP-2; and (iii) a dimer of epigallocatechin-3-O-gallate (EGCG), a polyphenol with attributed anticancer properties. Upon intravenous injection in a GBM mouse model, the bsAb-loaded NPs demonstrated higher tumor accumulation, greater inhibition of GBM growth and extended survival. Specifically, at day 56 post-treatment, survival rates showed a statistically significant improvement over controls. The therapeutic benefit observed in vivo was associated with reduction of proteins related to ferroptosis, B7-H3 downregulation, and increased intracranial infiltration of lymphocytes [ 73 ]. This illustrates the potential benefits of bsAb incorporation into rationally designed NPs.

A different strategy explored the incorporation of the approved bsAb blinatumomab into acid-labile polydopamine (PDA)-CaCO₃ NPs via electrostatic adsorption. These NPs were co-loaded with imiquimod (IMQ), a toll-like receptor 7 (TLR7) agonist. The payload was released in a controlled manner upon disintegration of the nanosystem in the acidic TME, while PDA conferred photothermal therapy capabilities. In a breast tumor model, the bsAb-loaded NPs significantly suppressed tumor growth without signs of systemic toxicity. Mechanistically, treatment with these NPs inhibited regulatory T-cell activity, promoted the infiltration of CD4⁺/CD8⁺ and IFN-γ⁺ cytotoxic T lymphocytes, enhanced IFN-γ secretion, and induced stronger T-cell activation, ultimately promoting a durable antitumor immune response [ 74 ].

Nanotechnology for improving current BsAb therapeutics. Critical considerations and future directions

Although still at an early stage of development, nanotechnology-based strategies using bsAbs have shown improved efficacy over free bsAbs, particularly by enabling combinatorial treatments that engage multiple antitumor mechanisms simultaneously. These include combining bsAbs with chemotherapeutics or photothermal agents to promote immunogenic cell death, thereby enhancing antigen release and immune activation. In parallel, bsAb- nanocarrier platforms have been engineered to incorporate immune modulators such as TLR agonists or checkpoint inhibitors, which synergize to potentiate T cell responses and overcome the immunosuppressive TME [ 70 , 71 , 74 ]. Moreover, tumor-responsive nanoplatforms capable of releasing payloads in response to microenvironmental cues (e.g., pH shifts, protease activity) enable dynamic adaptation to tumor conditions. Such adaptive designs enhance efficacy while simultaneously reducing systemic toxicity, positioning nanotechnology as a powerful tool to overcome antigen escape and microenvironmental barriers in solid tumors. While the complexity of these systems may complicate the assessment of individual component contribution, their synergistic potential holds promise for enhancing antitumor responses and overcoming resistance.

The hypothesis that NP encapsulation may prolong the circulation time of bsAbs is conceptually compelling; however, currently, there is not clear evidence supporting this advantage. Regarding safety, only a few studies have assessed the toxicity of therapeutic bsAb-loaded nanoplatforms, generally reporting no signs of organ damage or abnormal body weight [ 70 , 71 , 73 ]. Nevertheless, it remains unclear whether NP-mediated delivery could mitigate severe adverse effects commonly observed in bsAb clinical trials, such as the CRS and neurotoxicity. Future investigations should emphasize rational NP engineering and direct comparison with free bsAbs to determine whether these platforms can effectively extend bsAb circulation half-life and reduce off-target effects.

BsAb development has primarily focused on well-established cell surface antigens such as CD19, HER2 or EGFR, as well as components of the TME including fibroblast activation protein (FAP). While these approaches have demonstrated significant clinical promise, they are often associated by the risk of “on-target, off-tumor” toxicity, resulting from low-level antigen expression on normal tissues. This has driven researchers to explore more specific targets. The majority of tumor-specific antigens are localized intracellularly and, therefore, remain largely inaccessible to conventional antibody therapies. Recent developments in bsAb-based therapies have focused on targeting intracellular antigens or neoantigens presented as peptide/MHC-I complexes on the surface of tumor cells [ 75 ]. A key innovation in this area has been the development of immune-mobilizing monoclonal TCRs against cancer (ImmTACs), which combine an engineered, high-affinity TCR specific for peptide–MHC complexes with an anti-CD3 scFv to redirect T-cell activity toward tumor cells. Tebentafusp, the first ImmTAC approved for metastatic uveal melanoma, has validated this concept in the clinic by demonstrating durable survival benefit despite low target antigen density [ 76 ]. In parallel, TCR-mimetic bispecific antibodies (TCR-mimic bsAbs) have been generated to bind intracellularly derived peptide–MHC complexes with antibody-like specificity, extending the reach of bsAb therapies to non-surface tumor antigens such as mutated p53 or KRAS [ 77 , 78 ]. These strategies significantly expand the druggable tumor antigen repertoire, enabling the selective targeting of neoantigens and otherwise “undruggable” intracellular proteins. Collectively, ImmTACs and TCR-mimic bsAbs represent a new frontier in bsAb engineering, offering a route to precise, tumor-restricted therapies with transformative clinical potential. In parallel, nanotechnology is emerging as a key tool for the direct intracellular delivery of mAbs, overcoming traditional delivery barriers. This approach has shown success with mAbs targeting intracellular oncoproteins like mutant KRAS [ 79 ]. Building on these advances, bsAbs could be designed to simultaneously target multiple intracellular oncoproteins or signaling pathways, with nanocarriers facilitating their intracellular delivery in either protein or nucleic acid form. The RNA-based approach adds a new dimension to bsAb therapy and will be the focus of the following section.

mRNA nanocarriers encoding therapeutic BsAbs

The production of clinical-grade antibodies presents significant manufacturing challenges [ 80 ]. To overcome this limitation, in situ bsAb expression using nucleic acid-based formats is being actively investigated, using electroporation upon intramuscular injection of DNA [ 81 ], virus-based delivery approaches [ 82 , 83 ] and, more recently, mRNA-loaded NPs (Fig. 5 ). These strategies aim to enhance therapeutic efficacy, while reducing production complexity and cost, ultimately offering scalable alternatives to conventional recombinant protein manufacturing.

Fig. 5 mRNA-encoded bsAb: In situ expression and emerging applications in cell therapy. Comparison between conventional bsAb protein formats and mRNA-based delivery approaches. While recombinant bsAbs face manufacturing complexity and short half-life, mRNA-loaded nanocarriers such as lipid nanoparticles (LNPs), poly (β-amino ester) nanoparticles (PBAE NPs), or TransIT reagents enable in situ bsAb production. These can be applied systemically or, to ex vivo cell therapy strategies where current delivery approaches rely on viral or physical transfection methods. Created in https://BioRender.com

mRNA-NPs as vector for BsAb gene therapy

In 2017, Karikó, Türeci and Sahin were the first to provide compelling evidence for the feasibility of bsAb TCE-encoding mRNA. They developed three distinct TCE constructs directed against the TAAs claudin 6 and 18.2 (tight-junction proteins), and epithelial cell adhesion molecule. Following intravenous administration of mRNA encoding these bsAbs via the TransIT™ transfection reagent, expression was predominantly localized to the liver. In mice bearing subcutaneous human ovarian carcinoma xenografts, three weekly injections of 3 µg encapsulated mRNA induced complete tumor regression. In contrast, achieving comparable efficacy required ten injections of 4–7 µg of purified protein over the same period, thus validating superiority of the mRNA approach [ 84 ]. Building on this work, Stadler and Sahin developed mRNA-loaded LNPs encoding a TCE bsAb (CD3 x Claudin 6). This approach resulted in sustained liver expression and prolonged therapeutic bsAb concentrations in the bloodstream, with a half-life of 21.6 h compared to 12.2 h for the directly administered bsAb. In cynomolgus monkeys, half-lives ranged from 26.6 to 37.7 h. Weekly mRNA injections maintained therapeutic serum levels, driving regression of subcutaneous OV-90 xenografts in mice. BNT142 is now evaluated in phase I/II clinical trial for patients with claudin 6-positive advanced solid tumors ( NCT05262530 ) [ 85 ].

Additional studies have further demonstrated that in situ production of bsAbs using mRNA NPs offers superior antitumor efficacy, extended half-life, and reduced toxicity compared to direct administration of recombinant proteins. For instance, mRNA encoding anti-EpCAM x CD3 TCE delivered via poly (β-amino ester) conjugates coated with mannose-modified poly (glutamic acid) enabled targeted transfection of immune cells, enhanced T-cell infiltration, and effective TME remodelling in a model of peritoneal carcinomatosis [ 86 ]. Similarly, LNPs encoding TCEs such as B7H3 x CD3 (tested in melanoma and other solid tumors) [ 87 ], CD19 x CD3, and GPC3 x CD3 (evaluated in hepatocellular carcinoma) [ 88 ] exhibited prolonged protein expression, improved tumor control, and reduced dosing frequency compared to recombinant proteins in both murine and non-human primate models.

In addition to intravenous injection, intratumoral administration of mRNA-LNPs has proven effective in colorectal and ovarian cancer models. Weekly low-dose injections of EpCAM x CD3 [ 89 ], or HER2 x CD3 [ 90 ] mRNA led to strong tumor growth inhibition, suggesting that local delivery can overcome systemic barriers and ensure efficacy.

Beyond classical TCEs, mRNA strategies have expanded to encode bsAbs targeting immune checkpoints or chemokines. For instance, PD-L1 x PD-1 and PD-1 x CTLA-4 bsAbs delivered via LNPs achieved prolonged expression and enhanced efficacy in intestinal and colon cancer models [ 91 , 92 ]. Moreover, a liver-targeted approach using mRNA encoding a bsAb against the chemokines CCL2 and CCL5 yielded potent antitumor activity across multiple cancer models, including metastatic pancreatic and colorectal cancers [ 93 ].

Finally, co-delivery of two mRNA-encoded bsAbs (EGFR x CD3 and PD-L1 × 4-1BB) demonstrated synergistic tumor inhibition in a colorectal cancer model, highlighting the potential of multiplexed mRNA nanotherapies [ 94 ].

Although various carrier systems have been explored for RNA delivery, LNPs remain the gold-standard in gene therapy across multiple applications [ 95 ]. To date, in situ bsAb production has primarily relied on LNPs and PBAE-based NPs, as summarized in Table 3 .

Table 3 mRNA-NPs for BsAb in vivo production Nanocarrier mRNA encoded bsAb type Targets Duration of bsAb expression (versus recombinant protein) Tumor mouse model (route of administration) Ref. Polymer/lipid transfection reagent (TransIT) TCE CD3 x CLDN6 Pharmacologically relevant plasma levels for up to 144 h (versus < 24 h for protein) Ovarian carcinoma xenograft (IV) [ 84 ] LNP TCE CD3 x (CLDN6) 2 Half-life of 21.6 h (versus 12.2 h for protein) OV-90 xenograft PBMC humanized (IV) [ 85 ] PBAE NP with PGA-di-mannose coating TCE CD3 x EpCAM Detectable serum levels >48 h (versus < 24 h for protein) Ovarian cancer (IV) [ 86 ] LNP TCE CD3 x B7H3 Half-life of 73 h (versus 2 h for protein) Hematologic and solid tumor human xenografts (IV) [ 96 ] LNP TCE CD3 x CD19 and CD3 x GPC3 Detectable plasma levels for 168 h (direct comparison not included) Hepatocellular carcinoma (IV) [ 88 ] LNP TCE CD3 x EpCAM Not specified (direct comparison not included) Colorectal xenograft (ITU) [ 89 ] LNP TCE CD3 x HER2 Not specified (direct comparison not included) Ovarian cancer xenograft (ITU) [ 90 ] LNP Checkpoint inhibitor PD1 x PD-L1 Detectable plasma levels >35 days (versus 21 days for protein) Human PD-1 and PD-L1 knock -MC38 colon adenocarcinoma (IV) [ 91 ] LNP Checkpoint inhibitor CTLA4 x PD-1 Detectable serum levels >26 days (direct comparison not included) Colon cancer (IV) [ 92 ] LNP Signaling inhibitor CCL2 x CCL5 Detectable plasma levels for 72 h (direct comparison not included) Liver carcinoma, liver metastasis of pancreatic and colorectal cancers (IV) [ 93 ] Polymer/lipid transfection reagent (TransIT) TCE and co-stimulatory bsAb EGFR x CD3 and PD-L1 × 4-1BB Half-life of 18.9 h and 49.6 h (versus 0.6 h and 22.2 h for protein, respectively) Subcutaneous colorectal carcinoma xenograft (IV) [ 94 ] BsAb bispecific antibody, CCL chemokine (C-C motif) ligand, CD cluster of differentiation, CLDN6 claudin-6, CTLA-4 cytotoxic T-lymphocyte–associated antigen 4, epcam epithelial cell adhesion molecule, EGFR epidermal growth factor receptor, GPC3 glypican 3, HER2 human epidermal growth factor receptor 2, IV intravenous, ITU intratumoral, LNP lipid nanoparticle, MMP-2 matrix metalloproteinase-2, mRNA messenger RNA, PBAE poly(β-amino ester), PBMC peripheral blood mononuclear cell, PD-1 programmed death 1, PD-L1 programmed death ligand 1, PEG polyethylene glycol, PGA polyglutamic acid, TCE T cell engager

Nanotechnology enabling in situ-controlled production of bsAbs. Critical considerations and future directions

Gene therapy strategies employing mRNA-loaded NPs offer key advantages, including prolonged therapeutic effects and reduced systemic toxicity. These benefits are attributed to their capacity to achieve robust outcomes with lower and less frequent dosing compared to recombinant protein administration. However, the limited number of preclinical studies and variability in experimental designs make it currently difficult to draw definitive conclusions. Notably, reported mRNA-NP dosing regimens vary from 0.1 µg once weekly to 30 µg administered three times per week. This variability may also be influenced by multiple factors, including the delivery system, mRNA construct design, expression kinetics, administration route, and biological activity of the encoded protein. A comprehensive understanding of these parameters will be critical for successful clinical translation.

To date, most studies have utilized LNPs as delivery vehicles and relied on intravenous or intratumoral administration. However, work by Weissman and others has shown that the route of administration significantly influences the pharmacokinetics and expression profile of mRNA-encoded proteins [ 97 ]. These findings underscore the need to not only systematically investigate alternative delivery platforms, but also administration routes in the context of bsAb therapies.

In addition, advanced mRNA formats such as self-amplifying mRNA and circular RNA (circRNA) remain largely unexplored in bsAb applications. These constructs have shown promising results in improving expression kinetics and durability. For instance, circRNA encoding luciferase has achieved expression half-lives exceeding 80 h, nearly double as compared to linear mRNA [ 98 ], thereby implying significant potential for improved pharmacokinetics of mRNA-encoded bsAbs.

In the currently available literature, bsAb production is generally not targeted to a specific tissue; instead, achieving high overall levels of therapeutic protein is prioritized. This implies that their primary functional tropism towards the liver is not of concern, as virtually any cell may function as production and secretion center. This may in part explain the lack of clear selection patterns in terms of nanocarrier specifics, leaving the potent and extensively studied liver-tropic LNPs promising candidates. Particles with prominent extrahepatic tropism, such as Dan Siegwart’s lung or spleen SORT LNPs [ 99 ], have so far not been explored for the delivery of bsAb-encoding mRNA but may offer opportunities for specific applications. Site-specific production and secretion could result in higher local Ab concentrations at the target site, similar to intratumorally administered NPs.

Other innovative but unexplored strategies include the incorporation of mRNA encoding bsAbs into hydrogels or scaffolds to enable localized, controlled release [ 100 ]. Microneedle-based systems, such as mesoporous silica nanoneedles delivering minicircle DNA encoding an EpCAM x CD3 bsAb, have demonstrated effective gene delivery and antitumor activity in breast cancer models [ 101 ], suggesting these platforms could be adapted to bsAb mRNA-based strategies.

Overall, while mRNA-based in situ bsAb production is still an emerging area, the encouraging results obtained so far, and the wide range of yet-to-be-explored technologies suggest that this field holds substantial promise and offers ample opportunities for innovation and clinical advancement.

Future insights. Nanocarriers as potential vectors for BsAb adoptive cell therapies

Genetic information can also be introduced ex vivo into patient-derived cells, as demonstrated by adoptive cell therapies like CAR (chimeric antigen receptor) modified T cells. Clinically, this involves collecting patient cells, genetically modifying them (typically via viral vectors or electroporation), expanding them, and reinfusing them [ 102 ]. This strategy is now being extended to bsAbs by engineering cells to secrete bsAbs, thereby circumventing the need for continuous protein infusion (Fig. 5 ). Unlike direct gene transfer (Sect. 4.1), this method benefits from the intrinsic tumor-homing capacity of certain cells. Notably, certain cell types, such as T cells, offer dual functionality by acting both as bsAb producers and effector cells [ 2 , 103 ].

Following a pioneering study by Alvarez-Vallina’s group in 2003, demonstrating that ex vivo engineered human cells (embryonic kidney cells, HEK293) could secrete functionally active bsAbs in vivo [ 104 ], several methods to generate a variety of bsAb-secreting cell types with promising therapeutic effects are under active preclinical investigation. Among these, T cells engineered to secrete TCE antibodies, also known as STAb ( S ecretion of T -cell redirecting Ab s) therapy [ 103 ], is the most prominent. Preclinical STAb studies elicited significant positive responses for the treatment of hematological malignancies targeting CD19 [ 105 – 107 ], B-cell maturation antigen (BCMA) [ 108 ], CD123 [ 109 ], or CD1a [ 110 ]. Currently, two STAb first-in-human clinical trials in hematological cancers are actively enrolling patients. The same approach has been applied to solid tumors, including colon carcinoma targeting the CEA [ 111 ]. Comparative studies showed that STAb-T cells outperform second generation CAR-T cells by recruiting bystander, unmodified T cells, amplifying the therapeutic effects, and inducing immunologic memory [ 107 , 108 ].

Beyond T cells, mesenchymal stem cells (MSCs) have shown promise as bsAb producers due to their ease of expansion, extended lifespan, and transduction capabilities [ 112 ]. However, their immunosuppressive nature may facilitate tumor growth in vivo [ 113 ]. One strategy to mitigate this involves housing genetically modified MSCs in biomaterial scaffolds to create localized bsAb “biological minipumps,” thereby limiting systemic cell dissemination. Several studies have supported the safety, utility, and efficacy of this approach for the secretion of therapeutic proteins including bsAbs [ 112 ]. Plasma cells, known for their high secretory capacity [ 114 ], and other hematopoietic cells like NK cells [ 115 ] and macrophages [ 116 ] are also being explored for bsAb production.

All the bsAb-secreting cell types described above are currently generated ex vivo using viral vectors or electroporation. However, viral vector manufacturing is a complex, time-consuming, and costly process, representing a critical bottleneck for large-scale production and raisingconcerns such as insertional mutagenesis [ 117 ]. Electroporation is a batch-based process with limited scalability, substantial cell toxicity from electric pulses and reliance on specialized equipment [ 118 ]. Non-viral methods based on NPs could address these challenges, though their application for transfection of primary cells to create “secreting bsAb factories” has yet to be fully realized. NPs can be easily adapted for industrial-scale production. This offers a more cost-effective approach by reducing manufacturing complexity and minimizing the need for specialized facilities. Importantly, mRNA-NP-based systems circumvent the safety risks associated with viral integration and can be engineered to reduce toxicity compared to electroporation. Proof-of-concept studies in CAR-based immunotherapy have demonstrated success with nanocarriers, particularly LNPs, in generating CAR-T cells ex vivo with comparable activity to electroporation and lentiviral methods. Furthermore, NPs hold the potential to bypass ex vivo cell manipulation altogether by enabling direct in vivo engineering, an approach now advancing toward clinical evaluation [ 119 ]. Such technologies could ultimately help overcome the limitations of autologous and allogeneic therapies, including prolonged manufacturing timelines, high costs, and risk of graft-versus-host disease, paving the way for off-the-shelf treatments with straightforward redosing [ 120 , 121 ].

Therefore, we suggest future applications of nanotechnology-enabled approaches for the ex vivo and in vivo generation of cell-secreted bsAbs to provide safer, more scalable, and cost-effective platforms, thereby advancing these therapeutics towards more patient-friendly treatment modalities.

Challenges and outlook for clinical translation

The integration of nanotechnology with bsAbs technology offers complementary strategies to improve cancer therapy. Collectively, while preclinical studies demonstrate encouraging efficacy, clinical translation is in an early phase, with one ongoing trial (BNT142-01, a Phase I/II study of mRNA-LNPs encoding a CD3 × CLDN6 bsAb). Recent data from ASCO 2025 show that BNT142 has demonstrated first-in-human activity in CLDN6-positive solid tumors, including ovarian cancer, with a manageable safety profile [ 122 ]. Below, several challenges that could represent major obstacles to clinical implementation are discussed, as well as high-priority research gaps that need to be addressed.

Manufacturing, scalability, and regulatory hurdles

BsAbs exhibit greater structural complexity than conventional antibodies, which heightens the risk of chain mispairing, aggregation, and batch-to-batch variability [ 123 ]. Incorporating bsAbs into nanocarrier systems presents significant challenges, requiring precise conjugation and preservation of functional activity to ensure reproducibility. These technical demands become even more pronounced in advanced designs, such as stimuli-responsive or co-delivery platforms, raising concerns regarding scalability. Nevertheless, recent innovations including novel antibody formats, microfluidics-based manufacturing, continuous processing, and site-specific conjugation methods are progressively overcoming these barriers. For mRNA-encoded bsAbs, the inherently shorter production cycle [ 124 ] and regulatory precedents established during the development of COVID-19 vaccines provide a favorable translational landscape. Importantly, GMP-compliant, scalable, and protective nanocarrier systems must be integrated into the design process from the outset, thereby enabling promising concepts to advance efficiently into clinically applicable products.

Toward standardized characterization of bsAb–NP systems

Heterogeneity in experimental reporting represents a critical bottleneck. Key variables including ligand density, bsAb orientation and functionality, linker stability, and protein corona interactions critically influence biological performance, yet are often underreported. Establishing reproducibility and defining critical quality attributes will be decisive to align academic development with industrial and regulatory standards.

Managing risks in bsAb–nanomedicine translation

Clinical application of naked bsAbs has revealed risks of CRS and neurotoxicity, and it remains unclear whether nanocarrier delivery mitigates or simply modulates these toxicities. Encapsulation could minimize systemic peaks and extend half-life, but nanocarriers may introduce additional challenges including complement activation, hepatotoxicity, and long-term immunogenicity that could affect immune tolerance and response durability [ 125 ]. Locoregional or depot-based delivery of mRNA-encoded bsAbs is emerging as a promising strategy for solid tumors, where systemic administration is constrained by toxicity. Tissue-targeted LNPs and hydrogel-based systems further enhance this approach by maximizing local exposure while reducing systemic toxicities such as CRS and neurotoxicity. Together, these advances position mRNA-bsAbs as a compelling modality for solid tumors, provided that future studies integrate PK/PD biomarkers, adaptive trial designs, and long-term immune monitoring to balance efficacy with safety.

Bridging communities for clinical translation

The translation of bsAbs into nanocarrier formats is slow, partly due to limited communication between the therapeutic antibody and nanomedicine communities. In addition to creating new bsAb architectures, repurposing clinically validated bsAbs within delivery systems remains understudied. Only a few case studies exist, such as the use of blinatumomab loaded into CaCO₃/polydopamine nanoparticles to combine T cell engagement with local immunostimulation and photothermal therapy [ 74 ]. Concurrently, advances in platforms including mRNA-encoded bsAbs currently in clinical trials (BNT142 and CLDN6 x CD3) [ 85 , 122 ], and hydrogel or depot delivery of TCEs demonstrate that nanocarriers can enable sustained, tumor-localized activity while modulating systemic exposure. New concepts, such as bsAb-guided targeting of mRNA-LNPs, exemplify how antibody engineering and nanocarrier design can coevolve to improve tissue selectivity. A multidisciplinary framework uniting immuno-oncology, materials science, and regulatory science will be essential to standardizing CMC, PK/PD, and safety readouts and unlocking the full potential of bsAb-NP platforms. Reviews on antibody-functionalized nanocarriers for RNA therapeutics outline practical paths for such integration [ 126 ].

High-priority research gaps and future outlook

Optimization of bsAb–NC design: Control bsAb orientation, minimize non-specific interactions, enable controlled release, and achieve high-yield bsAb–mRNA delivery. AI-driven modeling and computational prediction tools will be instrumental in guiding these optimizations while anticipating toxicity risks. Standardized characterization: Establish robust analytical methods to quantify bsAb density, orientation, and stability, and define regulatory-quality attributes to ensure reproducibility and streamline clinical translation. Pharmacokinetic control: Apply rational nanocarrier engineering to extend bsAb bioactivity, optimize administration routes, and explore extrahepatic targeting strategies to maximize efficacy while minimizing systemic toxicity. Toxicity and stability assessment: Conduct rigorous safety evaluation, including risks of CRS or neurotoxicity, while leveraging innovative delivery approaches such as locoregional or depot systems. Equally critical is assessing long term immunogenicity and its implications for immune tolerance, which remains poorly understood. Sustainability and access: Broad adoption of bsAb–nanotechnology platforms will require scalable, reproducible, and cost-effective manufacturing pipelines to ensure accessibility across healthcare systems.

Collectively, addressing these research gaps will not only accelerate the bench‑to‑bedside translation of bsAb–nanotechnology platforms but also establish a foundation for next-generation immunotherapies that are safer, longer‑lasting, and broadly accessible. Importantly, advances in antibody engineering, mRNA delivery, and nanotechnology manufacturing pipelines are already converging, offering a unique window of opportunity. By embracing multidisciplinary collaboration and embedding regulatory foresight bsAb–NP systems can transcend the current limitations. If realized, this convergence has the potential to redefine precision oncology and deliver transformative treatments for patients.

Publisher’s note

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Elisa Battistini and Philipp Lapuhs contributed equally to this work.

Acknowledgements

LS would like to thank the Asociación Española Contra el Cáncer (AECC) and Fundación San Rafael for their support, funding, and encouragement of her research. Their contributions have been essential for the development of this work.

Authors’ contributions

EB, PL, MJA and LS conceptualized the review. All authors contributed to the writing of the manuscript. LS prepared all figures. LAV, MJA and LS contributed to critical revision and provided expert insights. All authors reviewed and approved the final version of the manuscript. EB and PL contributed equally to this work.

Funding

EB and PL acknowledge grants ED481A-2023-036 and ED481A-2022-237, respectively, ‘Programa de ayudas a la etapa predoctoral’ of Xunta de Galicia (Consellería de Cultura, Educación, Formación Profesional y Universidades o de la Agencia Gallega de Innovación) 2023 and 2022. AJ acknowledges a grant (05-IN606D-2023-2542597) from Xunta de Galicia “Doutoramentos industriais convocatoria 2023”. SGA receives support from the grant PREP2023-000545 funded by MCIN and by ESF. LAV laboratory is funded by the MCIN/AEI (PID2023-148429OB-I00, PID2020-117323RB- I00, PDC2021-121711-I00, CPP2022-009762, CPP2022-009765, CPP2023-010827), the Carlos III Health Institute (ISCIII) with European Regional Development Fund (FEDER) cofinancing (DTS20/00089, PMPTA22/00167); the Asociación Española contra el Cáncer (AECC) (PROYE19084ALVA and PRYGN234844ALVA), the CRIS Cancer Foundation (FCRIS-2021-0090 and FCRIS-2023-0070), the Fundación ‘‘La Caixa’’ (HR21-00761 project IL7R_LungCan), the Comunidad de Madrid (P2022/BMD-7225 Next Generation CART MAD), and the Fundación FERO (BBASELGAFERO2024.01). LRG is supported by an Industrial PhD fellowship from Comunidad de Madrid (IND2023/BMD-28821). MJA receive support from Xunta de Galicia (ED431C 2021/17 and ED431G 2019/02) and AEl (PID2023-150416OB-I00, PLEC2022-009298) funded by MCIN/AEI and the “European Union NextGeneration EU/PRTR”. LS is supported by INVES246233SANJ and SNRGS259729SANJ Fundación Científica Asociación Española Contra el Cáncer, and thanks Fundación San Rafael (PAI2024) for the support.

Data availability

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

MJA has financial interest in Libera Bio, Inc, a company related to immunotherapy. LAV has financial interests in Leadartis, Inc, and STAb Therapeutics, Inc companies related to immunotherapy.

References

1. Klein C Brinkmann U Reichert JM Kontermann RE The present and future of bispecific antibodies for cancer therapy Nat Rev Drug Discov 2024 23 301 19 10.1038/s41573-024-00896-6 38448606 Klein C, Brinkmann U, Reichert JM, Kontermann RE. The present and future of bispecific antibodies for cancer therapy. Nat Rev Drug Discov. 2024;23:301–19. 10.1038/s41573-024-00896-6. 38448606 10.1038/s41573-024-00896-6

2. Guo X Wu Y Xue Y Xie N Shen G Revolutionizing cancer immunotherapy: unleashing the potential of bispecific antibodies for targeted treatment Front Immunol 2023 14 1291836 10.3389/fimmu.2023.1291836 38106416 PMC10722299 Guo X, Wu Y, Xue Y, Xie N, Shen G. Revolutionizing cancer immunotherapy: unleashing the potential of bispecific antibodies for targeted treatment. Front Immunol. 2023;14:1291836. 10.3389/fimmu.2023.1291836. 38106416 10.3389/fimmu.2023.1291836 PMC10722299

3. Blanco B Domínguez-Alonso C Alvarez-Vallina L Bispecific immunomodulatory antibodies for cancer immunotherapy Clin Cancer Res 2021 27 5457 64 10.1158/1078-0432.CCR-20-3770 34108185 PMC9306338 Blanco B, Domínguez-Alonso C, Alvarez-Vallina L. Bispecific immunomodulatory antibodies for cancer immunotherapy. Clin Cancer Res. 2021;27:5457–64. 10.1158/1078-0432.CCR-20-3770. 34108185 10.1158/1078-0432.CCR-20-3770 PMC9306338

4. Beishenaliev A Loke YL Goh SJ Geo HN Mugila M Misran M Bispecific antibodies for targeted delivery of anti-cancer therapeutic agents: a review J Control Release 2023 359 268 86 10.1016/j.jconrel.2023.05.032 37244297 Beishenaliev A, Loke YL, Goh SJ, Geo HN, Mugila M, Misran M, et al. Bispecific antibodies for targeted delivery of anti-cancer therapeutic agents: a review. J Control Release. 2023;359:268–86. 10.1016/j.jconrel.2023.05.032. 37244297 10.1016/j.jconrel.2023.05.032

5. Hong Y Nam S-M Moon A Antibody-drug conjugates and bispecific antibodies targeting cancers: applications of click chemistry Arch Pharm Res 2023 46 131 48 10.1007/s12272-023-01433-6 36877356 Hong Y, Nam S-M, Moon A. Antibody-drug conjugates and bispecific antibodies targeting cancers: applications of click chemistry. Arch Pharm Res. 2023;46:131–48. 10.1007/s12272-023-01433-6. 36877356 10.1007/s12272-023-01433-6

6. Baeuerle PA Wesche H T-cell-engaging antibodies for the treatment of solid tumors: challenges and opportunities Curr Opin Oncol 2022 34 552 8 10.1097/CCO.0000000000000869 35880455 PMC9415207 Baeuerle PA, Wesche H. T-cell-engaging antibodies for the treatment of solid tumors: challenges and opportunities. Curr Opin Oncol. 2022;34:552–8. 10.1097/CCO.0000000000000869. 35880455 10.1097/CCO.0000000000000869 PMC9415207

7. Nathan P Hassel JC Rutkowski P Baurain J-F Butler MO Schlaak M Overall survival benefit with tebentafusp in metastatic uveal melanoma N Engl J Med 2021 385 1196 206 10.1056/NEJMoa2103485 34551229 Nathan P, Hassel JC, Rutkowski P, Baurain J-F, Butler MO, Schlaak M, et al. Overall survival benefit with tebentafusp in metastatic uveal melanoma. N Engl J Med. 2021;385:1196–206. 10.1056/NEJMoa2103485. 34551229 10.1056/NEJMoa2103485

8. Fenis A Demaria O Gauthier L Vivier E Narni-Mancinelli E New immune cell engagers for cancer immunotherapy Nat Rev Immunol 2024 24 7 471 86 10.1038/s41577-023-00982-7 38273127 Fenis A, Demaria O, Gauthier L, Vivier E, Narni-Mancinelli E. New immune cell engagers for cancer immunotherapy. Nat Rev Immunol. 2024;24(7):471–86. 10.1038/s41577-023-00982-7. 38273127 10.1038/s41577-023-00982-7

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

10. Park K Haura EB Leighl NB Mitchell P Shu CA Girard N Amivantamab in EGFR exon 20 insertion-mutated non-small-cell lung cancer progressing on platinum chemotherapy: initial results from the CHRYSALIS phase I study J Clin Oncol 2021 39 3391 402 10.1200/JCO.21.00662 34339292 PMC8791812 Park K, Haura EB, Leighl NB, Mitchell P, Shu CA, Girard N, et al. Amivantamab in EGFR exon 20 insertion-mutated non-small-cell lung cancer progressing on platinum chemotherapy: initial results from the CHRYSALIS phase I study. J Clin Oncol. 2021;39:3391–402. 10.1200/JCO.21.00662. 34339292 10.1200/JCO.21.00662 PMC8791812

11. Zhang T Lin Y Gao Q Bispecific antibodies targeting immunomodulatory checkpoints for cancer therapy Cancer Biol Med 2023 20 181 95 10.20892/j.issn.2095-3941.2023.0002 36971124 PMC10038071 Zhang T, Lin Y, Gao Q. Bispecific antibodies targeting immunomodulatory checkpoints for cancer therapy. Cancer Biol Med. 2023;20:181–95. 10.20892/j.issn.2095-3941.2023.0002. 36971124 10.20892/j.issn.2095-3941.2023.0002 PMC10038071

12. Keam SJ Cadonilimab. First approval Drugs 2022 82 1333 9 10.1007/s40265-022-01761-9 35986837 Keam SJ. Cadonilimab. First approval. Drugs. 2022;82:1333–9. 10.1007/s40265-022-01761-9. 35986837 10.1007/s40265-022-01761-9

13. Vaidya A Moore S Chatterjee S Guerrero E Kim M Farbiak L Expanding RNAi to kidneys, lungs, and spleen via selective organ targeting (SORT) SiRNA lipid nanoparticles Adv Mater 2024 36 e2313791 10.1002/adma.202313791 38973655 PMC11823468 Vaidya A, Moore S, Chatterjee S, Guerrero E, Kim M, Farbiak L, et al. Expanding RNAi to kidneys, lungs, and spleen via selective organ targeting (SORT) SiRNA lipid nanoparticles. Adv Mater. 2024;36:e2313791. 10.1002/adma.202313791. 38973655 10.1002/adma.202313791 PMC11823468

14. Dilliard SA Siegwart DJ Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs Nat Rev Mater 2023 8 282 300 10.1038/s41578-022-00529-7 36691401 PMC9850348 Dilliard SA, Siegwart DJ. Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs. Nat Rev Mater. 2023;8:282–300. 10.1038/s41578-022-00529-7. 36691401 10.1038/s41578-022-00529-7 PMC9850348

15. Marques AC Costa PJ Velho S Amaral MH Functionalizing nanoparticles with cancer-targeting antibodies: a comparison of strategies J Control Release 2020 320 180 200 10.1016/j.jconrel.2020.01.035 31978444 Marques AC, Costa PJ, Velho S, Amaral MH. Functionalizing nanoparticles with cancer-targeting antibodies: a comparison of strategies. J Control Release. 2020;320:180–200. 10.1016/j.jconrel.2020.01.035. 31978444 10.1016/j.jconrel.2020.01.035

16. Ho K-W Liu Y-L Liao T-Y Liu E-S Cheng T-L Strategies for non-covalent attachment of antibodies to pegylated nanoparticles for targeted drug delivery Int J Nanomed 2024 19 10045 64 10.2147/IJN.S479270 PMC11453133 39371476 Ho K-W, Liu Y-L, Liao T-Y, Liu E-S, Cheng T-L. Strategies for non-covalent attachment of antibodies to pegylated nanoparticles for targeted drug delivery. Int J Nanomed. 2024;19:10045–64. 10.2147/IJN.S479270. 10.2147/IJN.S479270 PMC11453133 39371476

17. Nikkhoi SK Rahbarizadeh F Ranjbar S Khaleghi S Farasat A Liposomal nanoparticle armed with bivalent bispecific single-domain antibodies, novel weapon in HER2 positive cancerous cell lines targeting Mol Immunol 2018 96 98 109 10.1016/j.molimm.2018.01.010 29549861 Nikkhoi SK, Rahbarizadeh F, Ranjbar S, Khaleghi S, Farasat A. Liposomal nanoparticle armed with bivalent bispecific single-domain antibodies, novel weapon in HER2 positive cancerous cell lines targeting. Mol Immunol. 2018;96:98–109. 10.1016/j.molimm.2018.01.010. 29549861 10.1016/j.molimm.2018.01.010

18. Wu S-C Chen Y-J Wang H-C Chou M-Y Chang T-Y Yuan S-S Bispecific antibody conjugated manganese-based magnetic engineered iron oxide for imaging of HER2/neu- and EGFR-expressing tumors Theranostics 2016 6 118 30 10.7150/thno.13069 26722378 PMC4679359 Wu S-C, Chen Y-J, Wang H-C, Chou M-Y, Chang T-Y, Yuan S-S, et al. Bispecific antibody conjugated manganese-based magnetic engineered iron oxide for imaging of HER2/neu- and EGFR-expressing tumors. Theranostics. 2016;6:118–30. 10.7150/thno.13069. 26722378 10.7150/thno.13069 PMC4679359

19. Kao C-H Wang J-Y Chuang K-H Chuang C-H Cheng T-C Hsieh Y-C One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles Biomaterials 2014 35 9930 40 10.1016/j.biomaterials.2014.08.032 25212525 Kao C-H, Wang J-Y, Chuang K-H, Chuang C-H, Cheng T-C, Hsieh Y-C, et al. One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles. Biomaterials. 2014;35:9930–40. 10.1016/j.biomaterials.2014.08.032. 25212525 10.1016/j.biomaterials.2014.08.032

20. Lin W-W Cheng Y-A Li C-C Ho K-W Chen H-J Chen I-JU Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy Sci Rep 2021 11 7598 10.1038/s41598-021-87271-2 33828191 PMC8027450 Lin W-W, Cheng Y-A, Li C-C, Ho K-W, Chen H-J, Chen I-JU, et al. Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy. Sci Rep. 2021;11:7598. 10.1038/s41598-021-87271-2. 33828191 10.1038/s41598-021-87271-2 PMC8027450

21. Cheng Y-A Chen I-J Su Y-C Cheng K-W Lu Y-C Lin W-W Enhanced drug internalization and therapeutic efficacy of pegylated nanoparticles by one-step formulation with anti-mPEG bispecific antibody in intrinsic drug-resistant breast cancer Biomater Sci 2019 7 3404 17 10.1039/c9bm00323a 31251311 Cheng Y-A, Chen I-J, Su Y-C, Cheng K-W, Lu Y-C, Lin W-W, et al. Enhanced drug internalization and therapeutic efficacy of pegylated nanoparticles by one-step formulation with anti-mPEG bispecific antibody in intrinsic drug-resistant breast cancer. Biomater Sci. 2019;7:3404–17. 10.1039/c9bm00323a. 31251311 10.1039/c9bm00323a

22. Moles E Howard CB Huda P Karsa M McCalmont H Kimpton K Delivery of pegylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia Sci Transl Med 2023 15 696 10.1126/scitranslmed.abm1262 37196067 Moles E, Howard CB, Huda P, Karsa M, McCalmont H, Kimpton K, et al. Delivery of pegylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia. Sci Transl Med. 2023;15:696. 10.1126/scitranslmed.abm1262. 10.1126/scitranslmed.abm1262 37196067

23. Howard CB Fletcher N Houston ZH Fuchs AV Boase NRB Simpson JD Overcoming instability of antibody-nanomaterial conjugates: next generation targeted nanomedicines using bispecific antibodies Adv Healthc Mater 2016 5 2055 68 10.1002/adhm.201600263 27283923 Howard CB, Fletcher N, Houston ZH, Fuchs AV, Boase NRB, Simpson JD, et al. Overcoming instability of antibody-nanomaterial conjugates: next generation targeted nanomedicines using bispecific antibodies. Adv Healthc Mater. 2016;5:2055–68. 10.1002/adhm.201600263. 27283923 10.1002/adhm.201600263

24. Cui J Ju Y Houston ZH Glass JJ Fletcher NL Alcantara S Modulating targeting of poly(ethylene glycol) particles to tumor cells using bispecific antibodies Adv Healthc Mater 2019 8 e1801607 10.1002/adhm.201801607 30868751 Cui J, Ju Y, Houston ZH, Glass JJ, Fletcher NL, Alcantara S, et al. Modulating targeting of poly(ethylene glycol) particles to tumor cells using bispecific antibodies. Adv Healthc Mater. 2019;8:e1801607. 10.1002/adhm.201801607. 30868751 10.1002/adhm.201801607

25. Lim M Fletcher NL Saunus JM McCart Reed AE Chittoory H Simpson PT Targeted hyperbranched nanoparticles for delivery of doxorubicin in breast cancer brain metastasis Mol Pharm 2023 20 6169 83 10.1021/acs.molpharmaceut.3c00558 37970806 PMC10699306 Lim M, Fletcher NL, Saunus JM, McCart Reed AE, Chittoory H, Simpson PT, et al. Targeted hyperbranched nanoparticles for delivery of doxorubicin in breast cancer brain metastasis. Mol Pharm. 2023;20:6169–83. 10.1021/acs.molpharmaceut.3c00558. 37970806 10.1021/acs.molpharmaceut.3c00558 PMC10699306

26. Janowicz PW Houston ZH Bunt J Fletcher NL Bell CA Cowin G Understanding nanomedicine treatment in an aggressive spontaneous brain cancer model at the stage of early blood brain barrier disruption Biomaterials 2022 283 121416 10.1016/j.biomaterials.2022.121416 35217483 Janowicz PW, Houston ZH, Bunt J, Fletcher NL, Bell CA, Cowin G, et al. Understanding nanomedicine treatment in an aggressive spontaneous brain cancer model at the stage of early blood brain barrier disruption. Biomaterials. 2022;283:121416. 10.1016/j.biomaterials.2022.121416. 35217483 10.1016/j.biomaterials.2022.121416

27. Su C-Y Chen M Chen L-C Ho Y-S Ho H-O Lin S-Y Bispecific antibodies (anti-mPEG/anti-HER2) for active tumor targeting of docetaxel (DTX)-loaded mPEGylated nanocarriers to enhance the chemotherapeutic efficacy of HER2-overexpressing tumors Drug Deliv 2018 25 1066 79 10.1080/10717544.2018.1466936 29718725 PMC6058516 Su C-Y, Chen M, Chen L-C, Ho Y-S, Ho H-O, Lin S-Y, et al. Bispecific antibodies (anti-mPEG/anti-HER2) for active tumor targeting of docetaxel (DTX)-loaded mPEGylated nanocarriers to enhance the chemotherapeutic efficacy of HER2-overexpressing tumors. Drug Deliv. 2018;25:1066–79. 10.1080/10717544.2018.1466936. 29718725 10.1080/10717544.2018.1466936 PMC6058516

28. Cheng W-J Lin S-Y Chen M Chen L-C Ho H-O Chuang K-H Active tumoral/tumor environmental dual-targeting by non-covalently arming with trispecific antibodies or dual-bispecific antibodies on docetaxel-loaded mPEGylated nanocarriers to enhance chemotherapeutic efficacy and minimize systemic toxicity Int J Nanomed 2021 16 4017 30 10.2147/IJN.S301237 PMC8203191 34140769 Cheng W-J, Lin S-Y, Chen M, Chen L-C, Ho H-O, Chuang K-H, et al. Active tumoral/tumor environmental dual-targeting by non-covalently arming with trispecific antibodies or dual-bispecific antibodies on docetaxel-loaded mPEGylated nanocarriers to enhance chemotherapeutic efficacy and minimize systemic toxicity. Int J Nanomed. 2021;16:4017–30. 10.2147/IJN.S301237. 10.2147/IJN.S301237 PMC8203191 34140769

29. Cheng W-J Lin S-Y Chuang K-H Chen M Ho H-O Chen L-C Combined docetaxel/pictilisib-loaded mPEGylated nanocarriers with dual HER2 targeting antibodies for synergistic chemotherapy of breast cancer Int J Nanomed 2022 17 5353 74 10.2147/IJN.S388066 PMC9677924 36419719 Cheng W-J, Lin S-Y, Chuang K-H, Chen M, Ho H-O, Chen L-C, et al. Combined docetaxel/pictilisib-loaded mPEGylated nanocarriers with dual HER2 targeting antibodies for synergistic chemotherapy of breast cancer. Int J Nanomed. 2022;17:5353–74. 10.2147/IJN.S388066. 10.2147/IJN.S388066 PMC9677924 36419719

30. Zhao Y Fletcher NL Gemmell A Houston ZH Howard CB Blakey I Investigation of the therapeutic potential of a synergistic delivery system through dual controlled release of camptothecin–doxorubicin Adv Ther 2020 3 1900202 10.1002/adtp.201900202 Zhao Y, Fletcher NL, Gemmell A, Houston ZH, Howard CB, Blakey I, et al. Investigation of the therapeutic potential of a synergistic delivery system through dual controlled release of camptothecin–doxorubicin. Adv Ther. 2020;3:1900202. 10.1002/adtp.201900202.

31. Zhang Y Gu X Huang L Yang Y He J Enhancing precision medicine: bispecific antibody-mediated targeted delivery of lipid nanoparticles for potential cancer therapy Int J Pharm 2024 654 123990 10.1016/j.ijpharm.2024.123990 38467208 Zhang Y, Gu X, Huang L, Yang Y, He J. Enhancing precision medicine: bispecific antibody-mediated targeted delivery of lipid nanoparticles for potential cancer therapy. Int J Pharm. 2024;654:123990. 10.1016/j.ijpharm.2024.123990. 38467208 10.1016/j.ijpharm.2024.123990

32. Logan A Howard CB Huda P Kimpton K Ma Z Thurecht KJ Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma J Control Release 2024 367 806 20 10.1016/j.jconrel.2024.02.007 38341177 Logan A, Howard CB, Huda P, Kimpton K, Ma Z, Thurecht KJ, et al. Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma. J Control Release. 2024;367:806–20. 10.1016/j.jconrel.2024.02.007. 38341177 10.1016/j.jconrel.2024.02.007

33. Amabile A Phelan M Yu Z Silva P Marks A Morla-Folch J Bispecific antibody targeting of lipid nanoparticles Preprint [bioRxiv] 2024 10.1101/2024.12.20.629467 Amabile A, Phelan M, Yu Z, Silva P, Marks A, Morla-Folch J, et al. Bispecific antibody targeting of lipid nanoparticles. Preprint [bioRxiv]. 2024. 10.1101/2024.12.20.629467.

34. Su Y-C Burnouf P-A Chuang K-H Chen B-M Cheng T-L Roffler SR Conditional internalization of pegylated nanomedicines by PEG engagers for triple negative breast cancer therapy Nat Commun 2017 8 15507 10.1038/ncomms15507 28593948 PMC5472176 Su Y-C, Burnouf P-A, Chuang K-H, Chen B-M, Cheng T-L, Roffler SR. Conditional internalization of pegylated nanomedicines by PEG engagers for triple negative breast cancer therapy. Nat Commun. 2017;8:15507. 10.1038/ncomms15507. 28593948 10.1038/ncomms15507 PMC5472176

35. Chen I-J Cheng Y-A Ho K-W Lin W-W Cheng K-W Lu Y-C Bispecific antibody (HER2 × mPEG) enhances anti-cancer effects by precise targeting and accumulation of mPEGylated liposomes Acta Biomater 2020 111 386 97 10.1016/j.actbio.2020.04.029 32417267 Chen I-J, Cheng Y-A, Ho K-W, Lin W-W, Cheng K-W, Lu Y-C, et al. Bispecific antibody (HER2 × mPEG) enhances anti-cancer effects by precise targeting and accumulation of mPEGylated liposomes. Acta Biomater. 2020;111:386–97. 10.1016/j.actbio.2020.04.029. 32417267 10.1016/j.actbio.2020.04.029

36. Parker CL McSweeney MD Lucas AT Jacobs TM Wadsworth D Zamboni WC Pretargeted delivery of PEG-coated drug carriers to breast tumors using multivalent, bispecific antibody against polyethylene glycol and HER2 Nanomedicine 2019 21 102076 10.1016/j.nano.2019.102076 31394261 PMC7224238 Parker CL, McSweeney MD, Lucas AT, Jacobs TM, Wadsworth D, Zamboni WC, et al. Pretargeted delivery of PEG-coated drug carriers to breast tumors using multivalent, bispecific antibody against polyethylene glycol and HER2. Nanomedicine. 2019;21:102076. 10.1016/j.nano.2019.102076. 31394261 10.1016/j.nano.2019.102076 PMC7224238

37. Ho K-W Chen I-JU Cheng Y-A Liao T-Y Liu E-S Chen H-J Double attack strategy for leukemia using a pre-targeting bispecific antibody (CD20 Ab-mPEG scFv) and actively attracting pegylated liposomal doxorubicin to enhance anti-tumor activity J Nanobiotechnol 2021 19 16 10.1186/s12951-020-00752-w PMC7796588 33422061 Ho K-W, Chen I-JU, Cheng Y-A, Liao T-Y, Liu E-S, Chen H-J, et al. Double attack strategy for leukemia using a pre-targeting bispecific antibody (CD20 Ab-mPEG scFv) and actively attracting pegylated liposomal doxorubicin to enhance anti-tumor activity. J Nanobiotechnol. 2021;19:16. 10.1186/s12951-020-00752-w. 10.1186/s12951-020-00752-w PMC7796588 33422061

38. Dietmair B Humphries J Mercer TR Thurecht KJ Howard CB Cheetham SW Targeted mRNA delivery with bispecific antibodies that tether LNPs to cell-surface markers Preprint [bioRxiv] 2024 10.1101/2024.10.17.618962 PMC11999258 40235853 Dietmair B, Humphries J, Mercer TR, Thurecht KJ, Howard CB, Cheetham SW. Targeted mRNA delivery with bispecific antibodies that tether LNPs to cell-surface markers. Preprint [bioRxiv]. 2024. 10.1101/2024.10.17.618962. 10.1016/j.omtn.2025.102520 PMC11999258 40235853

39. Sivaram AJ Wardiana A Howard CB Mahler SM Thurecht KJ Recent advances in the generation of antibody-nanomaterial conjugates Adv Healthc Mater 2018 7 1700607 10.1002/adhm.201700607 28961378 Sivaram AJ, Wardiana A, Howard CB, Mahler SM, Thurecht KJ. Recent advances in the generation of antibody-nanomaterial conjugates. Adv Healthc Mater. 2018;7:1700607. 10.1002/adhm.201700607. 10.1002/adhm.201700607 28961378

40. Van Lith SAM Van Duijnhoven SMJ Navis AC Leenders WPJ Dolk E Wennink JWH Legomedicine- a versatile chemo-enzymatic approach for the preparation of targeted dual-labeled Llama antibody–nanoparticle conjugates Bioconjug Chem 2017 28 539 48 10.1021/acs.bioconjchem.6b00638 28045502 PMC5330650 Van Lith SAM, Van Duijnhoven SMJ, Navis AC, Leenders WPJ, Dolk E, Wennink JWH, et al. Legomedicine- a versatile chemo-enzymatic approach for the preparation of targeted dual-labeled Llama antibody–nanoparticle conjugates. Bioconjug Chem. 2017;28:539–48. 10.1021/acs.bioconjchem.6b00638. 28045502 10.1021/acs.bioconjchem.6b00638 PMC5330650

41. Cheng Y-A Wu T-H Wang Y-M Cheng T-L Chen I-J Lu Y-C Humanized bispecific antibody (mPEG × HER2) rapidly confers pegylated nanoparticles tumor specificity for multimodality imaging in breast cancer J Nanobiotechnol 2020 18 118 10.1186/s12951-020-00680-9 PMC7457265 32854720 Cheng Y-A, Wu T-H, Wang Y-M, Cheng T-L, Chen I-J, Lu Y-C, et al. Humanized bispecific antibody (mPEG × HER2) rapidly confers pegylated nanoparticles tumor specificity for multimodality imaging in breast cancer. J Nanobiotechnol. 2020;18:118. 10.1186/s12951-020-00680-9. 10.1186/s12951-020-00680-9 PMC7457265 32854720

42. O’Brien MER Wigler N Inbar M Rosso R Grischke E Santoro A Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer Ann Oncol 2004 15 440 9 10.1093/annonc/mdh097 14998846 O’Brien MER, Wigler N, Inbar M, Rosso R, Grischke E, Santoro A, et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol. 2004;15:440–9. 10.1093/annonc/mdh097. 14998846 10.1093/annonc/mdh097

43. Barenholz Y Doxil ® -the first FDA-approved nano-drug: lessons learned J Control Release 2012 160 117 34 10.1016/j.jconrel.2012.03.020 22484195 Barenholz Y. Doxil ® -the first FDA-approved nano-drug: lessons learned. J Control Release. 2012;160:117–34. 10.1016/j.jconrel.2012.03.020. 22484195 10.1016/j.jconrel.2012.03.020

44. d’ Avanzo N Celia C Barone A Carafa M Di Marzio L Santos HA Immunogenicity of polyethylene glycol based nanomedicines: mechanisms, clinical implications and systematic approach Adv Ther 2020 3 1900170 10.1002/adtp.201900170 d’Avanzo N, Celia C, Barone A, Carafa M, Di Marzio L, Santos HA, et al. Immunogenicity of polyethylene glycol based nanomedicines: mechanisms, clinical implications and systematic approach. Adv Ther. 2020;3:1900170. 10.1002/adtp.201900170.

45. López-Estévez AM Lapuhs P Pineiro‐Alonso L Alonso MJ Personalized cancer nanomedicine: overcoming biological barriers for intracellular delivery of biopharmaceuticals Adv Mater 2024 36 2309355 10.1002/adma.202309355 38104275 López-Estévez AM, Lapuhs P, Pineiro‐Alonso L, Alonso MJ. Personalized cancer nanomedicine: overcoming biological barriers for intracellular delivery of biopharmaceuticals. Adv Mater. 2024;36:2309355. 10.1002/adma.202309355. 10.1002/adma.202309355 38104275

46. Zhang Z Sun H Giannino J Wu Y Cheng C Biodegradable zwitterionic polymers as PEG alternatives for drug delivery J Polym Sci 2024 62 10 2231 50 10.1002/pol.20230916 PMC11376432 39247254 Zhang Z, Sun H, Giannino J, Wu Y, Cheng C. Biodegradable zwitterionic polymers as PEG alternatives for drug delivery. J Polym Sci. 2024;62(10):2231–50. 10.1002/pol.20230916. 10.1002/pol.20230916 PMC11376432 39247254

47. Nogueira SS Schlegel A Maxeiner K Weber B Barz M Schroer MA Polysarcosine-functionalized lipid nanoparticles for therapeutic mRNA delivery ACS Appl Nano Mater 2020 3 10634 45 10.1021/acsanm.0c01834 Nogueira SS, Schlegel A, Maxeiner K, Weber B, Barz M, Schroer MA, et al. Polysarcosine-functionalized lipid nanoparticles for therapeutic mRNA delivery. ACS Appl Nano Mater. 2020;3:10634–45. 10.1021/acsanm.0c01834.

48. Liu L Pan D Chen S Martikainen M-V Kårlund A Ke J Systematic design of cell membrane coating to improve tumor targeting of nanoparticles Nat Commun 2022 13 6181 10.1038/s41467-022-33889-3 36261418 PMC9580449 Liu L, Pan D, Chen S, Martikainen M-V, Kårlund A, Ke J, et al. Systematic design of cell membrane coating to improve tumor targeting of nanoparticles. Nat Commun. 2022;13:6181. 10.1038/s41467-022-33889-3. 36261418 10.1038/s41467-022-33889-3 PMC9580449

49. Macário-Soares A Sousa‐Oliveira I Correia M Pires PC Sharma A Kumar Jha N Cell membrane and extracellular vesicle membrane‐coated nanoparticles: an envisaged approach for the management of skin conditions View 2024 5 20240043 10.1002/VIW.20240043 Macário-Soares A, Sousa‐Oliveira I, Correia M, Pires PC, Sharma A, Kumar Jha N, et al. Cell membrane and extracellular vesicle membrane‐coated nanoparticles: an envisaged approach for the management of skin conditions. View. 2024;5:20240043. 10.1002/VIW.20240043.

50. Xu F Jiang D Xu J Dai H Fan Q Fei Z Engineering of dendritic cell bispecific extracellular vesicles for tumor-targeting immunotherapy Cell Rep 2023 42 113138 10.1016/j.celrep.2023.113138 37738123 Xu F, Jiang D, Xu J, Dai H, Fan Q, Fei Z, et al. Engineering of dendritic cell bispecific extracellular vesicles for tumor-targeting immunotherapy. Cell Rep. 2023;42:113138. 10.1016/j.celrep.2023.113138. 37738123 10.1016/j.celrep.2023.113138

51. Jallinoja VIJ Houghton JL Current landscape in clinical pretargeted radioimmunoimaging and therapy J Nucl Med 2021 62 1200 6 10.2967/jnumed.120.260687 34016727 PMC8882889 Jallinoja VIJ, Houghton JL. Current landscape in clinical pretargeted radioimmunoimaging and therapy. J Nucl Med. 2021;62:1200–6. 10.2967/jnumed.120.260687. 34016727 10.2967/jnumed.120.260687 PMC8882889

52. Sharkey RM Karacay H Richel H McBride WJ Rossi EA Chang K Optimizing bispecific antibody pretargeting for use in radioimmunotherapy Clin Cancer Res 2003 9 S3897 913 14506188 Sharkey RM, Karacay H, Richel H, McBride WJ, Rossi EA, Chang K, et al. Optimizing bispecific antibody pretargeting for use in radioimmunotherapy. Clin Cancer Res. 2003;9:S3897–913. 14506188

53. Han X Zhou Y Liu W Precision cardio-oncology: understanding the cardiotoxicity of cancer therapy Npj Precision Onc 2017 1 31 10.1038/s41698-017-0034-x PMC5871905 29872712 Han X, Zhou Y, Liu W. Precision cardio-oncology: understanding the cardiotoxicity of cancer therapy. Npj Precision Onc. 2017;1:31. 10.1038/s41698-017-0034-x. 10.1038/s41698-017-0034-x PMC5871905 29872712

54. Géraud A Hueso T Laparra A Bige N Ouali K Cauquil C Reactions and adverse events induced by T-cell engagers as anti-cancer immunotherapies, a comprehensive review Eur J Cancer 2024 205 114075 10.1016/j.ejca.2024.114075 38733717 Géraud A, Hueso T, Laparra A, Bige N, Ouali K, Cauquil C, et al. Reactions and adverse events induced by T-cell engagers as anti-cancer immunotherapies, a comprehensive review. Eur J Cancer. 2024;205:114075. 10.1016/j.ejca.2024.114075. 38733717 10.1016/j.ejca.2024.114075

55. Haber L Olson K Kelly MP Crawford A DiLillo DJ Tavaré R Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning Sci Rep 2021 11 1 14397 10.1038/s41598-021-93842-0 34257348 PMC8277787 Haber L, Olson K, Kelly MP, Crawford A, DiLillo DJ, Tavaré R, et al. Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning. Sci Rep. 2021;11(1):14397. 10.1038/s41598-021-93842-0. 34257348 10.1038/s41598-021-93842-0 PMC8277787

56. Dang K Castello G Clarke SC Li Y Balasubramani A Boudreau A Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release J Immunother Cancer 2021 9 e002488 10.1136/jitc-2021-002488 34088740 PMC8183203 Dang K, Castello G, Clarke SC, Li Y, Balasubramani A, Boudreau A, et al. Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release. J Immunother Cancer. 2021;9:e002488. 10.1136/jitc-2021-002488. 34088740 10.1136/jitc-2021-002488 PMC8183203

57. Middelburg J Kemper K Engelberts P Labrijn AF Schuurman J van Hall T Overcoming challenges for CD3-bispecific antibody therapy in solid tumors Cancers (Basel) 2021 13 287 10.3390/cancers13020287 33466732 PMC7829968 Middelburg J, Kemper K, Engelberts P, Labrijn AF, Schuurman J, van Hall T. Overcoming challenges for CD3-bispecific antibody therapy in solid tumors. Cancers (Basel). 2021;13:287. 10.3390/cancers13020287. 33466732 10.3390/cancers13020287 PMC7829968

58. Wu L Seung E Xu L Rao E Lord DM Wei RR Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation Nat Cancer 2020 1 86 98 10.1038/s43018-019-0004-z 35121834 Wu L, Seung E, Xu L, Rao E, Lord DM, Wei RR, et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer. 2020;1:86–98. 10.1038/s43018-019-0004-z. 35121834 10.1038/s43018-019-0004-z

59. Ai Z Wang B Song Y Cheng P Liu X Sun P Prodrug-based bispecific antibodies for cancer therapy: advances and future directions Front Immunol 2025 16 1523693 10.3389/fimmu.2025.1523693 39911391 PMC11794264 Ai Z, Wang B, Song Y, Cheng P, Liu X, Sun P. Prodrug-based bispecific antibodies for cancer therapy: advances and future directions. Front Immunol. 2025;16:1523693. 10.3389/fimmu.2025.1523693. 39911391 10.3389/fimmu.2025.1523693 PMC11794264

60. McCue AC Demarest SJ Froning KJ Hickey MJ Antonysamy S Kuhlman B Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy MAbs 2024 16 2373325 10.1080/19420862.2024.2373325 38962811 PMC11225918 McCue AC, Demarest SJ, Froning KJ, Hickey MJ, Antonysamy S, Kuhlman B. Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy. MAbs. 2024;16:2373325. 10.1080/19420862.2024.2373325. 38962811 10.1080/19420862.2024.2373325 PMC11225918

61. Boustany LM LaPorte SL 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 2022 82 4288 98 10.1158/0008-5472.CAN-21-2483 36112781 PMC9664135 Boustany LM, LaPorte SL, Wong L, White C, Vinod V, Shen J, et al. A probody T cell-engaging bispecific antibody targeting EGFR and CD3 inhibits colon cancer growth with limited toxicity. Cancer Res. 2022;82:4288–98. 10.1158/0008-5472.CAN-21-2483. 36112781 10.1158/0008-5472.CAN-21-2483 PMC9664135

62. Gong N Han X Xue L Billingsley MM Huang X El-Mayta R Small-molecule-mediated control of the anti-tumour activity and off-tumour toxicity of a supramolecular bispecific T cell engager Nat Biomed Eng 2024 8 513 28 10.1038/s41551-023-01147-6 38378820 Gong N, Han X, Xue L, Billingsley MM, Huang X, El-Mayta R, et al. Small-molecule-mediated control of the anti-tumour activity and off-tumour toxicity of a supramolecular bispecific T cell engager. Nat Biomed Eng. 2024;8:513–28. 10.1038/s41551-023-01147-6. 38378820 10.1038/s41551-023-01147-6

63. Zhu M Wu B Brandl C Johnson J Wolf A Chow A Blinatumomab, a bispecific t-cell engager (BiTE ® ) for CD-19 targeted cancer immunotherapy: clinical pharmacology and its implications Clin Pharmacokinet 2016 55 1271 88 10.1007/s40262-016-0405-4 27209293 Zhu M, Wu B, Brandl C, Johnson J, Wolf A, Chow A, et al. Blinatumomab, a bispecific t-cell engager (BiTE ® ) for CD-19 targeted cancer immunotherapy: clinical pharmacology and its implications. Clin Pharmacokinet. 2016;55:1271–88. 10.1007/s40262-016-0405-4. 27209293 10.1007/s40262-016-0405-4

64. Zorzi A Linciano S Angelini A Non-covalent albumin-binding ligands for extending the circulating half-life of small biotherapeutics Medchemcomm 2019 10 1068 81 10.1039/c9md00018f 31391879 PMC6644573 Zorzi A, Linciano S, Angelini A. Non-covalent albumin-binding ligands for extending the circulating half-life of small biotherapeutics. Medchemcomm. 2019;10:1068–81. 10.1039/c9md00018f. 31391879 10.1039/c9md00018f PMC6644573

65. Cheng Q Zhang X-N Li J Chen J Wang Y Zhang Y Synthesis of bispecific antibody conjugates using functionalized poly-ADP-ribose polymers Biochemistry 2023 62 1138 44 10.1021/acs.biochem.2c00718 36821831 PMC10033384 Cheng Q, Zhang X-N, Li J, Chen J, Wang Y, Zhang Y. Synthesis of bispecific antibody conjugates using functionalized poly-ADP-ribose polymers. Biochemistry. 2023;62:1138–44. 10.1021/acs.biochem.2c00718. 36821831 10.1021/acs.biochem.2c00718 PMC10033384

66. Zhang W Li D Xu X Chen Y Shi X Pan Y A bispecific peptide-polymer conjugate bridging target-effector cells to enhance immunotherapy Adv Healthc Mater 2023 12 e2202977 10.1002/adhm.202202977 36878223 Zhang W, Li D, Xu X, Chen Y, Shi X, Pan Y, et al. A bispecific peptide-polymer conjugate bridging target-effector cells to enhance immunotherapy. Adv Healthc Mater. 2023;12:e2202977. 10.1002/adhm.202202977. 36878223 10.1002/adhm.202202977

67. Leconet W Liu H Guo M Le Lamer-Déchamps S Molinier C Kim S Anti-PSMA/CD3 bispecific antibody delivery and antitumor activity using a polymeric depot formulation Mol Cancer Ther 2018 17 1927 40 10.1158/1535-7163.MCT-17-1138 29891487 Leconet W, Liu H, Guo M, Le Lamer-Déchamps S, Molinier C, Kim S, et al. Anti-PSMA/CD3 bispecific antibody delivery and antitumor activity using a polymeric depot formulation. Mol Cancer Ther. 2018;17:1927–40. 10.1158/1535-7163.MCT-17-1138. 29891487 10.1158/1535-7163.MCT-17-1138

68. Wei P-S Chen Y-J Lin S-Y Chuang K-H Sheu M-T Ho H-O In situ subcutaneously injectable thermosensitive PEG-PLGA diblock and PLGA-PEG-PLGA triblock copolymer composite as sustained delivery of bispecific anti-CD3 ScFv T-cell/anti-EGFR fab engager (BiTEE) Biomaterials 2021 278 121166 10.1016/j.biomaterials.2021.121166 34634663 Wei P-S, Chen Y-J, Lin S-Y, Chuang K-H, Sheu M-T, Ho H-O. In situ subcutaneously injectable thermosensitive PEG-PLGA diblock and PLGA-PEG-PLGA triblock copolymer composite as sustained delivery of bispecific anti-CD3 ScFv T-cell/anti-EGFR fab engager (BiTEE). Biomaterials. 2021;278:121166. 10.1016/j.biomaterials.2021.121166. 34634663 10.1016/j.biomaterials.2021.121166

69. Alhallak K Sun J Wasden K Guenthner N O’Neal J Muz B Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy Leukemia 2021 35 2346 57 10.1038/s41375-021-01127-2 33479469 PMC8292428 Alhallak K, Sun J, Wasden K, Guenthner N, O’Neal J, Muz B, et al. Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy. Leukemia. 2021;35:2346–57. 10.1038/s41375-021-01127-2. 33479469 10.1038/s41375-021-01127-2 PMC8292428

70. Xu M Wen Y Liu Y Tan X Chen X Zhu X Hollow mesoporous ruthenium nanoparticles conjugated bispecific antibody for targeted anti-colorectal cancer response of combination therapy Nanoscale 2019 11 9661 78 10.1039/C9NR01904A 31065660 Xu M, Wen Y, Liu Y, Tan X, Chen X, Zhu X, et al. Hollow mesoporous ruthenium nanoparticles conjugated bispecific antibody for targeted anti-colorectal cancer response of combination therapy. Nanoscale. 2019;11:9661–78. 10.1039/C9NR01904A. 31065660 10.1039/c9nr01904a

71. Cheng W-J Chuang K-H Lo Y-J Chen M Chen Y-J Roffler SR Bispecific T-cell engagers non-covalently decorated drug-loaded pegylated nanocarriers for cancer immunochemotherapy J Control Release 2022 344 235 48 10.1016/j.jconrel.2022.03.015 35288168 Cheng W-J, Chuang K-H, Lo Y-J, Chen M, Chen Y-J, Roffler SR, et al. Bispecific T-cell engagers non-covalently decorated drug-loaded pegylated nanocarriers for cancer immunochemotherapy. J Control Release. 2022;344:235–48. 10.1016/j.jconrel.2022.03.015. 35288168 10.1016/j.jconrel.2022.03.015

72. Linderman SW DeRidder L Sanjurjo L Foote MB Alonso MJ Kirtane AR Enhancing immunotherapy with tumour-responsive nanomaterials Nat Rev Clin Oncol 2025 22 262 82 10.1038/s41571-025-01000-6 40050505 Linderman SW, DeRidder L, Sanjurjo L, Foote MB, Alonso MJ, Kirtane AR, et al. Enhancing immunotherapy with tumour-responsive nanomaterials. Nat Rev Clin Oncol. 2025;22:262–82. 10.1038/s41571-025-01000-6. 40050505 10.1038/s41571-025-01000-6

73. Fan R Chen C Mu M Chuan D Liu H Hou H Engineering MMP-2 activated nanoparticles carrying B7-H3 bispecific antibodies for ferroptosis-enhanced glioblastoma immunotherapy ACS Nano 2023 17 9126 39 10.1021/acsnano.2c12217 37097811 Fan R, Chen C, Mu M, Chuan D, Liu H, Hou H, et al. Engineering MMP-2 activated nanoparticles carrying B7-H3 bispecific antibodies for ferroptosis-enhanced glioblastoma immunotherapy. ACS Nano. 2023;17:9126–39. 10.1021/acsnano.2c12217. 37097811 10.1021/acsnano.2c12217

74. Li R Shi X Zhang J Liu B Shen J Liu H CaCO3-encapsulated polydopamine with an adsorbed TLR7 agonist for improved tumor photothermal immunotherapy Heliyon 2024 10 e33837 10.1016/j.heliyon.2024.e33837 39050425 PMC11268191 Li R, Shi X, Zhang J, Liu B, Shen J, Liu H, et al. CaCO3-encapsulated polydopamine with an adsorbed TLR7 agonist for improved tumor photothermal immunotherapy. Heliyon. 2024;10:e33837. 10.1016/j.heliyon.2024.e33837. 39050425 10.1016/j.heliyon.2024.e33837 PMC11268191

75. Xu G Luo Y Wang H Wang Y Liu B Wei J Therapeutic bispecific antibodies against intracellular tumor antigens Cancer Lett 2022 538 215699 10.1016/j.canlet.2022.215699 35487312 Xu G, Luo Y, Wang H, Wang Y, Liu B, Wei J. Therapeutic bispecific antibodies against intracellular tumor antigens. Cancer Lett. 2022;538:215699. 10.1016/j.canlet.2022.215699. 35487312 10.1016/j.canlet.2022.215699

76. Middleton MR McAlpine C Woodcock VK Corrie P Infante JR Steven NM Tebentafusp, a TCR/anti-CD3 bispecific fusion protein targeting gp100, potently activated antitumor immune responses in patients with metastatic melanoma Clin Cancer Res 2020 26 5869 78 10.1158/1078-0432.CCR-20-1247 32816891 PMC9210997 Middleton MR, McAlpine C, Woodcock VK, Corrie P, Infante JR, Steven NM, et al. Tebentafusp, a TCR/anti-CD3 bispecific fusion protein targeting gp100, potently activated antitumor immune responses in patients with metastatic melanoma. Clin Cancer Res. 2020;26:5869–78. 10.1158/1078-0432.CCR-20-1247. 32816891 10.1158/1078-0432.CCR-20-1247 PMC9210997

77. Douglass J Hsiue EH-C Mog BJ Hwang MS DiNapoli SR Pearlman AH Bispecific antibodies targeting mutant RAS neoantigens Sci Immunol 2021 6 57 10.1126/sciimmunol.abd5515 PMC8141259 33649101 Douglass J, Hsiue EH-C, Mog BJ, Hwang MS, DiNapoli SR, Pearlman AH, et al. Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol. 2021;6:57. 10.1126/sciimmunol.abd5515. 10.1126/sciimmunol.abd5515 PMC8141259 33649101

78. Ebrahimi S Lohnes BJ Khan SA Peipp M Bockamp E Klein C Targeting mutated KRAS by HLA-A*02:01 restricted anti-KRAS TCR-mimic CAR and bispecific T cell engager J Mol Med 2025 103 1231 47 10.1007/s00109-025-02585-2 40794198 PMC12449438 Ebrahimi S, Lohnes BJ, Khan SA, Peipp M, Bockamp E, Klein C, et al. Targeting mutated KRAS by HLA-A*02:01 restricted anti-KRAS TCR-mimic CAR and bispecific T cell engager. J Mol Med. 2025;103:1231–47. 10.1007/s00109-025-02585-2. 40794198 10.1007/s00109-025-02585-2 PMC12449438

79. López-Estévez AM Sanjurjo L Turrero Á Arriaga I Abrescia NGA Poveda A Nanotechnology-assisted intracellular delivery of antibody as a precision therapy approach for KRAS-driven tumors J Control Release 2024 373 277 92 10.1016/j.jconrel.2024.07.032 39019086 López-Estévez AM, Sanjurjo L, Turrero Á, Arriaga I, Abrescia NGA, Poveda A, et al. Nanotechnology-assisted intracellular delivery of antibody as a precision therapy approach for KRAS-driven tumors. J Control Release. 2024;373:277–92. 10.1016/j.jconrel.2024.07.032. 39019086 10.1016/j.jconrel.2024.07.032

80. Salih HR Jung G The challenges of translation EMBO Mol Med 2019 11 e10874 10.15252/emmm.201910874 31625285 PMC6895598 Salih HR, Jung G. The challenges of translation. EMBO Mol Med. 2019;11:e10874. 10.15252/emmm.201910874. 31625285 10.15252/emmm.201910874 PMC6895598

81. Bhojnagarwala PS O’Connell RP Park D Liaw K Ali AR Bordoloi D In vivo DNA-launched bispecific T cell engager targeting IL-13Rα2 controls tumor growth in an animal model of glioblastoma multiforme Mol Ther 2022 26 289 301 10.1016/j.omto.2022.07.003 PMC9418050 36090479 Bhojnagarwala PS, O’Connell RP, Park D, Liaw K, Ali AR, Bordoloi D, et al. In vivo DNA-launched bispecific T cell engager targeting IL-13Rα2 controls tumor growth in an animal model of glioblastoma multiforme. Mol Ther. 2022;26:289–301. 10.1016/j.omto.2022.07.003. 10.1016/j.omto.2022.07.003 PMC9418050 36090479

82. De Sostoa J Fajardo CA Moreno R Ramos MD Farrera-Sal M Alemany R Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager J Immunother Cancer 2019 7 1 15 10.1186/s40425-019-0505-4 30683154 PMC6347837 De Sostoa J, Fajardo CA, Moreno R, Ramos MD, Farrera-Sal M, Alemany R. Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager. J Immunother Cancer. 2019;7:1–15. 10.1186/s40425-019-0505-4. 30683154 10.1186/s40425-019-0505-4 PMC6347837

83. Basnet S Santos JM Quixabeira DCA Clubb JHA Grönberg-Vähä-Koskela SAM Arias V Oncolytic adenovirus coding for bispecific T cell engager against human MUC-1 potentiates T cell response against solid tumors Mol Ther Oncolytics 2023 28 59 73 10.1016/j.omto.2022.12.007 36699617 PMC9842968 Basnet S, Santos JM, Quixabeira DCA, Clubb JHA, Grönberg-Vähä-Koskela SAM, Arias V, et al. Oncolytic adenovirus coding for bispecific T cell engager against human MUC-1 potentiates T cell response against solid tumors. Mol Ther Oncolytics. 2023;28:59–73. 10.1016/j.omto.2022.12.007. 36699617 10.1016/j.omto.2022.12.007 PMC9842968

84. Stadler CR Bähr-Mahmud H Celik L Hebich B Roth AS Roth RP Elimination of large tumors in mice by mRNA-encoded bispecific antibodies Nat Med 2017 23 815 7 10.1038/nm.4356 28604701 Stadler CR, Bähr-Mahmud H, Celik L, Hebich B, Roth AS, Roth RP, et al. Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nat Med. 2017;23:815–7. 10.1038/nm.4356. 28604701 10.1038/nm.4356

85. Stadler CR Ellinghaus U Fischer L Bähr-Mahmud H Rao M Lindemann C Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human Claudin 6 Sci Transl Med 2024 16 1 13 10.1126/scitranslmed.adl2720 38776391 Stadler CR, Ellinghaus U, Fischer L, Bähr-Mahmud H, Rao M, Lindemann C, et al. Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human Claudin 6. Sci Transl Med. 2024;16:1–13. 10.1126/scitranslmed.adl2720. 10.1126/scitranslmed.adl2720 38776391

86. Hao S Inamdar VV Sigmund EC Zhang F Stephan SB Watson C BiTE secretion from in situ-programmed myeloid cells results in tumor-retained pharmacology J Control Release 2022 342 14 25 10.1016/j.jconrel.2021.12.029 34953983 PMC8840964 Hao S, Inamdar VV, Sigmund EC, Zhang F, Stephan SB, Watson C, et al. BiTE secretion from in situ-programmed myeloid cells results in tumor-retained pharmacology. J Control Release. 2022;342:14–25. 10.1016/j.jconrel.2021.12.029. 34953983 10.1016/j.jconrel.2021.12.029 PMC8840964

87. Chapoval AI Ni J Lau JS Wilcox RA Flies DB Liu D B7-H3: a costimulatory molecule for T cell activation and IFN-γ production Nat Immunol 2001 2 269 74 10.1038/85339 11224528 Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-γ production. Nat Immunol. 2001;2:269–74. 10.1038/85339. 11224528 10.1038/85339

88. Kai X, Zhang Y, Wei B, Tatang D, Angus S, Jin C et al. Tissue-targeted lipid nanoparticle delivery for mRNA encoding bispecific T-cell engager demonstrated potent antitumor effects on both hematological malignancies and solid tumors. J Immunother Cancer. 202;11:A1512–3. 10.1136/jitc-2023-SITC2023.1358. Meeting Abstract: SITC 38th Annual Meeting .

89. Golubovskaya V Sienkiewicz J Sun J Huang Y Hu L Zhou H mRNA-lipid nanoparticle (LNP) delivery of humanized EpCAM-CD3 bispecific antibody significantly blocks colorectal cancer tumor growth Cancers 2023 15 2860 10.3390/cancers15102860 37345198 PMC10216523 Golubovskaya V, Sienkiewicz J, Sun J, Huang Y, Hu L, Zhou H, et al. mRNA-lipid nanoparticle (LNP) delivery of humanized EpCAM-CD3 bispecific antibody significantly blocks colorectal cancer tumor growth. Cancers. 2023;15:2860. 10.3390/cancers15102860. 37345198 10.3390/cancers15102860 PMC10216523

90. Hu L Zhang S Sienkiewicz J Zhou H Berahovich R Sun J HER2-CD3-Fc bispecific antibody-encoding mRNA delivered by lipid nanoparticles suppresses HER2-positive tumor growth Vaccines 2024 12 808 10.3390/vaccines12070808 39066446 PMC11281407 Hu L, Zhang S, Sienkiewicz J, Zhou H, Berahovich R, Sun J, et al. HER2-CD3-Fc bispecific antibody-encoding mRNA delivered by lipid nanoparticles suppresses HER2-positive tumor growth. Vaccines. 2024;12:808. 10.3390/vaccines12070808. 39066446 10.3390/vaccines12070808 PMC11281407

91. Wu L Wang W Tian J Qi C Cai Z Yan W Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery Bioengineered 2021 12 12383 93 10.1080/21655979.2021.2003666 34895063 PMC8810065 Wu L, Wang W, Tian J, Qi C, Cai Z, Yan W, et al. Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery. Bioengineered. 2021;12:12383–93. 10.1080/21655979.2021.2003666. 34895063 10.1080/21655979.2021.2003666 PMC8810065

92. Zeng J Fang Y Zhang Z Lv Z Wang X Huang Q Antitumor activity of Z15-0-2, a bispecific nanobody targeting PD-1 and CTLA-4 Oncogene 2024 43 2244 52 10.1038/s41388-024-03066-5 38806619 PMC11245388 Zeng J, Fang Y, Zhang Z, Lv Z, Wang X, Huang Q, et al. Antitumor activity of Z15-0-2, a bispecific nanobody targeting PD-1 and CTLA-4. Oncogene. 2024;43:2244–52. 10.1038/s41388-024-03066-5. 38806619 10.1038/s41388-024-03066-5 PMC11245388

93. Wang Y Tiruthani K Li S Hu M Zhong G Tang Y mRNA delivery of a bispecific single-domain antibody to polarize tumor-associated macrophages and synergize immunotherapy against liver malignancies Adv Mater 2021 33 1 11 10.1002/adma.202007603 PMC8240965 33945178 Wang Y, Tiruthani K, Li S, Hu M, Zhong G, Tang Y, et al. mRNA delivery of a bispecific single-domain antibody to polarize tumor-associated macrophages and synergize immunotherapy against liver malignancies. Adv Mater. 2021;33:1–11. 10.1002/adma.202007603. 10.1002/adma.202007603 PMC8240965 33945178

94. Hangiu O Navarro R Frago S Rubio-Pérez L Tapia-Galisteo A Díez-Alonso L Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation Front Immunol 2025 15 1494206 10.3389/fimmu.2024.1494206 39835115 PMC11743637 Hangiu O, Navarro R, Frago S, Rubio-Pérez L, Tapia-Galisteo A, Díez-Alonso L, et al. Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation. Front Immunol. 2025;15:1494206. 10.3389/fimmu.2024.1494206. 39835115 10.3389/fimmu.2024.1494206 PMC11743637

95. Hou X Zaks T Langer R Dong Y Lipid nanoparticles for mRNA delivery Nat Rev Mater 2021 6 1078 94 10.1038/s41578-021-00358-0 34394960 PMC8353930 Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6:1078–94. 10.1038/s41578-021-00358-0. 34394960 10.1038/s41578-021-00358-0 PMC8353930

96. Huang C Duan X Wang J Tian Q Ren Y Chen K Lipid nanoparticle delivery system for Mrna encoding B7H3-redirected bispecific antibody displays potent antitumor effects on malignant tumors Adv Sci 2023 10 1 16 10.1002/advs.202205532 PMC9875623 36403209 Huang C, Duan X, Wang J, Tian Q, Ren Y, Chen K, et al. Lipid nanoparticle delivery system for Mrna encoding B7H3-redirected bispecific antibody displays potent antitumor effects on malignant tumors. Adv Sci. 2023;10:1–16. 10.1002/advs.202205532. 10.1002/advs.202205532 PMC9875623 36403209

97. Pardi N Tuyishime S Muramatsu H Kariko K Mui BL Tam YK Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes J Control Release 2015 217 345 51 10.1016/j.jconrel.2015.08.007 26264835 PMC4624045 Pardi N, Tuyishime S, Muramatsu H, Kariko K, Mui BL, Tam YK, et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J Control Release. 2015;217:345–51. 10.1016/j.jconrel.2015.08.007. 26264835 10.1016/j.jconrel.2015.08.007 PMC4624045

98. Wesselhoeft RA Kowalski PS Anderson DG Engineering circular RNA for potent and stable translation in eukaryotic cells Nat Commun 2018 9 2629 10.1038/s41467-018-05096-6 29980667 PMC6035260 Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018;9:2629. 10.1038/s41467-018-05096-6. 29980667 10.1038/s41467-018-05096-6 PMC6035260

99. Cheng Q Wei T Farbiak L Johnson LT Dilliard SA Siegwart DJ Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing Nat Nanotechnol 2020 15 313 20 10.1038/s41565-020-0669-6 32251383 PMC7735425 Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat Nanotechnol. 2020;15:313–20. 10.1038/s41565-020-0669-6. 32251383 10.1038/s41565-020-0669-6 PMC7735425

100. Zhong R Talebian S Mendes BB Wallace G Langer R Conde J Hydrogels for RNA delivery Nat Mater 2023 22 818 31 10.1038/s41563-023-01472-w 36941391 PMC10330049 Zhong R, Talebian S, Mendes BB, Wallace G, Langer R, Conde J, et al. Hydrogels for RNA delivery. Nat Mater. 2023;22:818–31. 10.1038/s41563-023-01472-w. 36941391 10.1038/s41563-023-01472-w PMC10330049

101. Cai J Jiang S Liao J Fan H Peng C Shi S Manganese-doped biostimulatory nanoneedle for MRI-visual bispecific antibody gene delivery and immunosuppression reversal as a cancer immunotherapy strategy Chem Eng J 2023 462 142242 10.1016/j.cej.2023.142242 Cai J, Jiang S, Liao J, Fan H, Peng C, Shi S, et al. Manganese-doped biostimulatory nanoneedle for MRI-visual bispecific antibody gene delivery and immunosuppression reversal as a cancer immunotherapy strategy. Chem Eng J. 2023;462:142242. 10.1016/j.cej.2023.142242.

102. Ayala Ceja M Khericha M Harris CM Puig-Saus C Chen YY CAR-T cell manufacturing: major process parameters and next-generation strategies J Exp Med 2024 221 e20230903 10.1084/jem.20230903 38226974 PMC10791545 Ayala Ceja M, Khericha M, Harris CM, Puig-Saus C, Chen YY. CAR-T cell manufacturing: major process parameters and next-generation strategies. J Exp Med. 2024;221:e20230903. 10.1084/jem.20230903. 38226974 10.1084/jem.20230903 PMC10791545

103. Blanco B Compte M Lykkemark S Sanz L Alvarez-Vallina L T cell-redirecting strategies to stab tumors: beyond cars and bispecific antibodies Trends Immunol 2019 40 243 57 10.1016/j.it.2019.01.008 30827461 Blanco B, Compte M, Lykkemark S, Sanz L, Alvarez-Vallina L. T cell-redirecting strategies to stab tumors: beyond cars and bispecific antibodies. Trends Immunol. 2019;40:243–57. 10.1016/j.it.2019.01.008. 30827461 10.1016/j.it.2019.01.008

104. Blanco B Holliger P Vile RG Alvarez-Vallina L Induction of human T lymphocyte cytotoxicity and Inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells J Immunol 2003 171 1070 7 10.4049/jimmunol.171.2.1070 12847281 Blanco B, Holliger P, Vile RG, Alvarez-Vallina L. Induction of human T lymphocyte cytotoxicity and Inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells. J Immunol. 2003;171:1070–7. 10.4049/jimmunol.171.2.1070. 12847281 10.4049/jimmunol.171.2.1070

105. Blanco B Ramírez-Fernández Á Bueno C Argemí-Muntadas L Fuentes P Aguilar-Sopeña Ó Overcoming CAR-mediated CD19 downmodulation and leukemia relapse with T lymphocytes secreting anti-CD19 T-cell engagers Cancer Immunol Res 2022 10 498 511 10.1158/2326-6066.CIR-21-0853 35362043 PMC7612571 Blanco B, Ramírez-Fernández Á, Bueno C, Argemí-Muntadas L, Fuentes P, Aguilar-Sopeña Ó, et al. Overcoming CAR-mediated CD19 downmodulation and leukemia relapse with T lymphocytes secreting anti-CD19 T-cell engagers. Cancer Immunol Res. 2022;10:498–511. 10.1158/2326-6066.CIR-21-0853. 35362043 10.1158/2326-6066.CIR-21-0853 PMC7612571

106. Liu X Barrett DM Jiang S Fang C Kalos M Grupp SA Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice Blood Cancer J 2016 6 e430 10.1038/bcj.2016.38 27258611 PMC5141353 Liu X, Barrett DM, Jiang S, Fang C, Kalos M, Grupp SA, et al. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice. Blood Cancer J. 2016;6:e430. 10.1038/bcj.2016.38. 27258611 10.1038/bcj.2016.38 PMC5141353

107. Velasquez MP Torres D Iwahori K Kakarla S Arber C Rodriguez-Cruz T T cells expressing CD19-specific engager molecules for the immunotherapy of CD19-positive malignancies Sci Rep 2016 6 27130 10.1038/srep27130 27255991 PMC4891739 Velasquez MP, Torres D, Iwahori K, Kakarla S, Arber C, Rodriguez-Cruz T, et al. T cells expressing CD19-specific engager molecules for the immunotherapy of CD19-positive malignancies. Sci Rep. 2016;6:27130. 10.1038/srep27130. 27255991 10.1038/srep27130 PMC4891739

108. Díez-Alonso L Falgas A Arroyo-Ródenas J Romencín PA Martínez A Gómez-Rosel M Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo Sci Transl Med 2024 16 734 10.1126/scitranslmed.adg7962 38354229 Díez-Alonso L, Falgas A, Arroyo-Ródenas J, Romencín PA, Martínez A, Gómez-Rosel M, et al. Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo. Sci Transl Med. 2024;16:734. 10.1126/scitranslmed.adg7962. 10.1126/scitranslmed.adg7962 38354229

109. Bonifant CL Szoor A Torres D Joseph N Velasquez MP Iwahori K CD123-engager T cells as a novel immunotherapeutic for acute myeloid leukemia Mol Ther 2016 24 1615 26 10.1038/mt.2016.116 27401038 PMC5113097 Bonifant CL, Szoor A, Torres D, Joseph N, Velasquez MP, Iwahori K, et al. CD123-engager T cells as a novel immunotherapeutic for acute myeloid leukemia. Mol Ther. 2016;24:1615–26. 10.1038/mt.2016.116. 27401038 10.1038/mt.2016.116 PMC5113097

110. Jiménez-Reinoso A Tirado N Martinez-Moreno A Díaz VM García-Peydró M Hangiu O Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers J Immunother Cancer 2022 10 e005333 10.1136/jitc-2022-005333 36564128 PMC9791403 Jiménez-Reinoso A, Tirado N, Martinez-Moreno A, Díaz VM, García-Peydró M, Hangiu O, et al. Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers. J Immunother Cancer. 2022;10:e005333. 10.1136/jitc-2022-005333. 36564128 10.1136/jitc-2022-005333 PMC9791403

111. Compte M Blanco B Serrano F Cuesta AM Sanz L Bernad A Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA x anti-CD3 diabodies from lentivirally transduced human lymphocytes Cancer Gene Ther 2007 14 380 8 10.1038/sj.cgt.7701021 17218946 Compte M, Blanco B, Serrano F, Cuesta AM, Sanz L, Bernad A, et al. Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA x anti-CD3 diabodies from lentivirally transduced human lymphocytes. Cancer Gene Ther. 2007;14:380–8. 10.1038/sj.cgt.7701021. 17218946 10.1038/sj.cgt.7701021

112. Compte M Cuesta AM Sánchez-Martín D Alonso-Camino V Vicario JL Sanz L Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds Stem Cells 2009 27 753 60 10.1634/stemcells.2008-0831 19096041 PMC2729675 Compte M, Cuesta AM, Sánchez-Martín D, Alonso-Camino V, Vicario JL, Sanz L, et al. Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds. Stem Cells. 2009;27:753–60. 10.1634/stemcells.2008-0831. 19096041 10.1634/stemcells.2008-0831 PMC2729675

113. Djouad F Plence P Bony C Tropel P Apparailly F Sany J Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals Blood 2003 102 3837 44 10.1182/blood-2003-04-1193 12881305 Djouad F, Plence P, Bony C, Tropel P, Apparailly F, Sany J, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood. 2003;102:3837–44. 10.1182/blood-2003-04-1193. 12881305 10.1182/blood-2003-04-1193

114. Hill TF Narvekar P Asher G Camp N Thomas KR Tasian SK Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing Preprint [bioRxiv] 2023 10.1101/2023.08.24.554523 PMC11405176 38959896 Hill TF, Narvekar P, Asher G, Camp N, Thomas KR, Tasian SK, et al. Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing. Preprint [bioRxiv]. 2023. 10.1101/2023.08.24.554523.

115. Zhang M Lam K-P Xu S Natural killer cell engagers (NKCEs): a new frontier in cancer immunotherapy Front Immunol 2023 14 1207276 10.3389/fimmu.2023.1207276 37638058 PMC10450036 Zhang M, Lam K-P, Xu S. Natural killer cell engagers (NKCEs): a new frontier in cancer immunotherapy. Front Immunol. 2023;14:1207276. 10.3389/fimmu.2023.1207276. 37638058 10.3389/fimmu.2023.1207276 PMC10450036

116. Gardell JL Matsumoto LR Chinn H DeGolier KR Kreuser SA Prieskorn B Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma J Immunother Cancer 2020 8 e001202 10.1136/jitc-2020-001202 33122397 PMC7597484 Gardell JL, Matsumoto LR, Chinn H, DeGolier KR, Kreuser SA, Prieskorn B, et al. Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma. J Immunother Cancer. 2020;8:e001202. 10.1136/jitc-2020-001202. 33122397 10.1136/jitc-2020-001202 PMC7597484

117. Lee NK Chang JW Manufacturing cell and gene therapies: challenges in clinical translation Ann Lab Med 2024 44 314 23 10.3343/alm.2023.0382 38361427 PMC10961620 Lee NK, Chang JW. Manufacturing cell and gene therapies: challenges in clinical translation. Ann Lab Med. 2024;44:314–23. 10.3343/alm.2023.0382. 38361427 10.3343/alm.2023.0382 PMC10961620

118. Mir LM Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments Methods Mol Biol 2014 1121 3 23 10.1007/978-1-4614-9632-8_1 24510808 Mir LM. Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments. Methods Mol Biol. 2014;1121:3–23. 10.1007/978-1-4614-9632-8_1. 24510808 10.1007/978-1-4614-9632-8_1

119. Mullard A In vivo CAR T cells move into clinical trials Nat Rev Drug Discov 2024 10.1038/d41573-024-00150-z 39284972 Mullard A. In vivo CAR T cells move into clinical trials. Nat Rev Drug Discov. 2024. 10.1038/d41573-024-00150-z. 39284972 10.1038/d41573-024-00150-z

120. Depil S Duchateau P Grupp SA Mufti G Poirot L Off-the-shelf allogeneic CAR T cells: development and challenges Nat Rev Drug Discov 2020 19 185 99 10.1038/s41573-019-0051-2 31900462 Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. Off-the-shelf allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19:185–99. 10.1038/s41573-019-0051-2. 31900462 10.1038/s41573-019-0051-2

121. Hunter TL Bao Y Zhang Y Matsuda D Riener R Wang A In vivo CAR T cell generation to treat cancer and autoimmune disease Science 2025 388 1311 7 10.1126/science.ads8473 40536974 Hunter TL, Bao Y, Zhang Y, Matsuda D, Riener R, Wang A, et al. In vivo CAR T cell generation to treat cancer and autoimmune disease. Science. 2025;388:1311–7. 10.1126/science.ads8473. 40536974 10.1126/science.ads8473

122. Yap TA Hernando-Calvo AH Calvo E Moreno V Marquez R Papadopoulos KP First-in-human phase I/II trial evaluating BNT142, a first-in-class mRNA encoded, bispecific antibody targeting Claudin 6 (CLDN6) and CD3, in patients (pts) with CLDN6-positive advanced solid tumors J Clin Oncol 2025 43 16suppl 2501 2501 10.1200/JCO.2025.43.16_suppl.2501 Yap TA, Hernando-Calvo AH, Calvo E, Moreno V, Marquez R, Papadopoulos P KP, et al. First-in-human phase I/II trial evaluating BNT142, a first-in-class mRNA encoded, bispecific antibody targeting Claudin 6 (CLDN6) and CD3, in patients (pts) with CLDN6-positive advanced solid tumors. J Clin Oncol. 2025;43(16suppl):2501–2501. 10.1200/JCO.2025.43.16_suppl.2501. Meeting Abstract: 2025 ASCO Annual Meeting I.

123. Ingavat N Dzulkiflie N Liew JM Wang X Leong E Loh HP Investigation on environmental factors contributing to bispecific antibody stability and the reversal of self-associated aggregates Bioresour Bioprocess 2024 11 82 10.1186/s40643-024-00796-y 39177850 PMC11343937 Ingavat N, Dzulkiflie N, Liew JM, Wang X, Leong E, Loh HP, et al. Investigation on environmental factors contributing to bispecific antibody stability and the reversal of self-associated aggregates. Bioresour Bioprocess. 2024;11:82. 10.1186/s40643-024-00796-y. 39177850 10.1186/s40643-024-00796-y PMC11343937

124. Shi Y Shi M Wang Y You J Progress and prospects of mRNA-based drugs in pre-clinical and clinical applications Sig Transduct Target Ther 2024 9 322 10.1038/s41392-024-02002-z PMC11564800 39543114 Shi Y, Shi M, Wang Y, You J. Progress and prospects of mRNA-based drugs in pre-clinical and clinical applications. Sig Transduct Target Ther. 2024;9:322. 10.1038/s41392-024-02002-z. 10.1038/s41392-024-02002-z PMC11564800 39543114

125. Fortune A Aime A Raymond D Kumar S Nanotechnology in medicine: a double-edged sword for health outcomes Health Nanotechnol 2025 1 9 10.1186/s44301-025-00008-2 Fortune A, Aime A, Raymond D, Kumar S. Nanotechnology in medicine: a double-edged sword for health outcomes. Health Nanotechnol. 2025;1:9. 10.1186/s44301-025-00008-2.

126. Nabih NW Hassan HAFM Preis E Schaefer J Babker A Abbas AM Antibody-functionalized lipid nanocarriers for RNA-based cancer gene therapy: advances and challenges in targeted delivery Nanoscale Adv 2025 10.1039/d5na00323g 40880603 PMC12371570 Nabih NW, Hassan HAFM, Preis E, Schaefer J, Babker A, Abbas AM, et al. Antibody-functionalized lipid nanocarriers for RNA-based cancer gene therapy: advances and challenges in targeted delivery. Nanoscale Adv. 2025. 10.1039/d5na00323g. 40880603 10.1039/d5na00323g PMC12371570