Bispecific and multispecific immune engagers for redirecting innate and adaptive immunity against hematologic cancers

Abstract

Bispecific antibodies (BsAbs) and multispecific antibodies (MsAbs) represent a transformative class of immunotherapeutics in oncology. These engineered molecules possess the unique ability to simultaneously target two or more distinct antigens, thereby facilitating precise immune cell engagement and disrupting multiple signaling pathways. This comprehensive review focuses on their pivotal role in redirecting both innate (natural killer cells, phagocytes) and adaptive (T cells) immunity, with a particular emphasis on their application against hematologic cancers. Significant clinical successes, exemplified by the FDA approval of agents such as blinatumomab, underscore the profound impact these therapies have had on patient outcomes. Despite their promise, inherent challenges persist, including managing treatment-related toxicities, overcoming tumor resistance mechanisms, and optimizing pharmacokinetic profiles. Ongoing research, however, continues to drive innovative strategies to address these hurdles, highlighting the immense potential of immune engagers to deliver more personalized, scalable, and highly effective cancer treatments in the future.

Introduction

The landscape of cancer therapy has undergone a profound transformation with the advent of immunotherapy, marking a paradigm shift beyond conventional modalities such as surgery, radiation, and chemotherapy [1–3]. This evolution is rooted in the recognition of the immune system’s intrinsic capacity to identify and eliminate malignant cells. While traditional monoclonal antibodies (mAbs) have made significant contributions, their efficacy is often limited by targeting a single antigen, which can lead to the development of resistance mechanisms through pathway switching or the upregulation of alternative receptors by tumor cells [4–6]. This often necessitates the use of combination therapies to achieve sustained therapeutic benefit.

In response to these limitations, bispecific and multispecific antibodies have emerged as a significant advancement in therapeutic antibody engineering[6]. These sophisticated molecules are designed to simultaneously bind two or more distinct epitopes or antigens, offering a more comprehensive and precise treatment approach than their monoclonal counterparts[4, 6]. This integrated approach, where a single molecule achieves the effect of combination therapy, holds the potential to enhance therapeutic efficacy, overcome resistance, and reduce off-target effects by ensuring synchronous targeting and potentially synergistic actions at the cellular level.

At their core, these agents function as “immune cell engagers” (ICEs), engineered constructs specifically designed to bridge immune effector cells—such as T cells, natural killer (NK) cells, and phagocytes—to tumor cells[7, 8]. This physical linkage effectively redirects the immune system’s potent anti-tumor capabilities directly to the malignant cells. A critical advantage of T-cell-redirecting engagers is their ability to induce T-cell activation and tumor killing in a major histocompatibility complex (MHC)-independent manner [9, 10]. This mechanism directly addresses a crucial immune evasion strategy employed by many hematologic malignancies, where tumor cells often downregulate MHC expression to escape T-cell recognition[7, 10]. This fundamental advantage positions immune engagers as particularly potent in contexts where conventional T-cell recognition might be impaired [11].

The ability of bispecific antibodies and T-cell engagers to be readily available as “off-the-shelf” therapies presents a substantial logistical advantage over autologous CAR-T cell therapies. Unlike CAR-T, which requires a complex, multi-week manufacturing process prone to failure and significant delays, immune engagers can be administered immediately upon diagnosis [7, 9, 10, 12, 13]. Furthermore, compared to small molecule inhibitors (e.g., tyrosine kinase inhibitors) that target specific intracellular enzymatic pockets and are frequently limited by the rapid emergence of secondary resistance mutations, immune engagers leverage the broad cytotoxic power of the immune system to mechanically lyse tumor cells independent of specific intracellular signaling pathways. This MHC-independent and signaling-independent mechanism positions immune engagers as a robust alternative for patients who have exhausted conventional targeted therapies [14, 15].

Hematologic cancers, which often originate and disseminate within primary or secondary lymphoid organs, pose unique challenges due to their sophisticated mechanisms of immune evasion[16, 17]. These mechanisms include immunological ignorance, where the cancer fails to alert immune sensing mechanisms; active immune suppression, often involving the recruitment of regulatory T cells (T-regs) and myeloid-derived suppressor cells (MDSCs); a relatively low mutational burden, which may limit the generation of neoantigens; and alterations in antigen presentation pathways[16, 18]. Understanding these complex interactions is crucial for developing effective immunotherapeutic approaches. This review provides a comprehensive examination of the fundamental biology, diverse molecular formats, and mechanisms by which immune engagers redirect both innate and adaptive immunity. It further delves into their clinical progress in hematologic malignancies, discusses associated toxicities and strategies for their management, and explores future directions for these innovative therapeutics.

Fundamental principles of immune engagers

Bispecific and multispecific antibody formats and design principles

Bispecific antibodies (BsAbs) are engineered immunoglobulins capable of simultaneously binding to two distinct epitopes or antigens, a significant departure from conventional monoclonal antibodies (mAbs) which target only a single antigen [4, 19]. Extending this concept, multispecific antibodies (MsAbs) are designed to target three or more antigens, offering enhanced therapeutic potential [19, 20]. This field has experienced rapid growth, with over 100 different molecular formats emerging, each offering unique functional properties[21].

These diverse formats can broadly be classified based on the presence or absence of an Fc region:

• Fc-containing (IgG-like) formats These molecules retain the crystallizable fragment (Fc) region, which confers a longer serum half-life and can mediate Fc-dependent effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) [4, 22]. Often, these formats incorporate purposeful mutations, such as the “Knob-into-Hole” (KiH) technology, to enhance correct heavy chain pairing and ensure proper assembly of asymmetric structures. These can be symmetric or asymmetric in their overall structure [23].

• Fc-devoid (Fragment-based) formats These constructs typically lack the Fc region, comprising minimal antigen-binding fragments such as single-chain variable fragments (scFv) or variable domains from camelid heavy-chain-only antibodies (VHH) [1, 24]. Their smaller size can facilitate enhanced tissue penetration, but the absence of an Fc region generally results in a shorter serum half-life due to rapid renal clearance [25]. Flexible peptide linkers are often introduced to connect the different binding moieties [1].

Several key formats exemplify the ingenuity in this field:

• Bispecific T-cell Engagers (BiTEs) A prominent Fc-devoid format, BiTEs consist of two scFvs connected by a flexible linker. One scFv typically binds to the T cell-specific CD3 complex, while the second targets a tumor-associated antigen (TAA) [25–27]. Blinatumomab, targeting CD19 and CD3, is a seminal example of this class [25, 28].

• Dual-affinity re-targeting (DARTs) These are a type of diabody format, characterized by variable domains of two antigen-binding specificities linked to two independent polypeptide chains, which are stabilized by a C-terminal disulfide bridge [29]. Like BiTEs, DARTs generally lack an Fc domain [29].

• Tandem diabodies (TandAbs) These combine two scFvs for each target, connected by a single polypeptide chain, designed to maintain bivalent avidity and possess a molecular weight that exceeds the renal clearance threshold, thereby extending their half-life compared to smaller fragments [29, 30].

• Trispecific antibodies These molecules integrate the antigen-binding domains of three or more mAbs into a single framework [7, 26, 31]. Examples include designs utilizing “Knob-into-Hole” technology combined with scFv fusions, as seen in novel neutralizing antibodies against sarbecoviruses[32]. Other symmetrical designs aim to target multiple immune receptors and TAAs simultaneously, such as trispecific NK cell engagers [33]. In hematologic malignancies, trispecific antibodies like JNJ-5322 (CD3 x BCMA x GPRC5D) have shown impressive results in phase 1 trials for relapsed/refractory multiple myeloma, with high response rates and manageable toxicities, serving as a background example of advanced multispecific design principles [32].

• Heavy chain-only antibodies (HCAbs)/Single-domain antibodies (VHH) Derived from camelid antibodies, these functional VH regions do not associate with light chains and serve as versatile building blocks for multispecific constructs due to their small size and high tolerance to fusion with other molecules [34, 35].

The design of immune engagers is an intricate process, often involving an iterative optimization cycle. Initial Fc-devoid formats, while potent, faced limitations such as short half-lives and the need for frequent, often continuous, dosing [21]. This led to the development of Fc-containing or half-life-extended (HLE) formats to improve pharmacokinetics [7, 36]. However, even these advancements present trade-offs; for instance, prolonged exposure from HLE formats might be undesirable if toxicity emerges [25]. This ongoing refinement suggests that the optimal molecular format is highly context-dependent, tailored to specific disease biology, target expression, and the desired therapeutic window, driving the continuous emergence of new designs [21, 37] (Table 1).

Table 1.

Key bispecific and multispecific antibody formats

Format name	Key structural features	Valency/specificity	Advantages	Disadvantages	Representative examples	Refs.
BiTE (Bispecific T-cell Engager)	Fc-devoid; two scFv fragments linked by a flexible peptide linker	Bispecific (e.g., CD3 x TAA)	Small size, enhanced tissue penetration, MHC-independent T-cell activation	Short half-life (1–4 h), requires continuous infusion, potential for systemic toxicity	Blinatumomab (CD19xCD3)	[25]
DART (Dual-Affinity Re-Targeting)	Fc-devoid; variable domains of two specificities on two polypeptide chains, stabilized by disulfide bridge	Bispecific (e.g., CD3 x TAA)	High stability, avoids in vitro T-cell gene transduction needs	Short half-life, rapid clearance	Flotetuzumab (CD123xCD3)	[9]
TandAb (Tandem Diabody)	Fc-devoid; two scFv fragments for each target, connected by single polypeptide	Bivalent bispecific	Maintains bivalent avidity, molecular weight exceeds renal clearance threshold	Complex assembly, potential for aggregation	AFM-11 (CD19xCD3 for ALL, terminated)	[29]
IgG-like Bispecific Antibody (e.g., KiH)	Fc-containing; full-length IgG structure with engineered heavy chains (e.g., Knob-into-Hole)	Bispecific or Trispecific	Long serum half-life (~ 7 days), retains Fc-mediated effector functions, easier purification (Protein A)	Potential for immunogenicity, complex engineering for correct pairing	Teclistamab (BCMAxCD3), Elranatamab (BCMAxCD3), Talquetamab (GPRC5DxCD3), Tri-1/Tri-2 (SARS-CoV-2),Tri-1/Tri-2 (SARS-CoV-2, background example)	[9]
HLE-BiTE (Half-Life-Extended BiTE)	Fc-containing; BiTE fused to an Fc domain or albumin	Bispecific (e.g., CD3 x TAA)	Extended serum half-life, reduced dosing frequency	Potential for prolonged exposure-related toxicity, increased size	AMG 701 (BCMAxCD3)	[25]
Trispecific Antibodies	Integrates binding domains of three mAbs; various formats (e.g., scFv fusions, symmetrical IgG-like)	Trispecific	Enhanced binding affinity, broader neutralization, potential to prevent escape mutants, synergistic effects	Increased production complexity, potential for immunogenicity	HC16-HCP (EGFR/PD-L1/CD16a), Tri-1/Tri-2 (SARS-CoV-2), HC16-HCP (EGFR/PD-L1/CD16a, background example for solid tumors), Tri-1/Tri-2 (SARS-CoV-2, background example); JNJ-5322 (CD3 x BCMA x GPRC5D for MM)	[32]
HCAbs/VHH (Heavy Chain-Only/Single-Domain Antibodies)	Smallest functional antibody fragments, functional VH regions without light chains	Monospecific (as building blocks)	Small size, high tissue penetration, tolerates fusion to other molecules, ideal as modular building blocks for multispecifics	Poor biophysical characteristics when expressed alone, may require engineering for stability	Nanobody-based CD38-specific hcAbs for MM	[38]

All examples are for hematologic malignancies unless otherwise specified as background for non-hematologic indications. BCMA B-cell maturation antigen; GPRC5D G-protein-coupled receptor family C group 5 member D; ALL acute lymphoblastic leukemia; MM multiple myeloma

Chemical Conjugation Strategies: While genetic engineering remains the dominant method for production, chemical conjugation offers a versatile alternative for constructing bispecific and multispecific antibodies. Chemical techniques, such as click chemistry and bio-orthogonal conjugation, allow for the precise coupling of distinct antibody fragments (e.g., Fab or scFv) or the attachment of non-proteinaceous payloads. Recent advancements in site-specific bioconjugation have enabled the synthesis of homogenous bispecific constructs with defined stoichiometry, bypassing some of the chain-pairing issues inherent in recombinant co-expression [39, 40]. These methods expand the toolkit for antibody engineering, particularly for generating novel formats that may be difficult to express biologically.

Developability considerations are paramount for the successful translation of these molecules into therapeutics [41]. Fc-devoid formats, while offering better biodistribution, are often prone to aggregation, leading to issues with solubility and stability [23]. Linkers, crucial for connecting different moieties, can introduce immunogenicity or alter pharmacokinetic properties[23, 41]. Engineered sequence elements or impurities from manufacturing can also increase the risk of anti-drug antibody (ADA) generation, potentially diminishing therapeutic efficacy [23, 42]. Furthermore, polyspecificity (off-target binding) and polyreactivity (nonspecific binding) are undesirable attributes that can lead to off-target toxicity [23, 43, 44]. The structural heterogeneity of BsAbs/MsAbs, especially with complex formats, poses significant challenges for high-volume production and purification, often requiring custom processes and bespoke analytical assays to distinguish correctly paired products from impurities[45]. The field is moving beyond simple dual-antigen binding to creating highly sophisticated, multi-functional molecules. These next-generation engagers are designed not just to bridge cells or block pathways, but to actively sculpt the tumor microenvironment, provide costimulation, or deliver localized immune-modulatory signals, effectively acting as “mini-immune systems” in a single construct. This increasing complexity necessitates the adoption of advanced computational design and machine learning tools to predict and control their assembly and function [34].

Mechanisms of action: bridging immune effector cells to cancer cells

The core principle underpinning the function of immune engagers is the physical linkage of immune effector cells to target tumor cells [7, 46] This strategic redirection harnesses the immune system’s anti-tumor potential, effectively overcoming critical barriers in cancer treatment such as immune evasion and MHC downregulation [7].

The fundamental mechanism involves the formation of an immunological synapse. This occurs when the immune engager simultaneously binds to a tumor-associated antigen (TAA) expressed on the surface of the cancer cell and an activating receptor present on the immune effector cell, such as CD3 on T cells or CD16a on natural killer (NK) cells [21, 47]. This precise molecular bridge brings the effector cell into intimate contact with the malignant target. Upon formation of this synapse, the engaged immune cells undergo activation and proliferation, leading to the release of potent cytotoxic molecules, including perforin and granzymes, which subsequently induce apoptosis and lysis of the tumor cells [9, 48]. A critical advantage, particularly for T-cell-redirecting engagers, is their ability to induce T-cell activation and tumor killing in a major histocompatibility complex (MHC)-independent manner [9, 49]. This mechanism is crucial as it bypasses common immune escape strategies employed by tumor cells, which often involve the downregulation of MHC expression to evade recognition by endogenous T cells [7, 50].

Beyond direct immune cell engagement, some multispecific antibodies are designed to simultaneously block multiple signaling pathways implicated in disease progression [51]. This provides a broader and potentially more effective therapeutic approach by interfering with redundant or compensatory survival mechanisms within the tumor [4, 52]. For example, amivantamab, an approved bispecific antibody, targets both EGFR and MET receptors. While it can engage immune cells via its Fc domain, its primary bispecific utility lies in disrupting multiple oncogenic signaling pathways simultaneously to inhibit tumor growth and promote cell death [4, 50]. Note that amivantamab is primarily used in solid tumors (e.g., non-small cell lung cancer) and serves here as a background example of dual signaling blockade; hematologic-specific examples include CD20xCD3 BiAbs that combine T-cell engagement with signaling disruption in B-cell malignancies [4, 53].

The efficacy of these bispecific agents can be influenced by several factors, including their functional affinity, the expression levels of target receptors, the ratio of effector to target cells, and the presence of soluble target antigens [21, 54]. The direct engagement and activation of immune cells by engagers introduce a unique pharmacokinetic/pharmacodynamic (PK/PD) complexity not typically observed with traditional targeted therapies [55]. The observed “nonintuitive dose response relationships” indicate that optimal dosing is not simply a matter of drug concentration but rather a dynamic interplay with the immune system and the tumor microenvironment. This necessitates sophisticated modeling and careful dose escalation strategies to balance therapeutic efficacy with potential toxicity, often requiring continuous infusion or step-up dosing regimens [9]. This highlights that immune engagers are not merely molecular bridges but multifaceted immune modulators, actively reprogramming the tumor microenvironment and bypassing key immune escape pathways, leading to a more profound anti-tumor effect (Fig. 1).

Fig. 1.

Mechanisms of immune engager therapies. A Immune Cell Bridging: An immunological synapse is formed when a bispecific antibody (e.g., a T-cell engager) simultaneously binds a tumor-associated antigen (TAA) on the cancer cell and the CD3 receptor on a T cell. This physical linkage triggers the release of perforin and granzymes (cytotoxic granules), leading to tumor cell lysis. B Dual Signaling Blockade: Bispecific antibodies (e.g., amivantamab) can simultaneously bind two different receptors on the tumor cell surface (e.g., EGFR and MET). This inhibits redundant oncogenic signaling pathways while potentially engaging immune effector cells via the Fc region (ADCC), resulting in inhibited tumor growth. Note: Examples in (B) are from solid tumors as background; hematologic equivalents include CD20xCD3 for B-cell signaling disruption

Redirecting adaptive immunity: T-cell engagers

Mechanism of T-cell engagement

T-cell engagers (TCEs) represent a cornerstone of bispecific antibody therapy, primarily leveraging the CD3 complex on T cells as their central immune effector target[21]. CD3 is a universal marker for T cells and an indispensable component of the T-cell receptor (TCR) complex, making it an ideal handle for redirecting T-cell cytotoxicity [56].

The fundamental action of TCEs involves the precise formation of an artificial immunological synapse [57], as described in Section I.B. This engagement triggers robust T-cell activation, proliferation, and the subsequent release of a cascade of pro-inflammatory cytokines [21, 58]. Following activation, these redirected T cells induce tumor cell death through the release of cytotoxic granules containing perforin and granzymes, leading to target cell apoptosis [9].

The potent T-cell activation, while crucial for efficacy, is inherently linked to the primary safety challenges observed with TCEs, namely cytokine release syndrome (CRS) and neurotoxicity [25, 59] This highlights a delicate balance where the therapeutic strength of T-cell engagers is also their main safety concern, necessitating sophisticated dose optimization strategies and concurrent management protocols. To mitigate systemic toxicity and enhance tumor-localized T-cell activation, the relative affinity of the engager for the TAA compared to CD3 can be precisely optimized [11, 21]. For instance, a lower affinity for CD3, as seen with Tnb-486 (a CD19xCD3 bispecific antibody), has been associated with a reduced incidence of neurotoxicity [9]. This strategic tuning of binding affinities is a critical design principle that directly impacts the therapeutic window, aiming to achieve a favorable safety profile by limiting off-target T-cell activation and systemic cytokine release, thereby allowing for higher, more effective doses to be be administered within the tumor microenvironment [60] (Fig. 2).

Fig. 2.

Mechanism of T-cell engagers (TCEs). A Bispecific T-cell Engager (BiTE) facilitates the interaction between a cytotoxic T cell and a tumor cell. The anti-CD3 arm binds to the T-cell receptor complex, while the anti-TAA arm binds to the antigen on the tumor cell. This bridging induces T-cell activation, proliferation, and the secretion of pro-inflammatory cytokines (e.g., IFN-γ, TNF-α). Consequently, the T cell releases cytotoxic granules containing perforin and granzymes, inducing apoptosis in the target tumor cell independent of MHC presentation. Note: All mechanisms shown are applicable to hematologic malignancies; no non-hematologic examples

Clinical applications in hematologic malignancies

Bispecific and multispecific immune engagers have demonstrated remarkable clinical activity across various hematologic malignancies, offering novel therapeutic avenues, particularly in relapsed/refractory settings.

Multiple myeloma (MM)

In multiple myeloma, B-cell maturation antigen (BCMA) has emerged as a prime therapeutic target due to its high and selective expression on malignant plasma cells[9]. Several BCMAxCD3 bispecific antibodies have achieved significant clinical milestones:

• Teclistamab Approved for relapsed/refractory MM (RRMM), teclistamab demonstrated an overall response rate (ORR) of 63.0% and a complete response (CR) in 39.4% of patients in the MajesTEC-1 trial [9, 61, 62]. Common adverse events included grade 1–2 cytokine release syndrome (CRS), cytopenias, and infections[9].

• Elranatamab This agent has shown promise in RRMM, achieving an ORR of 61.0% and CR in 27.6% of patients in the MagnetisMM-3 trial, with CRS being the most common side effect[9, 63].

• Linvoseltamab In the LINKER-MM1 trial, linvoseltamab achieved an ORR of 64% at a higher dose (200 mg) in triple-refractory MM [9].

• ABBV-383 A phase 1 trial reported an ORR of 57% and CR in 29% of RRMM patients, notably without requiring step-up dosing [9, 64]. Other BCMAxCD3 BiTEs under investigation include AMG 420 and AMG 701 [65].

Beyond BCMA, G-protein-coupled receptor family C group 5 member D (GPRC5D) is another promising target, with primary expression on malignant plasma cells and restricted expression on normal tissues [9, 66].

• Talquetamab This GPRC5DxCD3 bispecific antibody reported ORRs of 64–70% in heavily pretreated RRMM patients in the MonumenTAL-1 trial [9, 67]. Unique adverse events included skin and nail disorders [9].

• RG6234 Demonstrated an ORR of 71.4% in RRMM patients in a phase 1 trial, including those with prior BCMA-targeting bispecific antibody therapy [9].

Fc Receptor Homolog 5 (FcRH5) is also being explored as a target, given its enrichment on malignant plasma cells [65]. Cevostamab (anti-FcRH5) has shown treatment responses in patients previously exposed to various therapies [9].

Acute lymphoblastic leukemia (ALL)

Blinatumomab (CD19xCD3) has pioneered the field of bispecific antibody development and is a cornerstone therapy for B-cell precursor ALL. It is approved for both relapsed/refractory B-cell precursor ALL and minimal residual disease (MRD)-positive B-ALL [7, 68].

• Efficacy The TOWER trial demonstrated significant improvements in overall survival (7.7 months vs. 4 months) and complete remission rates (34% vs. 16%) compared to chemotherapy in relapsed/refractory B-ALL [25]. It also achieved complete MRD response in 78% of MRD-positive B-ALL patients, leading to improved overall and relapse-free survival [25].

• Safety Generally favorable, with primary toxicities including infection, hematologic toxicity, and neurotoxicity, though with a lower incidence of CRS compared to other bispecific antibodies [25]. Neurotoxicity, while more frequent, is often transient [9].

Long-term outcomes for immune engagers are beginning to emerge, particularly for blinatumomab. Final analysis of the TOWER study confirmed a persistent overall survival benefit compared to chemotherapy. More notably, in the BLAST study evaluating patients with minimal residual disease (MRD), blinatumomab induced a complete MRD response in 78% of patients, which translated into a median overall survival of 36.5 months and an estimated 5-year overall survival rate of 43% in complete responders [69].

In non-Hodgkin lymphoma, updated 3-year follow-up data for mosunetuzumab in relapsed/refractory follicular lymphoma showed an estimated 36-month overall survival rate of 82.4%, with durable remissions persisting after the completion of fixed-duration therapy [70]. Similarly, in multiple myeloma, long-term follow-up from the MajesTEC-1 trial of teclistamab (median follow-up 30.4 months) reported a median overall survival of 22.2 months, with responses deepening over time [71]. These data underscore that unlike conventional salvage chemotherapy, which often serves merely as a bridge to transplant, immune engagers have the potential to induce durable, long-term remissions.

Non-hodgkin lymphoma (NHL)

For non-Hodgkin lymphoma, CD20 is a highly attractive target due to its expression on malignant B cells and minimal risk of myelosuppression [9].

• CD20xCD3 bispecific antibodies Glofitamab, mosunetuzumab, epcoritamab, and odronextamab have shown significant activity across various NHL subtypes [9, 72].

• In relapsed/refractory follicular lymphoma (RRFL), mosunetuzumab and odronextamab achieved CR rates of 60% and 75%, respectively[9]. For diffuse large B-cell lymphoma (DLBCL), CR rates ranged from 37–39% [9].

• Safety Low-grade CRS is the most common toxicity, with severe CRS being rare[9] Severity can be mitigated by step-up dosing and premedication [9]. These agents generally show a lower incidence and severity of immune effector cell-associated neurotoxicity syndrome (ICANS) compared to CAR-T therapy [9].

CD19-targeting bispecific antibodies have also been evaluated. While blinatumomab showed substantial efficacy, its development for NHL was halted due to high rates of potentially severe neurological events [9, 73]. In contrast, Tnb-486 (CD19xCD3) demonstrated high CR rates in RRFL with lower ICANS and CRS incidence, likely attributed to its low-affinity CD3 moiety [9]. The higher incidence of ICANS with CD19xCD3 bispecific antibodies is potentially linked to on-target off-tumor toxicity from CD19 expression on pericytes and vascular smooth muscles lining the blood-brain barrier [9, 74].

Acute myeloid leukemia (AML)

Acute myeloid leukemia presents unique challenges for bispecific antibody development, primarily due to the limited number of ubiquitously expressed target antigens and concerns about on-target off-tumor toxicities arising from overlapping antigen expression on hematopoietic stem cells [9].

• CD33 targeting CD33 is expressed on immature myeloid blasts and leukemic stem cells, but also on hematopoietic stem cells, which increases the risk of myelosuppression [9].While some CD33xCD3 BiTE clinical trials were terminated, JNJ-67,561,244 and AMV564 are currently in clinical development [9, 74].

• CD123 targeting CD123 is widely expressed on leukemic stem cells and myeloid blasts [9]. Flotetuzumab (CD123xCD3 DART) and XmAb14045 (CD123xCD3 BiAb) have shown anti-leukemic activity [9, 73]. AFM28 (CD123xCD16A) and Vibecotamab (CD123xCD3) have also demonstrated promising efficacy in heavily pretreated relapsed/refractory AML [75].

• CLL-1 (CLEC12A) targeting C-type lectin-like molecule-1 (CLL-1) is expressed on leukemic stem cells and myeloid blasts but not on hematopoietic stem cells, making it a particularly promising target [9, 73].

• FLT-3 targeting Fms-like tyrosine kinase 3 (FLT-3) is expressed on AML cells in most patients and is mutated in approximately 30% [76]. CLN-049 (CD3xFLT3 BiTE) is currently in phase 1 clinical trials [9].

The selection of a tumor-associated antigen is paramount, as a truly “tumor-specific” antigen is rare; some level of off-target expression on healthy tissues is common [25]. Therefore, the therapeutic window for immune engagers is often defined by the differential expression levels between malignant and normal tissues [46]. This drives the continuous search for novel targets with highly restricted expression and the development of strategies to mitigate on-target, off-tumor toxicities. The success of these agents in heavily pretreated patients is paving the way for immune engagers to become a cornerstone of cancer therapy, potentially shifting from salvage options to frontline treatments in select cases, or reducing the need for more intensive therapies like allogeneic stem cell transplantation [9, 77]. However, these future possibilities must be tempered by current limitations, including high rates of infections due to hypogammaglobulinemia, mechanisms of relapse (e.g., antigen loss), and the lack of long-term follow-up data beyond 2–3 years for most agents [78–80].

FDA/EMA approved T-cell engagers and their efficacy

The rapid clinical translation of bispecific antibodies has led to several approvals, particularly for hematologic malignancies. As of recent reviews, a growing number of these agents have received regulatory approval, underscoring their transformative impact (Table 2).

Table 2.

FDA/EMA approved immune engagers for hematologic malignancies

Drug name (Trade name)	Target antigens	Disease indication	Key clinical efficacy data (ORR/CR/OS/MRD Negativity)	Common toxicities	Refs.
Blinatumomab (Blincyto)	CD19xCD3	Relapsed/Refractory B-cell Precursor ALL; MRD-positive B-cell Precursor ALL	R/R ALL: OS 7.7 vs. 4 months (chemo); CR 34% vs. 16% (chemo) [25] MRD-positive ALL: 78% complete MRD response [25].	CRS (lower incidence), Neurotoxicity (more frequent, transient), Infection, Hematologic toxicity	[25]
Teclistamab (Tecvayli)	BCMAxCD3	Relapsed/Refractory Multiple Myeloma (RRMM)	ORR 63.0%, CR 39.4% (MajesTEC-1 trial)	CRS (grade 1–2 common), Cytopenias, Infections	[9]
Elranatamab (Elrexfio)	BCMAxCD3	Relapsed/Refractory Multiple Myeloma (RRMM)	ORR 61.0%, CR 27.6% (MagnetisMM-3 trial)	CRS (most common), Hematologic toxicity	[9]
Talquetamab (Talvey)	GPRC5DxCD3	Relapsed/Refractory Multiple Myeloma (RRMM)	ORR 64–70% (MonumenTAL-1 trial)	CRS (77–80%, primarily grade 1–2), Hematologic toxicity, Skin and nail disorders	[9]
Mosunetuzumab (Lunsumio)	CD20xCD3	Relapsed/Refractory Follicular Lymphoma (RRFL)	RRFL: CR rates 60%. DLBCL: CR rates 37–39% (with glofitamab, epcoritamab)	Low-grade CRS (most common), Infections, ICANS (lower incidence than CAR-T)	[9]
Epcoritamab (Epkinly)	CD20xCD3	Relapsed/Refractory Diffuse Large B-cell Lymphoma (RR DLBCL)	DLBCL: CR rates 37–39%	Low-grade CRS (most common), Infections, ICANS (lower incidence than CAR-T)	[9]
Glofitamab (Columvi)	CD20xCD3	Relapsed/Refractory Diffuse Large B-cell Lymphoma (RR DLBCL)	DLBCL: CR rates 37–39%, better durability	Low-grade CRS (most common), Infections, ICANS (lower incidence than CAR-T)	[9]

Approval dates and specific efficacy data may vary slightly based on the most recent updates from regulatory bodies

Harnessing innate immunity: NK cell and phagocyte engagers

While T-cell engagers have dominated the clinical landscape, there is a growing recognition of the critical role of innate immunity in anti-tumor responses. Strategies to harness natural killer (NK) cells and phagocytes through immune engagers offer complementary approaches, particularly valuable in contexts where adaptive immune responses are compromised or tumor cells employ specific evasion mechanisms. These approaches are still early in development compared to T-cell engagers, with less clinical information available, reflecting their current limitations but also their promise for future expansion in hematologic malignancies.

NK cell engagers (NKCEs): mechanisms and targets

Natural Killer (NK) cells are essential components of the innate immune system, possessing the inherent capacity for direct killing of cancer cells and the ability to amplify anti-tumor immune responses through the secretion of cytokines [81]. A key advantage of NK cells is their effectiveness against tumors with low immunogenicity or those that have downregulated MHC expression, as NK cell activation does not require antigen presentation via MHC molecules.3.

NK cell engagers (NKCEs) are engineered constructs designed to bridge NK cells to tumor cells. This is achieved by simultaneously binding to a tumor-associated antigen (TAA) on the cancer cell and an activating receptor on the NK cell [7]. This interaction facilitates the formation of an immune synapse, leading to the release of cytotoxic granules and cytokines like IFN-γ, which effectively lyse tumor cells [7].

Key activating receptors on NK cells that are commonly targeted by NKCEs include:

• CD16a (FcγRIII) This is a primary target for NKCEs, as its engagement facilitates antibody-dependent cell-mediated cytotoxicity (ADCC) [7]. Many approved therapeutic antibodies are known to exert their anti-tumor effects, in part, through CD16a-mediated ADCC [33, 82].

• NKG2D, NKp30, NKp46, NKG2C These are other crucial activating receptors on NK cells that can be engaged by NKCEs to induce potent cytotoxicity against tumor cells[7].

The field of NKCEs is evolving with different formats:

• Bispecific killer cell engagers (BiKEs) These are typically composed of two single-chain variable fragments (scFv), one targeting an NK cell activation receptor (e.g., CD16a) and the other binding a TAA on cancer cells [7, 82].

• Trispecific killer cell engagers (TriKEs) Building on the bispecific design, TriKEs incorporate a third domain, often a cytokine such as IL-15, to further enhance NK cell proliferation, survival, and cytotoxic activity [7, 83].

Clinical progress in this area includes AFM13, an NKCE that targets CD30 on tumor cells and CD16a on NK cells, which has shown promising results in clinical trials for Hodgkin lymphoma and other CD30-positive solid tumors[7]. Furthermore, symmetrical trispecific NK cell engagers designed to target EGFR, PD-L1, and CD16a have been developed, demonstrating the ability to mediate potent ADCC effects[33]. In acute myeloid leukemia, AFM28 (CD123xCD16A) has also shown promising efficacy in heavily pretreated relapsed/refractory patients [75]. The Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist (BT7480) also represents a novel approach to activate CD137-expressing immune cells, including NK cells, within the tumor microenvironment to elicit anti-tumor responses [84].

Phagocyte cell engagers (PCEs): mechanisms and targets

Phagocytes, including macrophages and dendritic cells, play a critical role in the immune response by eliminating tumor cells through engulfment and by presenting antigens to activate adaptive immunity [7]. Phagocyte cell engagers (PCEs) are designed to activate these cells to eliminate tumor cells [7, 85].

A key mechanism targeted by PCEs involves blocking the CD47-SIRPα axis [7, 86]. Tumor cells frequently overexpress CD47, which acts as a “Don’t Eat Me” signal by binding to SIRPα on macrophages and dendritic cells, thereby inhibiting phagocytosis[7]. PCEs are designed to block this interaction, thereby restoring macrophage-mediated clearance of tumor cells [7, 86]. Beyond direct phagocytosis, blocking this axis can also reprogram immunosuppressive M2 macrophages into pro-inflammatory M1 macrophages, further boosting the overall anti-tumor immune response[7].

Emerging therapies also include dendritic cell engagers, which aim to link dendritic cells with T cells to enhance immune responses. For example, some strategies focus on bridging cDC1 cells with PD-1 + T cells to strengthen immune synapses and improve antigen presentation[7]. In hematologic malignancies, PCEs remain early-stage, with limited clinical data. Examples include CD47 blockers like magrolimab (anti-CD47 mAb) in phase 3 trials for AML and MDS, showing enhanced phagocytosis when combined with azacitidine [87]. Bispecific macrophage engagers (BiMEs) targeting CD47 and tumor antigens are in preclinical development for MM and lymphomas [88].

Emerging clinical potential in hematologic malignancies

The development of NKCEs and PCEs holds significant clinical potential, particularly for hematologic malignancies. These innate immune engagers are highly relevant for cancers that exhibit low immunogenicity or MHC downregulation, as their mechanisms are largely independent of MHC presentation, offering a distinct advantage over therapies solely reliant on T-cell recognition [7].

This diversification of immune engagement strategies represents a crucial recognition that a truly comprehensive immune redirection must harness both adaptive and innate arms of the immune system. Relying solely on T-cell engagement may leave critical immune evasion pathways unaddressed, especially given tumor heterogeneity and the development of adaptive resistance [89]. Therefore, NKCEs and PCEs offer a complementary approach to T-cell engagers, particularly in cases where T-cell responses are impaired or tumor cells develop resistance to T-cell-mediated killing [11].

While T-cell engagers currently dominate the commercial landscape, NKCEs and PCEs are rapidly progressing, with numerous candidates in clinical trials [7, 8]. Research trends indicate a relatively greater emphasis on hematologic cancers for NKCEs, including acute myeloid leukemia (AML) and multiple myeloma (MM), compared to solid tumors [7, 90]. The distinct mechanisms and advantages of innate and adaptive immune engagers suggest a strong rationale for their combinatorial use. For instance, PCEs could enhance antigen presentation to T cells, while NKCEs could provide immediate cytotoxicity and overcome MHC downregulation, thereby creating a more robust and sustained anti-tumor response [91]. This synergy could address resistance mechanisms and improve the depth and durability of response, representing a key future direction for the field (Fig. 3).

Fig. 3.

Harnessing innate immunity: NK cell and phagocyte engagers. A NK Cell Engagers (NKCEs): Bispecific or trispecific constructs bind the CD16a receptor on Natural Killer (NK) cells and a tumor antigen. This triggers Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and cytokine release. B Phagocyte Cell Engagers (PCEs): These agents block the “Don’t Eat Me” signal by antagonizing the CD47-SIRPα interaction. The Fc region or a second binding arm engages the macrophage, promoting phagocytosis of the tumor cell and shifting the macrophage phenotype from M2 (immunosuppressive) to M1 (pro-inflammatory). Note: All mechanisms shown are applicable to hematologic malignancies; examples like AFM13 (CD30xCD16a for HL) and magrolimab (CD47 blocker for AML/MDS)

Challenges and strategies to enhance immune engager therapy

Despite the remarkable successes of bispecific and multispecific immune engagers, their clinical development and widespread adoption are accompanied by a series of inherent challenges. Addressing these hurdles is crucial for maximizing their therapeutic potential.

Pharmacokinetic and developability considerations

Optimizing the pharmacokinetic (PK) profile and ensuring the developability of immune engagers are critical for their successful translation into effective therapeutics.

• Half-life and dosing regimens Fc-devoid formats, such as early BiTEs, are characterized by short serum half-lives (typically 1–4 h)[21]. This necessitates continuous intravenous infusion, which poses significant logistical challenges for patients and healthcare systems [45]. To overcome this, half-life-extended (HLE) BiTEs have been engineered by incorporating an Fc domain or fusing to albumin, thereby prolonging serum half-life to approximately 7 days and allowing for less frequent dosing [7]. However, prolonged systemic exposure, while convenient, might be undesirable in the event of observed toxicity, requiring careful management [25]. Deviations from native IgG structures in engineered formats can also inherently decrease overall stability [23, 92].

• Immunogenicity The complex engineering involved in creating novel antibody formats, including the introduction of new sequence elements or linkers, can increase the risk of eliciting an undesired immune response in patients, leading to the generation of anti-drug antibodies (ADAs) [23]. ADAs can diminish therapeutic efficacy, alter PK, and cause adverse reactions [93]. Therefore, rigorous in silico prediction tools and in vitro assays (e.g., HLA binding assays, T-cell activation assays, B-cell assays) are employed early in the development pipeline to assess and mitigate immunogenicity risk [23]. Product quality attributes, such as aggregation, also significantly influence immunogenic potential [23].

• Specificity and off-target toxicity Polyspecificity (off-target binding) and polyreactivity (nonspecific binding) are undesirable attributes that can alter efficacy, clearance, and tissue distribution, potentially leading to significant off-target toxicities [23, 44]. A notable example is the neurotoxicity observed with CD19xCD3 bispecific antibodies, which is thought to be partly due to CD19 expression on pericytes and vascular smooth muscles lining the blood-brain barrier [9, 94]. The scarcity of truly tumor-specific antigens means that some level of expression on healthy tissues is a common challenge [25, 44]. To address this, in silico tools and in vitro specificity assays (e.g., human cell microarrays, hydrophobic interaction chromatography) are utilized to predict and identify potential off-target binding[23]. Furthermore, combinatorial targeting of two different tumor antigens can enhance specificity[25]. Innovative strategies like “probodies,” which are designed to be activated only within the tumor microenvironment by tumor-associated proteases, represent an emerging approach to reduce systemic toxicity by localizing therapeutic activity [25, 95].

• Stability (Thermostability, Chemical, Colloidal) The engineering of non-native antibody structures can compromise their inherent stability. This includes thermostability (resistance to unfolding upon heat), chemical stability (integrity of covalent bonds), and colloidal stability (tendency to remain dispersed in solution without aggregating)[23] Liabilities in these areas can lead to aggregation, degradation, and impact shelf-life and activity. Fc-devoid scFv domains, due to their increased flexibility, are particularly prone to aggregation and reduced colloidal stability. Engineering solutions, such as the introduction of disulfide bonds and rational protein design, are employed to improve thermostability. Forced degradation studies and advanced analytical methods like size-exclusion chromatography (SEC) and SDS-PAGE are crucial for monitoring chemical stability and identifying fragmentation [23, 46].

• Manufacturability and production The structural heterogeneity of bispecific and multispecific antibodies, especially with more complex formats, presents significant manufacturing challenges. Incorrectly paired variants are undesirable impurities that are often difficult to distinguish from the desired product and purify effectively [45]. This necessitates advanced cell line development, vector design, and sophisticated purification methods, including LC/MS, to distinguish mispaired variants [23]. Developing bioassays that accurately and reproducibly reflect the dual or multi-mechanisms of action is also a substantial challenge in quality control [23]. In an effort to overcome manufacturing costs and storage/clearance issues, in vivo gene therapy approaches, utilizing modified oncolytic viruses or synthetic nucleic acids, are being explored to enable in situ expression of immune engagers directly at the tumor site [45].

The design of immune engagers is a complex multi-objective optimization problem. Engineers must navigate a “trade-off matrix” where improving one aspect, such as extending half-life, may inadvertently compromise another, such as the toxicity profile or manufacturing complexity. This necessitates a holistic developability assessment from the earliest stages, integrating in silico, in vitro, and in vivo data, to identify the optimal balance for a given therapeutic goal. Furthermore, the field is increasingly moving towards “smart” immune engagers that are not only designed for specific targets but also for controlled delivery and activation. This aims to maximize the therapeutic index by localizing activity and minimizing systemic exposure, representing a significant leap in precision medicine and potentially overcoming some of the most persistent hurdles in cancer immunotherapy.

Managing treatment-related toxicities

The potent immune activation elicited by bispecific and multispecific engagers, while central to their efficacy, can also lead to significant treatment-related toxicities. Effective management of these adverse events is crucial for patient safety and sustained therapeutic benefit.

Cytokine release syndrome (CRS)

• Mechanism CRS is a common acute toxicity associated with T-cell-engaging bispecific antibodies (T-BiSp), driven by robust T-cell activation and an exaggerated systemic inflammatory response [25].

• Clinical manifestations CRS can range in severity from mild fever and constitutional symptoms to severe hypotension, hypoxia, and multi-organ dysfunction [9] Most cases are generally low-grade [9].

• Management strategies Proactive management is key. Dose optimization strategies, such as step-up dosing (SUD), involve administering lower initial doses followed by gradual escalation to attenuate the cascade of cytokine release and reduce overall CRS risk [21]. Pretreatment with corticosteroids (e.g., dexamethasone), antipyretics (e.g., acetaminophen), and antihistamines is commonly employed [9]. In B-cell lymphomas, administration of B-cell depleting agents prior to T-BiSp may also decrease CRS risk [96]. For severe CRS, the IL-6-blocking monoclonal antibody tocilizumab is a critical rescue agent, often administered prophylactically or reactively [9]. Kinase inhibitors (e.g., PI3K, JAK, and mTOR inhibitors) are also being explored for their potential to suppress cytokine release downstream of TNFα signaling [96]. Intensive monitoring and comprehensive supportive care are paramount throughout treatment [9].

Neurotoxicity (Immune effector cell-associated neurotoxicity syndrome—ICANS)

• Mechanism ICANS can arise as a complication of CRS or manifest as an independent toxicity [9, 97]. A higher incidence of ICANS with some CD19xCD3 bispecific antibodies is potentially linked to on-target off-tumor toxicity, given CD19 expression on pericytes and vascular smooth muscles lining the blood-brain barrier [9, 98].

• Symptoms Clinical manifestations can include seizures, confusion, tremors, dysphasia/aphasia, and ataxia [9, 97]. These symptoms are often transient [9].

• Management Management strategies for ICANS are similar to those for CRS, including the use of corticosteroids and tocilizumab [9, 98].

Infections

• Causes Infections are common and can be severe, resulting from multifactorial causes including the underlying refractory disease, prior immunosuppressive therapies, neutropenia, hypogammaglobulinemia (due to B-cell impairment), and T-cell exhaustion induced by prolonged immune activation [9, 99].

• Prevention Prophylactic intravenous immunoglobulin (IVIG) and antimicrobials (antibiotics, antivirals, antifungals) are often recommended to mitigate infection risk [9].

Hematologic toxicity

• Manifestations Patients may experience anemia, thrombocytopenia, and neutropenia [9, 100].

• Management These toxicities are typically managed with supportive measures, such as blood product transfusions and granulocyte colony-stimulating factors [9].

The very mechanism that drives the therapeutic efficacy of immune engagers—potent T-cell activation—also underlies their primary toxicities, particularly CRS and neurotoxicity[9, 46]. This creates a narrow therapeutic window that necessitates precise and proactive management. Effective control of toxicities is not merely a matter of patient safety; it is intrinsically linked to maximizing therapeutic efficacy, as uncontrolled adverse events can lead to treatment interruptions or discontinuation, thereby compromising patient outcomes. This underscores the need for a proactive, multidisciplinary approach to monitoring and intervention, making the “art” of clinical pharmacology as crucial as the “science” of molecular design. The extensive clinical experience gained from CAR-T cell therapies has provided invaluable lessons in recognizing and managing immune-related adverse events, particularly CRS and ICANS. This pre-existing knowledge base has significantly accelerated the clinical development and safer implementation of bispecific and multispecific engagers, allowing for more informed risk mitigation strategies and patient care protocols from the outset.

Overcoming resistance mechanisms

Despite their significant clinical successes, hematologic malignancies can develop various resistance mechanisms to immune engager therapies. Understanding and counteracting these mechanisms are critical for improving long-term outcomes.

Antigen escape

• Mechanism The administration of immune engagers can impose strong selective pressures on tumor clones expressing the target antigen [46]. This inadvertently confers a selective advantage to subclones that lack or have downregulated the target antigen, leading to their expansion and disease relapse [21].

• Strategy To circumvent antigen escape, combinatorial strategies are being explored, including the development of multispecific antibodies (e.g., trispecific antibodies) that target multiple distinct tumor antigens simultaneously [21]. Another approach involves upregulating target antigen expression, for instance, using gamma-secretase inhibitors to increase BCMA expression on multiple myeloma cells [9, 101].

T-cell exhaustion

• Mechanism Prolonged administration of T-cell engagers can lead to continuous T-cell stimulation, resulting in T-cell exhaustion. This state is characterized by an anergic phenotype, impaired cytotoxic function, and altered cytokine production, ultimately promoting tumor survival and disease progression [9].

• Strategy Combining immune engagers with immune checkpoint inhibitors (ICIs), such as anti-PD-1, anti-LAG-3, anti-TIM-3, or anti-TIGIT antibodies, can help overcome T-cell exhaustion and restore T-cell activation and effector functions [21, 102] Additionally, designing engagers that activate co-stimulatory receptors on T cells (e.g., CD28, 4-1BB, OX40) is a promising strategy to enhance T-cell proliferation and sustained activity [7, 103].

Immunosuppressive tumor microenvironment (TME)

• Mechanism The tumor microenvironment in hematologic malignancies can be profoundly immunosuppressive, characterized by the presence and activity of regulatory T cells (T-regs), myeloid-derived suppressor cells (MDSCs), and the upregulation of various immune checkpoint molecules [9, 104]. This hostile environment leads to T-cell dysfunction or anergy, hindering effective anti-tumor responses [16].

• Strategy Modulating the TME with immunomodulatory agents (e.g., IMiDs like lenalidomide or thalidomide), combining engagers with ICIs, or utilizing agents that can reprogram immunosuppressive cells (e.g., phagocyte cell engagers that convert M2 macrophages to pro-inflammatory M1 macrophages) can significantly improve therapeutic responses [7, 105].

Genetic abnormalities and altered intracellular signaling

• Mechanism Specific genetic abnormalities observed in certain leukemias (e.g., AML and ALL) or alterations in intracellular signaling pathways (e.g., disrupted interferon-gamma signaling) can confer intrinsic resistance to immune engager therapies [9].

• Strategy A deeper understanding of these unique resistance pathways is required [16]. Combinatorial approaches that target multiple signaling pathways simultaneously may prove beneficial in overcoming such intrinsic resistance.

Cancer cells exhibit an inherent ability to adapt to immune attack, a process known as adaptive immune resistance, manifested through mechanisms like antigen escape and T-cell exhaustion [106]. This adaptability, conceptually framed by the “three Cs” (camouflage, coercion, cytoprotection) of immune evasion [107], underscores that therapeutic strategies must anticipate and counteract this inherent tumor plasticity. This means moving beyond single-target approaches to multi-target engagers and combination therapies that simultaneously address multiple resistance pathways, effectively engaging in a co-evolutionary arms race with the tumor to maintain therapeutic advantage. Furthermore, future success in immune engager therapy, particularly in more challenging hematologic malignancies, will increasingly depend on strategies that “normalize” or “reprogram” the tumor microenvironment [108]. This involves not just killing tumor cells but actively reversing immunosuppression, enhancing antigen presentation, and fostering a pro-inflammatory, anti-tumor immune milieu, thereby creating a more permissive environment for the engagers to exert their full therapeutic potential.

Limitations and long-term considerations

While immune engagers have shown impressive short-term efficacy, long-term data remain limited, with most follow-up capped at 2–3 years. Relapse rates can be high (up to 50% in some studies), often due to antigen loss or T-cell exhaustion. Infections are a major concern, exacerbated by hypogammaglobulinemia from B-cell depletion, occurring in 70–80% of patients on CD19- or BCMA-targeted therapies and increasing risks of opportunistic pathogens like PCP or VZV. Prophylactic IVIG reduces severe infection rates by up to 90%, but does not fully mitigate risks. Lack of extended data hinders understanding of durable remissions versus late relapses [78, 80].

Novel engineering and combination strategies

The field of immune engagers is characterized by continuous innovation in molecular engineering and strategic combination therapies, aimed at overcoming current limitations and expanding their clinical utility.

Multi-antigenic targeting

• Rationale To overcome antigen escape, address tumor heterogeneity, and achieve broader anti-tumor activity [9, 109].

• Strategies The development of trispecific antibodies that bind to three distinct antigens represents a significant advancement [7] Clinical trials have shown that combinations of bispecific antibodies targeting different antigens, such as BCMA and GPRC5D in multiple myeloma, can yield enhanced overall response rates [9].

Co-stimulatory engagement

• Rationale To enhance T-cell activation, proliferation, and cytotoxicity, and to overcome T-cell exhaustion [7].

• Strategies Designing “Simultaneous Multiple Immune Targeting Engagers (SMiTEs)” that incorporate co-stimulatory signals (e.g., 4-1BB, OX40, CD28) in addition to TAA and CD3 binding [7, 8]. For example, BT7480, a Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist, demonstrates robust CD137 agonism, leading to profound reprogramming of the tumor immune microenvironment [76].

Checkpoint inhibitory T-cell engagers (CiTEs)

• Rationale To combine T-cell redirection with immune checkpoint blockade to overcome immunosuppression within the tumor microenvironment [7].

• Strategies Engineering engagers that simultaneously bind a TAA, CD3, and an immune checkpoint molecule (e.g., PD-1/PD-L1, LAG-3) [21, 110].

Novel delivery methods

• Rationale To improve drug delivery, reduce systemic toxicity, and maintain sustained therapeutic concentrations at the tumor site[45].

Strategies

• Half-life-extended (HLE) BiTEs As previously discussed, incorporating an Fc domain to prolong serum half-life, reducing dosing frequency [7].

• Secreted BiTEs (In vivo production) Genetically modified cells (e.g., CAR T cells, oncolytic viruses) or synthetic nucleic acids (mRNA, plasmid DNA) can be engineered to express and release BiTEs directly at the tumor site [111]. This approach localizes therapeutic activity, potentially reduces systemic toxicity, and may overcome manufacturing costs and storage/clearance issues associated with purified recombinant proteins [7].

• Conditional engagers These “logic-gated” approaches are designed to be activated only by specific factors within the tumor microenvironment (e.g., tumor-associated proteases for “probodies”), thereby minimizing on-target off-tumor toxicity [25, 112].

Combination with conventional therapies

• Rationale To enhance efficacy, overcome resistance, and achieve synergistic effects with existing standard-of-care treatments.

• Strategies Combining immune engagers with chemotherapy, targeted therapies (e.g., tyrosine kinase inhibitors in Philadelphia chromosome-positive ALL), or other immunotherapies (e.g., CAR-T cells, monoclonal antibodies like daratumumab) [9, 113].

The field is increasingly adopting a modular, “Lego-like” approach to antibody engineering, where various “building blocks” (Fab, scFv, Fc, VHH, cytokines) are combined to create diverse and multi-functional formats[4]. This modularity facilitates the rapid prototyping and optimization of complex molecules, including trispecifics, co-stimulatory engagers, and CiTEs [7]. This design philosophy, significantly aided by advancements in computational protein design and machine learning, is accelerating the development of highly customized and sophisticated immune engagers tailored to specific tumor biology and patient needs [11]. The future of immune engagers also lies in blurring the lines between traditional biologics and advanced cell/gene therapies. By leveraging in vivo production or “switchable” systems, researchers aim to combine the advantages of potent, localized cell-mediated immunity with the convenience and scalability of off-the-shelf antibody therapeutics, potentially creating a new class of highly effective and accessible treatments [114] (Table 3).

Table 3.

Emerging targets and investigational immune engagers in hematologic cancers

Target Antigen(s)	Immune Engager Type	Disease Indication	Current Development Stage	Rationale/Potential Advantage	Refs.
GPRC5D	Bispecific (CD3)	Multiple Myeloma	Approved (Talquetamab), Phase 1/2 (RG6234)	High expression on MM cells, restricted normal tissue expression, addresses BCMA antigen escape	[9]
FcRH5	Bispecific (CD3)	Multiple Myeloma	Phase 1 (Cevostamab)	Enriched on malignant plasma cells, maintained expression in RRMM	[9]
CD38	Bispecific (CD3)	Multiple Myeloma	Phase 1 (ISB 1342)	Established MM target, potential for combination with other agents	[9]
CD33	Bispecific (CD3)	Acute Myeloid Leukemia	Phase 1/2 (JNJ-67561244, AMV564)	Expressed on myeloid blasts and LSCs, but also HSCs (toxicity concern)	[9]
CD123	Bispecific (CD3, CD16A)	Acute Myeloid Leukemia	Phase 1/2 (XmAb14045, APVO436, MGD024, Vibecotamab, AFM28)	Widely expressed on leukemic stem cells and myeloid blasts	[9]
CLL-1 (CLEC12A)	Bispecific (CD3)	Acute Myeloid Leukemia	Preclinical/Early Phase	Expressed on LSCs and myeloid blasts, but not HSCs (reduced myelosuppression risk)
FLT-3	Bispecific (CD3)	Acute Myeloid Leukemia	Phase 1 (CLN-049) 6	Mutated in ~ 30% of AML, expressed on LSCs	[9]
CD137 (4-1BB)	Bispecific (TAA x CD137)	Various Cancers	Preclinical/Phase 1 (BT7480) 45	Co-stimulatory engagement to enhance T-cell activation and overcome exhaustion	[7]
LAG-3, TIM-3, TIGIT	Checkpoint Inhibitory TCEs (CiTEs)	Various Cancers	Preclinical/Early Phase	Overcome T-cell exhaustion and immunosuppression in TME 2	[9]
Multiple TAAs (e.g., CD19 & CD22)	Trispecific/Combinatorial Bispecifics	ALL, NHL	Preclinical/Early Phase	Counteract antigen escape and target tumor heterogeneity	[9, 115]

Conclusion and future outlook

Bispecific and multispecific immune engagers have profoundly transformed the treatment landscape of hematologic malignancies, moving beyond the limitations of conventional therapies and even offering distinct advantages over cell-based approaches like CAR-T cells [7]. The field has witnessed remarkable achievements, particularly in redirecting adaptive immunity through T-cell engagers, with several agents now approved for challenging diseases such as acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin lymphoma [25]. The emerging success in harnessing innate immunity via NK cell and phagocyte engagers further broadens the therapeutic arsenal.

Despite these advancements, persistent challenges remain. These include the inherent adaptability of cancer cells leading to antigen escape, the phenomenon of T-cell exhaustion under prolonged stimulation, the pervasive immunosuppressive tumor microenvironment, and the management of treatment-related toxicities such as cytokine release syndrome and neurotoxicity [9].

The future directions for immune engager therapies are focused on addressing these challenges through several innovative avenues:

• Rational design and advanced engineering Continued advancements in protein engineering, increasingly leveraging computational tools and machine learning, are crucial for designing more stable, specific, and multi-functional molecules with optimized pharmacokinetic and pharmacodynamic profiles [23]. This will enable the creation of highly customized engagers that precisely interact with specific tumor biology.

• Multi-pronged immune engagement The field is moving towards engaging multiple immune cell types (T cells, NK cells, phagocytes) and simultaneously targeting multiple antigens or pathways. This multi-pronged approach is essential to overcome resistance mechanisms, address tumor heterogeneity, and achieve deeper, more durable responses[7].

• Personalized and precision approaches The increasing understanding of tumor biology and immune evasion mechanisms will facilitate biomarker-driven patient selection and the development of tailored immunotherapy strategies, ensuring that the right engager is delivered to the right patient at the right time [9].

• Earlier intervention and combination therapies Given their demonstrated efficacy in relapsed/refractory settings, there is an expanding role for immune engagers in earlier disease stages, where tumor burden is lower and the immune microenvironment may be more permissive. Furthermore, synergistic combinations with other therapeutic modalities, including chemotherapy, targeted therapies, and other immunotherapies, hold significant promise to enhance efficacy and prevent the emergence of resistance [9].

• Novel delivery systems The development of in vivo production methods and conditional activation strategies represents a significant step towards improving the therapeutic index and patient convenience. These innovations aim to localize therapeutic activity to the tumor site while minimizing systemic exposure and associated toxicities [45].

In conclusion, the trajectory of bispecific and multispecific immune engagers points towards a future where these sophisticated molecules will serve as a cornerstone of next-generation cancer immunotherapies. Through continued scientific rigor and innovative engineering, they are poised to significantly improve outcomes for patients battling hematologic malignancies, offering hope for more effective and enduring remissions.

Author contributions

Mustafa T. Ardah, Waleed K. Abdulsahib, Ihsan Khudhair Jasim, H. Malathi, Pradeepta Sekhar Patro, D. Alex Anand, Gunjan Mukherjee, Aashna Sinha, and Shakhrijakhon Aminqulov contributed to the conception, design, and drafting of the manuscript. All authors read and approved the final version of the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.