Homogeneous antibody-drug conjugates with dual payloads: potential, methods and considerations

ABSTRACT

The development of site-specific dual-payload antibody-drug conjugates (ADCs) represents a potential advancement in targeted cancer therapy, enabling the simultaneous delivery of two distinct drugs into the same cancer cells to overcome payload resistance and enhance therapeutic efficacy. Here, we examine various methodologies for achieving site-specific dual-payload conjugation, including the use of multi-functional linkers, canonical amino acids, non-canonical amino acids, and enzyme-mediated methods, all of which facilitate precise control over payload attachment while ensuring homogeneity. We explore the implications of different conjugation techniques on drug-to-antibody ratios and the ratios of the two payloads, as well as their impact on process complexity and manufacturability. Additionally, we address the potential advantages of dual-payload ADCs compared to ADCs combined with traditional chemotherapy or single-payload ADC/ADC combinations. By evaluating these innovative methods, we aim to provide a comprehensive understanding of the current landscape in dual-payload ADC development and outline emerging directions necessary for further advancement of this promising therapeutic strategy.

Introduction

Antibody-drug conjugates (ADCs) have gained substantial attention due to their potential for targeted delivery of cytotoxins to cancer cells. This approach minimizes systemic toxicity, enhances therapeutic efficacy, and ultimately broadens the therapeutic index. The first generation of ADCs was created by randomly conjugating the linker payload to solvent-exposed lysine residues, resulting in heterogeneity in both the number of payloads and their distribution on the antibodies.^1–3 These heterogeneous product mixtures revealed a relatively narrow therapeutic index due to variations in linker payload stability, internalization, and pharmacokinetics (PK), which led to different efficacy and safety profiles across various species.^4–6 Second-generation ADCs, inspired by THIOMAB™, feature precise control over the location and number of payloads, demonstrating an improved therapeutic index in preclinical studies.^7,8 Over the past 10 years, the considerable efforts devoted to developing site-specific conjugation methods have yielded numerous options for ADC development.^9–12

There is a critical need to safely enhance the potency of ADCs for targeting of low target antigen-expressing and heterogeneous tumors and to overcome the formation of drug resistance and relapse.^13–16 Various emerging single-agent conjugate modalities, including bispecific ADCs, probody – drug conjugates, immune-stimulating ADCs, and protein-degrader ADCs have been reviewed as next-generation ADC platforms to tackle these challenges.^17,18 Several excellent reviews have outlined the rationale for combining different classes of cytotoxic compounds and immune-activating molecules and other compounds targeting diverse signaling pathways involved in tumorigenesis.^19,20 In contrast to combining chemotherapies or a single-payload ADC with chemotherapy, or the combination of two single-payload ADCs, a dual-payload ADC leverages two payloads with distinct mechanisms of action (MOA) to target tumor cells simultaneously, enhancing therapeutic efficacy by overcoming drug resistance mechanisms and activating immune response^{17,18,21–25} (Figure 1). This review examines various reported methodologies for achieving site-specific dual-payload conjugation, highlighting the advantages and limitations of each approach in terms of flexibility for achieving different drug-to-antibody ratios (DAR) of individual and two combined payloads. It also discusses process development complexities and the potential advantages of dual-payload ADCs.

Figure 1.

Three different combination therapy strategies. In contrast to ADC with chemotherapy, or the combination of two single payload ADCs, dual-payload ADCs deliver the two payloads with distinct MOAs to same tumor cells simultaneously. Three classes of methods to make homogeneous dual-payload ADCs are summarized in this review.

Homogeneous dual-payload conjugation has been exemplified by the introduction of additional orthogonal chemistry through linker design or combinations of orthogonal, site-specific, single-payload conjugation methods. The methods for creating homogeneous dual-payload ADCs can be divided into three classes, as shown in Figure 1, based on the methods of introduction of orthogonality. The first class includes methods that require branched multifunctional linkers and encompasses two categories: one involves branched multifunctional linkers as adapters, consisting of a primary functional group for conjugation to proteins and a second to covalently link two or more functional groups, enabling orthogonal conjugation chemistries for attaching payloads. The other category uses the direct synthesis of a linker with two or more payloads, utilizing only one type of conjugation chemistry. The second class includes methods that use two amino acids, canonical and/or non-canonical amino acids with different chemical activities. The third class includes methods using two residues or tags recognized by enzymes with orthogonal activities for conjugation, or a combination of amino acids and enzyme-mediated conjugation methods.

Potential of dual-payload ADCs to overcome drug resistance resulting from ADC therapy

Clinical data have demonstrated that resistance to a topoisomerase I inhibitor (Topo1i)-based ADC can lead to resistance to other Topo1i-based ADCs, regardless of the target antigen. For example, the Phase I TROPION-PanTumor01 study (NCT03401385) found that only 15% (2 of 14) of patients with prior exposure to Topo1i ADCs, such as sacituzumab govitecan (Trop2, SN-38 Topo1i payload), trastuzumab deruxtecan (Her2, DXd Topo1i payload), and patritumab deruxtecan (Her3, DXd Topo1i payload), had a confirmed overall response to datopotamab deruxtecan (Trop2, DXd Topo1i payload). In contrast, 40% (12 of 30) of Topo1i-naïve patients achieved a confirmed overall response.²⁶ Additionally, data indicated that switching between sacituzumab govitecan and trastuzumab deruxtecan or vice versa consistently resulted in lower response rates and shorter progression-free survival (PFS)^27,28 (Figure 2).

Figure 2.

Impact of sequential ADC treatment on efficacy in clinical trials. (a) Payload resistant to Topo1i linker payloads regardless of antigen targets limits ADC efficacy. (b) Switching payload classes maintains ADC efficacy irrespective of antigen targets.

These findings suggest that the anti-tumor activity of Topo1i-based ADCs is often limited by the development of resistance to the payload itself, a phenomenon known as cross-payload resistance.²⁶ Once resistance to a Topo1i payload develops, it substantially reduces the effectiveness of subsequent treatments with other ADCs that use the same cytotoxic agent, even when targeting distinct tumor antigens. This persistent low response rate underscores the clinical challenge of cross-payload resistance and highlights the critical need for strategies to overcome this limitation in the clinical application of Topo1i-based ADC therapies.

In contrast, switching to an ADC with a payload that utilizes a different mechanism of action (MOA) – such as transitioning from a microtubule inhibitor-based payload to a Topo1i-based payload – can help maintain therapeutic efficacy. For example, the DESTINY-Breast02 Phase 3 trial (NCT03523585) of trastuzumab deruxtecan (HER2 targeting, Topo1i-based payload) demonstrated a 70% confirmed overall response rate with a PFS of 17.8 months, compared to only a 29% response rate and 6.9-month PFS with physician’s choice treatment. Notably, most of the patients in this trial had previously been treated with trastuzumab emtansine (HER2 targeting, maytansine microtubule inhibitor payload).^29,30 In addition, transitioning from sacituzumab govitecan to enfortumab vedotin (Nectin4 targeting, microtubule inhibitor payload) or vice versa has shown significant improvements in both PFS and overall survival (OS).^31,32

These findings suggest that ADCs employing different cytotoxic agents can bypass the specific resistance pathways associated with Topo1i-based therapies, providing more durable and clinically meaningful responses. By leveraging different payloads with distinct MOAs, clinicians can optimize treatment strategies, potentially offering patients continued efficacy despite prior resistance to a specific payload.

Combinations of chemotherapies are often used to address tumor heterogeneity and acquired resistance in cancer treatment.^33,34 It is common practice to investigate the effectiveness of combining ADCs with various anticancer drugs, including traditional chemotherapies, molecularly targeted agents, and immunotherapies.^35,36 As an extension of these concepts, dual-payload ADCs are designed to enhance the therapeutic efficacy of single-payload ADCs while maintaining their safety profiles. Dual-payload ADCs are single therapies designed to deliver two payloads directly into the same cancer cells. By conjugating an antibody with two distinct payloads, ADCs can target cancer cells through diverse and often synergistic mechanisms. The DAR and the ratio of the two payloads are critical parameters in the development of dual-payload ADCs. Achieving precise control over these attributes is particularly challenging with heterogeneous ADC conjugation technologies. In contrast, well-controlled and precise ADC technologies provide a more effective solution, enabling accurate payload distribution while preserving ADC stability and optimizing PK. The advancement of site-specific conjugation technology generated opportunities to develop homogeneous dual-payload ADCs as an advanced therapeutic strategy.

One dual-payload ADC approach is the delivery of multiple cytotoxins with distinct MOAs. This approach benefits relapsed patients who have progressed after a prior single-payload ADC treatment, but it may also benefit treatment-naïve patients by proactively addressing the onset of acquired resistance to each payload. For instance, one payload may be a microtubule-targeting agent, such as maytansinoids or auristatins. These agents disrupt microtubule function, which is crucial for cell division, leading to mitotic arrest and subsequent apoptosis of the cancer cells. The second payload might target topoisomerases, such as camptothecin or its derivatives.^37–39 Cancer cells often develop resistance to single-agent therapies.^33,40,41 Combining these inhibitors targeting different hallmarks of tumorigenesis may help to overcome resistance mechanisms, as cells that are resistant to one agent may still be vulnerable to the other. Combining a microtubule inhibitor with a topoisomerase inhibitor on the same ADC holds the potential to enhance its efficacy and durability of response. Such combinations not only amplify the direct cytotoxic effects on the cancer cells, but can also increase immunogenic cell death (ICD), which can further stimulate the immune system to recognize and attack the tumor.⁴²

Another dual-payload approach involves delivering a second, potentiating payload to enhance the efficacy of the primary payload, potentially re-sensitizing resistant tumors in patients who have stopped responding to treatment with a single payload ADC. An effective strategy for this is the combination of topoisomerase inhibitors with DNA damage response inhibitors (DDRis).^43,44 DDRis target pathways involved in detecting and repairing DNA damage. When used in conjunction with Topo1i, DDRis can further compromise the cell’s ability to repair the damage induced by the topoisomerase inhibitors. For example, poly (adenosine diphosphate-ribose) polymerase inhibitors, can prevent the repair of DNA breaks caused by topoisomerase inhibitors, resulting in synthetic lethality. This approach takes advantage of the cancer cell’s impaired repair mechanisms, making it more susceptible to treatment. Cancer cells often develop resistance to topoisomerase inhibitors through enhanced DNA repair mechanisms or activation of alternative repair pathways. By inhibiting these repair mechanisms with DDRis, the combination therapy can overcome such resistance, rendering the cancer cells more vulnerable to the effects of topoisomerase inhibitors.⁴⁵

ADCs with immune agonist payload, such as toll-like receptors or stimulator of interferon genes (STINGs), were shown to elicit a robust immune response within the tumor microenvironment.^46–48 Additionally, combining cytotoxic agents with immune agonists represents a promising approach that was shown to increase immune cell infiltration into the tumor microenvironment and to enhance overall anti-tumor activity in preclinical models.^23,49 This dual mechanism not only facilitates direct killing of tumor cells through the cytotoxic payload, but can also activate the immune system to specifically target the patient’s tumor. Immunostimulating ADCs present known challenges, particularly toxicity associated with Fc-mediated uptake.^50,51 A dual-payload ADC conjugated to both an immune agonist and a cytotoxin, combined with a silent Fc region, may help mitigate this issue. Optimizing the ratio of immune agonist to cytotoxin in preclinical studies is crucial to balancing efficacy and toxicity, thereby enhancing the therapeutic index.

Combination therapy has proven effective in both preclinical and clinical settings. Although synergy and additivity are desirable for dual-payload ADCs, specifically for the combination of synthetic lethality design and immunostimulating dual-payload ADCs, they may not be required to enhance efficacy. Analysis of clinical data has shown that enhanced efficacy observed in many US Food and Drug Administration-approved drug combinations can often be attributed to independent drug actions and interpatient variability, even in the absence of demonstrable synergistic or additive effects.⁵²

In summary, the integration of different payload types within ADCs holds substantial potential to improve cancer treatment outcomes. These approaches aim to maximize therapeutic efficacy, overcome resistance mechanisms, and harness the immune system to target and destroy cancer cells more effectively.

Methods for creating homogeneous dual-payload ADCs

Branched multifunctional linkers

Multifunctional linkers feature three or more branches. One branch is used for the attachment to antibodies or other protein scaffolds. The other branches can either link to reactive functional groups to enable alternative conjugation chemistries for attaching payloads with different MOAs or directly link to the payloads themselves (Table 1).

Table 1.

Homogeneous dual-payload conjugation methods enabled by branched multifunctional linker.

Dual conjugation strategy	Protein conjugation chemistry	Adapter conjugation to linker payload-1	Adapter conjugation to linker payload-2	Protein modality	Reference
Selective deprotection of branched linkers	interchain cysteine	cysteine/maleimide	acetamidomethyl protected cysteine/maleimide	IgG	25
Branched linkers with orthogonal conjugation handles	engineered cysteine	alkyne/azide	ketone/aminooxy	IgG	53
	interchain cysteine	lysine/NHS ester	tetrazine/TCO	cys-minibody	54
	disulfide bridging	tetrazine/TCO	azide/BCN	IgG	55
	disulfide bridging	cyclopropene/tetrazine	azide/BCN	IgG	56
	disulfide bridging	cyclooctyne/azide	alkyne/azide	IgG	57
	mTG	azide/DBCO	tetrazine/TCO	IgG	24,58
	mTG	cyclopentadiene/maleimide	furan/maleimide	IgG	59
	mTG	cysteine/maleimide	azide/DBCO	IgG	60
Branched linkers with pre-loaded payloads	mTG	n.a.	n.a.	IgG	61,62
	engineered cysteine	n.a.	n.a.	IgG	63

A group of scientists from SEAGEN designed an adaptor with two distinct cysteine protecting groups, allowing for selective deprotection and subsequent conjugation to different linker payloads.²⁵ One of the cysteines is protected by a reducible disulfide group that can be easily reduced using ris (2-carboxyethyl)phosphine (TCEP). The other cysteine is protected by an acetamidomethyl group, which is resistant to TCEP reduction but can be selectively unmasked under mild aqueous conditions using mercury acetate (Figure 3a). The adapter, with its two orthogonal protection groups, was initially conjugated to the interchain thiols of a CD30-targeting antibody using maleimide chemistry. In this review, we defined the use of reduced interchain disulfide bonds to generate DAR8 as a homogeneous conjugation technology, whereas applying the same method to generate lower DAR species is considered a heterogeneous technology due to uneven drug distribution. After introducing the adapter, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) linker payloads were conjugated to the antibody in a two-step deprotection-conjugation process. The resulting dual-payload conjugate, containing a total of 16 drug molecules, were evenly split between MMAE and MMAF and demonstrated superior efficacy in a xenograft model with multi-drug resistant-positive DEL-BVR cells compared to ADCs carrying either MMAE or MMAF alone. Furthermore, the dual-payload ADC exhibited greater efficacy than single-payload ADCs in heterogeneous tumor models, emphasizing the significance of bystander effect of MMAE, but not MMAF, in the context of dual payloads.

Figure 3.

Homogeneous dual-payload conjugation methods enabled by branched multifunctional linkers. The linkers contain multifunctional groups to allow attachment of the linker to antibodies and installment of payloads. (a) Orthogonality introduced by differential deprotection of cysteine groups. (b) Alkyne/carbonyl functional groups for payload conjugation introduced to an antibody through maleimide chemistry. (c) Three different multifunctional linker designs to employ disulfide bridging. (d) Branched linkers introduced to antibodies using mTG. Deglycosylation is optional depending on the primary conjugation handle. (e) Payloads are attached to the branched linker before conjugation. The primary conjugation method is mTG conjugation. (f) Trifunctional linker with maleimide as primary conjugation handle bridging a cytotoxic agent, a radiolabeling group, and TCO. Amino acids in IgGs that are involved in conjugation are depicted. Reactive functional groups in multifunctional linkers are highlighted in gray for primary conjugation chemistry, orange and blue for linker payload installment. Stars in blue or orange represent payloads.

Orthogonal chemistry can be incorporated directly into multifunctional adaptors, providing convenience in the conjugation processes (Figure 3b). Building on the broad application of engineered cysteine conjugation methods, MedImmune developed a heterotrifunctional linker featuring maleimide, azide, and ketone groups. This design allows the linker to be attached to THIOMAB, followed by the conjugation of two different payloads using azide-alkyne click chemistry and oxime ligation, respectively.⁵³ A dual-payload ADC containing a pyrrolobenzodiazepine (PBD) dimer and MMAE achieved an overall conjugation efficiency of over 90% across three steps. The trifunctional adaptor was first conjugated to the engineered cysteine. Subsequently, the adaptor-functionalized antibody was conjugated to MMAE via oxime ligation. After the free drug removal, the alkyne group was subjected to copper-catalyzed azide – alkyne cycloaddition (CuAAC) reaction to attach the PBD dimer. The resulting dual-payload ADC exhibited comparable in vitro cell killing activity compared to the ADC conjugated with PBD dimer alone, likely due to the significantly higher potency of the PBD dimer compared to MMAE. Despite the failure of the dual-payload ADC to induce additive or synergistic effects in cell killing, the heterotrifunctional adapter offers a flexible platform for preparing dual mechanistic drug conjugates in a site-specific manner. A similar heterotrifunctional adapter containing maleimide, Boc-protected amine, and tetrazine has been reported for fluorescence imaging applications.⁵⁴

Disulfide bridging leverages interchain disulfides to generate site-specific ADCs.⁶⁴ Building on existing disulfide bridging conjugation handles, such as dibromomaleimide^65–67 and divinylpyrimidine,^56,68,69 two groups have successfully developed antibody dual-payload conjugates by incorporating orthogonal click-chemistry handles into the adapter.^55,56 The process involves reducing the antibody to expose interchain cysteines for disulfide bridging to install the adapter, followed by linker payload conjugation in a single-step reaction (Figure 3c). The third group of researchers used dibromopyridazinedione for thiol bridging. The two branches with alkyne and bicyclononyne (BCN) groups exhibit different reactivity, which enables selective conjugation, with one payload attached to BCN via strain-promoted alkyne-azide cycloaddition (SPAAC), followed by a second payload conjugated to the alkyne using CuAAC.⁵⁷

Derived from microbial transglutaminase (mTG) conjugation technology, which does not require protein engineering when using the Q295 site on the heavy chain (HC) of IgGs,^70,71 the trifunctional adapter design using a primary amine and two additional orthogonal conjugation chemistries combines the rapid kinetics of click chemistry with the convenience of mTG-mediated, enzymatic conjugation (Figure 3d). Walker and colleagues reported using such a strategy to create a dual-conjugate ADC targeting HER2,⁵⁸ one arm being DM1 and another arm being a polypropylene glycol (PEG) intended as a genetic method to reduce the hydrophobicity of the ADC. Another group also utilized mTG as the primary conjugation method to the Q295 site. The trifunctional linker contained a furan group and a cyclopentadinene group, both of which are reactive to maleimide linker payloads.⁵⁹ Accessing the Q295 site for mTG conjugation typically requires deglycosylation or mutation of the N297 site to prevent glycosylation. Wehrmüller and colleagues discovered that overall, positively charged peptides with a lysine, such as RAK, enabled mTG conjugation to Q295 without the need for deglycosylation. They further expanded the RAK linker to include branches with cysteine and azide groups, allowing the creation of a dual radio-immunoconjugate containing both a cytotoxin and a metal chelator.⁶⁰

One limitation of the aforementioned modular adaptors and most of the site-specific conjugation chemistry approaches is that the DAR is constrained by the number of available conjugation handles on the antibodies. Scientists from the University of Texas reported adjusting the DAR by increasing the number of branches of the adapters⁷² (Figure 3d). Applying the same concept, the addition of a tetrazine arm created an adapter system that enabled the generation of a panel of homogeneous dual-payload ADCs with total DARs of 2  +  2, 4  +  2, and 2  +  4.²⁴ The flexibility in DAR adjustment is advantageous for fine-tuning the physicochemical properties, efficacy, and toxicity profiles of ADCs, based on the disease target and the combination of payloads. The study elegantly demonstrated the advantage of dual-payload ADCs with fine-tuned payload ratios by showing that the 4  +  2 mmAE/MMAF dual-payload ADC had superior efficacy compared to both the DAR6 mmAE ADC and the co-administration of two DAR4 single-payload ADCs. The imaging study supported the hypothesis that co-administration of two single-payload ADCs targeting the same antigen can lead to target antigen-binding competition, resulting in reduced efficiency in the delivery of each payload.

A similar approach to overcoming the limitations of available conjugation sites is to use branched linker payloads, which have been widely used to increase the DAR. Compared with the branched adapters discussed above, the use of branched linker payloads eliminates one conjugation step and simplifies the conjugation process. However, the total DAR and ratio of the two payloads are important product attributes for ADCs with dual payloads, where each DAR and each ratio of the two payloads requires a new linker payload synthesis. Merck & Co. Inc. and Synaffix BV have demonstrated the potential of the approach to double the DAR by using mTG conjugation⁷³ and GlycoConnect technology,⁷⁴ respectively. Monoclonal antibodies (mAbs) produced in CHO cells with remodeled glycans for conjugation lose Fc gamma receptor (FcγR) functionality. However, FcγRs are not essential for the primary MOA of ADCs. Additionally, the interaction with FcγRs on non-target immune cells may lead to unintended uptake and degradation of ADCs in immune cells.⁷⁵ Dual-payload ADCs can be produced using the same design principle of changing one of the payloads to a payload with a different MOA (Figure 3e). Both Araris Biotech AG and LigaChem Biosciences have used the branched linker payload to make multi-payload ADCs.^61,76 Using this approach, Araris Biotech AG created a DAR4 dual-payload ADC by appending an exatecan and an exatecan derivative with lower cell permeability. Their preclinical data demonstrated higher payload accumulation in tumors, better efficacy, and lower toxicity.⁶²

Sadowsky and coworkers designed a trifunctional linker bridging a radiolabeling group, trans-cyclooctyne (TCO) and cytotoxic agent in a manner that could attenuate cytotoxic activity and allow tracking of the intact labeled species over extended periods in vivo.⁶³ In the design, a PBD dimer was added between the primary conjugation handle and the branch point via a novel self-immolative para-aminobenzyl (PAB) linker to a radiometal chelating group, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and TCO group for decaging. The PBD dimer is released and becomes active upon dosing of tetrazine and the reaction with TCO-PAB-DOTA (Figure 3f).

Amino acids with orthogonal activities

Many site-specific conjugation technologies utilize amino acids with selective chemistry as conjugation handles. Cysteine conjugation is one of the widely used chemistries due to its high nucleophilicity among the 20 common natural amino acids.⁷⁷ Lysine, the second most nucleophilic amino acid after cysteine, is also highly reactive, making it a focal point for site-specific conjugation research. Site-specific lysine conjugation is an active research area and is achievable through modulating the protein microenvironment or selection of conjugation partners.⁷⁸ Non-canonical amino acids (ncAA) such as ketone containing para-acetylphenylalanine (pAcF)^79,80 and azide-containing para-azidomethyl phenylalanine (pAMF)^81,82 have been successfully applied in ADC development. As discussed below, production of homogeneous dual-payload ADCs has been demonstrated by combining two canonical amino acids, two non-canonical amino acids, or one from each category (Table 2).

Table 2.

Homogeneous dual-payload conjugation methods enabled by two amino acids incorporated in proteins.

Dual conjugation strategy	Liner payload-1 conjugation	Linker payload-2 conjugation	Protein modality	Reference
Canonical animo acids	engineered cysteine/methylsulfone phenyloxadiazole linker	selenocysteine/iodoacetamide linker	scFv-Fc; IgG	83,84
	lysine/β-lactam linker	arginine/phenylglyoxal linker	DVD-IgG	85
Non-canonical amino acids	azide/DBCO	tetrazine/TCO	scFv; Fab	86
	pAcF	azide	IgG	87
	5-hydroxtryptophan/diazo	azide/DBCO	IgG	88
	engineered cysteine/maleimide linker	para-acetylphenylalanine/aminooxyl linker	Fab	89
	pAMF/DBCO linker	pAMF/DBCO linker	IgG	This review
	pAMF/DBCO linker	pAcF/aminooxy linker	IgG	This review
Canonical and non-canonical	pAMF/DBCO linker	cysteine/maleimide	IgG	This review

Canonical amino acids

ADCs created using engineered cysteine, termed TDC (THIOMAB ADC), represent the first reported homogeneous ADC with an improved therapeutic index, largely attributed to enhanced linker-payload stability achieved through strategic site selection, which minimizes systemic toxicity.⁷ This innovation set a precedent, inspiring subsequent identification of alternative engineered cysteine sites for drug development purposes.^90–93 Engineered cysteines are widely used due to their versatility, and the incorporation of an additional conjugation handle on antibodies already containing engineered cysteines has become a common approach to achieve homogeneous ADC with dual payloads. Selenocysteine, known as the 21st natural amino acid, is a unique candidate for such conjugation strategies because it is present in several enzymes. By substituting the sulfur atom in cysteine with selenium, selenocysteine attains a pKa of 5.2, making it a more reactive nucleophile than cysteine’s thiolate (pKa 8.3). Despite its potential, selenocysteine’s utility as a conjugation handle was historically limited due to challenges in its incorporation. However, recent advancements in recoding technology have enabled its incorporation into antibodies.^94,95 Combining selenocysteine with engineered cysteine to facilitate homogeneous dual-payload conjugation was first demonstrated on a HER2-targeting scFv-Fc⁸³(Figure 4a). The THIO-SELENOMAB conjugate was subjected to a mild reduction process, revealing the capped selenocysteine and engineered cysteine groups. This facilitated a sequential conjugation strategy where an iodoacetamide linker was first attached to selenocysteine at acidic pH, followed by the conjugation of cysteine at basic pH using a methylsulfone phenyloxadiazole (ODA) linker, noted for its greater stability than the traditional maleimide linker.⁹⁶ This method was later expanded to attach both a PNU and a MMAE linker payload to a HER2-targeting antibody. The resulting dual-payload ADC exhibited potency comparable to the PNU ADC, indicating dominant cytotoxic effect from the PNU warhead. Cell morphology studies revealed the presence of both DNA damaging and tubulin inhibition MOAs in the dual-payload conjugate.⁸⁴

Figure 4.

Homogeneous dual-payload conjugation methods enabled by amino acids with orthogonal activities. (a) Stepwise conjugation methods through engineered cysteine and selenocysteine. (b) Dual-DVD antibody with lysine and engineered arginine in the Fv region with distinct reactivity. (c) Incorporation of two ncAAs using orthogonal aaRS/tRNA pairs or single dual-functional ncAA. (d) Combination of pAcF and engineered cysteine. (e) Combination of pre-conjugated LC and introduction of pAMF for second payload conjugation during HC synthesis and ADC assembly. (e) pAcF and pAMF incorporation by decoupled LC production and IgG synthesis using Xpress® technology. (g) Combination of pAMF and interchain cysteine conjugation methods. Amino acids in IgG that are involved in conjugation are depicted. Reactive functional groups from amino acids in protein or linker payloads are highlighted in orange and blue. Stars in blue or orange represent payloads.

First-generation ADCs largely relied on solvent-exposed lysine residues on antibody surfaces for attachment of linker payloads. However, due to a lack of selectivity, linker payloads were randomly attached to any accessible lysine, resulting in product heterogeneity.^1–3 The aldolase antibody h38C2 offers a unique solution by conferring distinct properties of a lysine residue buried in a hydrophobic pocket. This lysine has a pKa of approximately 6, making it more nucleophilic at physiological pH, capable of catalyzing aldol and retro-aldol reactions, and selectively reactive with 1,3-diketone and β-lactam derivatives.^97,98 Building on these distinctive properties, a site-specific conjugation platform was developed to generate homogeneous ADCs by grafting the nucleophilic lysine domain from the inner Fv domain onto a dual variable domain antibody (DVD-IgG).⁹⁷ Using this platform, researchers created a homogeneous dual-payload conjugation strategy by engineering a heterodimeric DVD-IgG with orthogonal reactivity. In this design, a nucleophilic lysine is present on one arm of the inner Fv domain, while an arginine replaces the lysine on the other arm, introducing a second, distinct reactive site.⁸⁵ This dual site-specific conjugation process allows the arginine to selectively conjugate to a phenylglyoxal linker, which is inert to the β-lactam linker. Meanwhile, the lysine is conjugated to the β-lactam linker (Figure 4b). While nucleophilic lysine may exhibit cross-reactivity with the phenylglyoxal linker when present in large excess, this was shown to be minimized by decreasing the linker-to-protein ratio. Due to the modular nature of DVD-IgG, this dual payload, site-specific conjugation method could be expanded to produce a variety of ADCs, each with a different outer Fv domain targeting specific epitopes. Though the approach has limited orthogonal attachment sites, this limitation can be readily addressed by incorporating a branched linker, which offers additional conjugation flexibility, as discussed below. A dual-payload conjugate containing MMAE and DM1 was produced by the combination of lysine conjugation and inter-chain cysteine conjugation.⁹⁹ Although the resulting MMAE/DM1 dual-payload conjugate was heterogenous, the adoption of site-specific lysine conjugation methods⁷⁸ and fully conjugated inter-chain cysteine conjugate (DAR8) is a promising method yet to be demonstrated.

Non-canonical amino acids

The incorporation of ncAAs has proven to be a transformative approach for developing second-generation ADCs that are both homogeneous and highly stable.¹⁰⁰ One widely used method for incorporating ncAAs involves the amber stop codon suppression, which requires engineering an orthogonal aminoacyl-tRNA synthetase (aaRS) and its tRNA pair.^82,101 Using ncAAs also simplifies the conjugation process. Traditional cysteine conjugation, for instance, requires reduction to expose thiol groups, while enzyme-mediated conjugations often involve two steps. In contrast, ncAAs allow for direct and specific conjugation, reducing process development complexity. Consequently, this approach enhances manufacturability and ensures greater consistency in product quality, which is essential for scalability and therapeutic efficacy and safety. The potential of ncAAs for ADC development has been recognized by the biopharmaceutical industry, with demonstrated feasibility at manufacturing scale. Notable examples include Sutro Biopharma’s STRO-002 anti-folate receptor alpha (FolRα) ADC⁸¹ and Ambrx Biopharma’s ARX517 anti-prostate-specific membrane antigen (PSMA) ADC,¹⁰² both of which illustrate the practical application of ncAA-based strategies in commercial ADC production.

The site-specific incorporation of multiple distinct ncAAs has shown potential in creating antibody dual-payload conjugates using orthogonal aaRS/tRNA pairs capable of suppressing different nonsense codons (Figure 4c). Osgood et al. discovered that tryptophanyl aaRS/tRNA pair from E. coli is an efficient opal codon (TGA) suppressor. Together with orthogonal E. coli leucyl aaRS/tRNA pairs as amber suppressor, 5-hydroxytryptophan (5-HTP) and the azide-containing amino acid LCA were incorporated into positions 121 and 198 of the trastuzumab HC, respectively. Subsequently, a dual-payload ADC was generated using diazo-MMAF and dibenzocyclooctyne (DBCO)-PNU159682 in a one-pot reaction.⁸⁸ Using similar strategies, Xiao et al. combined the orthogonal ocher (TAA codon) suppressor pyrrolysyl aaRS/tRNA pair with the amber (TAG codon) suppressing E. coli tyrosyl aaRS/tRNA pair to incorporate two different ncAA, pAcF and azido-lysine, into distinct sites of a full-length anti-HER2 antibody. The antibody was subsequently conjugated to auristatin and the fluorophore Alexa 488 to obtain a defined antibody-drug-fluorophore conjugate.⁸⁷

The practical application of incorporating two ncAA is constrained by the low incorporation efficiency and operational complexity using conventional manufacturing processes. To overcome these limitations, a scientific team recently advanced this approach by developing dual-functional ncAAs, pTAF (4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine) and mTAF (3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine), which enables highly efficient, orthogonal, and specific conjugation (Figure 3c). The versatility of pTAF was demonstrated across three antibody fragments, one scFv and two Fabs, used in both imaging and ADC discovery applications.⁸⁶ Further, the team successfully achieved concurrent incorporation of mTAF and pAcF in a scFv, offering an alternative method for multi-payload conjugations. Notably, the efficiency of double ncAA incorporation was 70%, with a production titer of about 20 mg/L. It is desirable to further improve the titer while maintaining ncAA incorporation efficiency, providing flexibility to increase the DAR and adjust the ratio of the two payloads as needed.

Another research team demonstrated homogeneous dual-payload conjugation by combining ncAA with engineered cysteines.⁸⁹ Using trastuzumab Fab as a model protein, they engineered a cysteine on light chain (LC) (A124C) and incorporated pAcF on HC (A121pAcF). A stepwise approach was developed to create a homogeneous dual-payload conjugate with Alexa 568 and Alexa 488. First, aminooxy fluorescein was conjugated at pH 4.0, followed by maleimide conjugation after adjusting the pH to 6.0 (Figure 4d). The resulting dual-dye conjugate exhibited fluorescein signal intensities comparable to those of the respective single-dye conjugates, indicating that the dual dye conjugation was as efficient as individual single-dye conjugation reactions.

Farràs et al. recently introduced a site-specific conjugation method leveraging the independent production of HC and LC.¹⁰³ In this approach, HC and LC were produced separately in two cell lines. Purified LC was initially conjugated to MMAE at the C-terminal interchain cysteine, then assembled in vitro with prefabricated HC to create a DAR2 ADC. This method suggested the potential for homogeneous dual-payload conjugation, where distinct cytotoxins could be attached to LC and HC prior to ADC assembly. However, challenges such as low titers from separate HC and LC production and process complexity limit large-scale application of the method. Sutro Biopharma recently reported that using prefabricated LCs enhances antibody production titer in the XpressCF + ^Ⓡ platform.¹⁰⁴ LC is produced in E. coli, purified, and subsequently added to the cell-free reaction for HC synthesis, resulting in IgG assembly. Integration of prefabricated LC with cell-free HC synthesis not only boosts antibody production titer but also provides a scalable platform for homogeneous dual-payload conjugates. In this method, pAMF is incorporated into LC during its prefabrication. After purification, the pre-conjugated LC is used as a reagent in cell-free reaction, where pAMF is incorporated into the HC. The LC pre-conjugated antibody is then purified and conjugated to a second payload via SPAAC (Figure 4e). This approach only requires a single amber suppression system, including aaRS, tRNA, and a ncAA. However, handling cytotoxic pre-conjugated LC during antibody production introduces additional safety considerations. To improve this process, pAcF is incorporated into the LC during prefabrication instead of pAMF. The pAcF-containing LC is then used in a cell-free system where pAMF is introduced into HC. Following antibody purification, the payloads with aminooxy and DBCO linkers are conjugated in a one-pot, single-step reaction (Figure 2f). The feasibility of this conjugation route was demonstrated with aminooxy-Alexa 647 and DBCO-Alexa 555 conjugated to an antibody containing pAcF on the LC and pAMF on the HC. The dual dye conjugate appeared purple, a combination of Alexa 555 (pink) and Alexa 647 (blue), and resolved into distinct LC and HC bands upon reduction when visualized on SDS-PAGE (Figure 5a). Subsequently, a prototype FolRα-targeting immunostimulant ADC (iADC) was generated, bearing two immunostimulants on LC and four cytotoxins on HC via the one-pot, single-step process. Subunit LC-MS analysis of the iADC confirmed the specificity of oxime ligation and SPAAC, with only targeted conjugates detected (Figure 5b), achieving over 90% conjugation efficiency on both LC and HC. This flexible approach allows for varying locations and quantities of pAcF and pAMF on HC and LC, enabling diverse scaffold configurations such as 2  +  2, 4  +  4, 2  +  4, 4  +  2, 2  +  6, and 6  +  2 (Figure 5d). E. coli-derived mAbs are aglycosylated and lack FcγR functionality. For the tumor-targeted immunostimulating ADCs, such as STING agonist ADCs, research has shown that STING signaling within tumor cells contributes directly to antitumor activity.¹⁰⁵ Therefore, Fc-mediated uptake is not required. The cytotoxin facilitates direct tumor cell killing, while the immune agonist activates immune cells to enhance the overall antitumor response.

Figure 5.

Example of homogeneous dual-payload conjugation enabled by combination of pAcF/pAMF or pAMF/interchain cysteine. (a) An antibody containing pAcF on LC and pAMF on HC was conjugated to aminooxy-Alexa 647 alone (lane 1), DBCO-Alexa555 alone (lane 2), aminooxy-Alexa647 and DBCO-Alexa555 (lane 3). Lanes 4–6 were reduced samples in lane 1–3. (b) Conjugation efficiency and orthogonality of oxime ligation and SPAAC was confirmed by subunit LC-MS analysis. (c) Subunit LC-MS analysis confirmed conjugation of DBCO linker payload-1 (LP1) to pAMF on HC followed by conjugation of maleimide linker payload 2 (LP2) to interchain cysteine to produce a DAR12 (4 LP1, 8 LP2) dual-payload ADC. Panels i, ii and iii are deconvoluted mass spectra of LC region of unconjugated IgG, after DBCO LP conjugation, and after maleimide conjugation, respectively. Panels iv, v and vi are deconvoluted mass spectra of HC region of unconjugated IgG, after DBCO LP conjugation, and after maleimide conjugation respectively. (d) A wide range of scaffolds with different DAR and payload ratio can be generated using combination of pAcF and pAMF, the conjugation method is illustrated in Figure 2f.

Non-canonical amino acid and cysteine

Dual-payload conjugates were produced using a combination of pAMF and interchain cysteine conjugation (Figure 4g). Initially, a DBCO-containing linker payload was conjugated to the antibody, followed by maleimide conjugation. Both SPAAC and maleimide conjugation exhibit rapid kinetics, allowing the conjugation reaction to reach near completion within hours (Figure 5c). While the DAR of the maleimide linker payload remains fixed, the overall DAR can be adjusted by modulating the number of pAMF sites (Table 4). Additionally, by alternating the DBCO and maleimide linker payloads, diverse scaffold configurations, including 8  +  2, 8  +  4, 8  +  8, 4  +  8, and 2  +  8, can be achieved. The generation of a library of 19 dual-payload ADCs using non-canonical cyclopentadiene-containing amino acids and interchain cysteine residues on trastuzumab was demonstrated. A range of DAR from 1.0 to 7.1 was achieved through cysteine conjugation.¹¹⁴ This work is worth mentioning as it explores a variety of drug combinations involving MMAE, alvespimycin, aldoxorubicin, vinblastine, and doxorubicin, although these conjugates are not considered homogeneous.

Table 4.

Summary of process complexity and flexibility to adjust DAR and payload ratio of the methods that have been demonstrated on IgG.

Dual conjugation strategy	Branched linker/dual payload conjugation method	Non-traditional expression systems (Yes-1; no-1)	Protein engineering requirement (Yes,1; no-0)	Conjugation			Range of DAR and payload ratio	Reference
				Number of reaction steps	Number of purification steps	Column purification requirement (Yes-1; no-0)
Multi-modality linkers	Interchain cysteine (adapter)	0	0	6	3	0	8  +  8	25
	Eng. cysteine (adapter)	0	1	5	4	1	2  +  2; 4  +  4; 6  +  6	53
	Interchain cysteine (adapter)	0	0	6	4	0	8  +  8	54
	Disulfide bridging (adapter)	0	0	4	2	1	4  +  4	55
	Disulfide bridging (adapter)	0	0	3	2	1	4  +  4	56
	Disulfide bridging (adapter)	0	0	4	2	1	4  +  4	57
	mTG (adapter)	0	0	4	3	2	2  +  2; 2  +  4; 4  +  2	24,58
	mTG (adapter)	0	0	4	3	2	2  +  2	59
	mTG (adapter)	0	0	3	2	1	2  +  2	60
	mTG/eng. cysteine (linker payload)	0	0	2	2	1	2  +  2	62,63,73
Amino acids	Lys/Arg	0	1	1	1	0	1  +  1	85
	in vivo ncAA incorporation	1	1	1	1	0	1  +  1	86–88
	pAMF/pAMF	1	1	4	3	0	2  +  2/4/6/8; 4  +  2/4/6/8	This review
	pAMF/pAcF	1	1	1	1	0	2  +  2/4/6/8; 4  +  2/4/6/8	This review
	pAMF/cys.	1	1	3	1	0	8  +  2/4/6/8	This review
Enzyme mediated	Eng. cys/LAP tag	0	1	3	2	1	2  +  2/4/6	106
	mTG/eng. cys	0	1	6	3	1	2  +  2/4/6	107
	Sortase/butelase	0	1	1	1	1	2  +  2	108
	hFGE/scFGE	0	1	12	5	1	2  +  2	109
	Glycan engineering/tyrosinase	0	1	4	3	1	2  +  2	110
	Glycan engineering/affinity-based conjugation	0	0	2	1	1	2  +  2	111
	eSrtA(2A–9)/eSrtA(4S–9)	0	1	2	2	1	2  +  2	112,113
	Eng. endoS/sortase	0	1	1	1	1	4  +  4	49

Enzyme-mediated methods

mTG,^6,70,71 sortase A,¹¹⁵ and formylglycine-generating enzymes (FGE)^109,116,117 are extensively used in the pharmaceutical industry to produce homogeneous ADCs. Emerging enzyme-mediated conjugation methods, such as those involving butelase 1,¹¹⁸ and lipoate-acid ligase A^119,120 show promise for broader applications. However, enzyme-mediated conjugation typically requires incorporation of the conjugation tag at the protein C-terminus,¹²¹ which limits the labeling efficiency, variability of the conjugation sites and the flexibility to adjust the ratio of dual payloads. Additionally, this approach often involves a two-step conjugation process for each enzymatic reaction, including attachment of an adapter and followed by conjugation to the payload of interest, which can lead to lower overall ADC product yield.

Combinations of different enzyme-mediated conjugation methods to produce homogeneous dual-payload ADCs have been explored by several research groups. As discussed below, these orthogonal enzymatic activities can derive from naturally occurring enzymes or can be introduced through engineered enzymes (Table 3).

Table 3.

Homogenous dual conjugation methods enabled by enzymatic conjugation tags.

Dual conjugation strategy	Linker payload-1 conjugation	Linker payload-2 conjugation	Protein modality	Reference
Two conjugation tags recognized by natural enzymes or natural enzyme with amino acid conjugation handle	Q295/primary amine linker (mTG)	LAP TAG/lipoate acid linker (lipoate acid ligase)	IgG	106
	Q295/primary amine linker (mTG)	engineered cysteine/maleimide linker	IgG	107
	LPXTG/polyglycine linker (sortase)	NHV/X(C/I/L/V) linker (butalase 1)	IgG	108
	CTPSR (hFGE)/HIPS-azide linker	CTAGR (scFGE)/HIPS-azide linker	IgG	109
	azide (glycan engineering)/BCN linker	o-quinone (tyrosinase)/TCO linker	IgG	110
	glycan engineering (endoS2 mutant) to conjugate linker payload to N297	affinity based conjugation to lysine 248	IgG	111
Two conjugation tags recognized by engineered enzymes	LAXTG/polyglycine linker (eSrtA(2A–9))	LPXTG/polyglycine linker (eSrtA(4S–9))	IgG	112,113
	glycan engineering (endoS2 mutant)	LAXTG/polyglycine linker (engineered Sortase)	IgG	49

The natural enzyme

mTG was combined with lipoate-acid ligase (LpIA) to create homogeneous dual-payload ADCs. LpIA recognizes a 13-amino acid sequence named LplA acceptor peptide (LAP). Thornlow et al. demonstrated the specificity of both LpIA-mediated and mTG-mediated conjugation methods.¹⁰⁶ They first conjugated a lipoate acid trans-cyclooctyne (TCO) linker to the LAP tag at the C-terminus of the LC with > 85% conjugation efficiency. Subsequently, mTG was used to introduce a PEG3-azide linker at the Q295 position on the HC of trastuzumab. Following desalting to remove excess TCO and azide linkers, two fluorescent dyes, rhodamine with an extracellular protease-sensitive matrix metalloproteinase-2 cleavable linker and fluorescein with an intracellular protease-sensitive cathepsin B cleavable linker, were site-specifically conjugated to the adaptors containing azide and TCO, respectively (Figure 6a). Incubation of the dual dye conjugates with HER2-expressing SK-BR-3 cells demonstrated the extracellular release of rhodamine and the intracellular release of fluorescein. With distinct extracellular and intracellular release triggers, this proof-of-concept study highlighted the potential for delivering drugs with differential spatial release from a single conjugate for cooperative drug combinations efficacies.

Figure 6.

Homogeneous dual-payload conjugation mediated by enzymes with orthogonal activities of (a) mTG and lipoate ligase, (b) mTG and engineered cysteine, (c) sortase and butelase 1, (d) hFGE and MtFGE. Reactive functional groups from amino acids in protein and linker payloads are highlighted in orange and blue. Stars in blue or orange represent payloads. Homogeneous dual-payload conjugation mediated by enzymes with orthogonal activities of (e) glycoengineering and tyrosinase, (f) glycoengineering and affinity tag guided site-specific conjugation method, (g) engineered sortases, (i) glycoengineering and sortase. Reactive functional groups from amino acids in protein and linker payloads are highlighted in orange and blue. Stars in blue or orange represent payloads. Blue square: N-acetylgalactosamine; green cycle: mannose; yellow cycle: galactose; red triangle: fucose.

Researchers at Pfizer reported a dual-payload conjugation method utilizing mTG in conjunction with engineered cysteines.¹⁰⁷ The goal of this research was to develop a solid-phase conjugation method to facilitate high-throughput production of dual-payload conjugates. mAbs with engineered cysteine residues were first deglycosylated to expose the Q295 site for mTG conjugation. The IgGs were then immobilized on a protein A (ProA) resin, followed by conjugation with a BCN-containing adapter. Afterward, the immobilized antibody underwent reduction and reoxidation, resulting in the introduction of two chemically orthogonal reactive groups, strained alkyne (BCN) and thiol, on the antibody, primed for dual-payload conjugation (Figure 6b). In this study, BODIPY-maleimide and Cy5.5-azide were conjugated to the linkers in a single-step reaction, achieving overall labeling efficiencies of 95% at the Q295 site and 80% at the engineered cysteine 183 on the LC. This method was further expanded to generate dual-labeled, site-specific antibody and Fab conjugates.

The small size of the sortag and the synthetic accessibility of polyglycine-linked substrates have enabled the use of sortase A (SrtA) in various applications, including the synthesis of ADCs,¹¹⁵ protein – protein conjugates,¹²² protein – lipid conjugates,¹²³ and protein – surface conjugates.¹²⁴ Harmand and colleagues successfully demonstrated the feasibility of homogeneous dual-payload ADC using sortase and butelase 1 that recognizes a three-amino-acid peptide (NHV) and ligates it to incoming nucleophiles composed of two amino acids (X(C/I/L/V)) attached to a payload of choice¹⁰⁸ (Figure 6c).

FGE catalyze the targeted oxidation of a cysteine residue within the conserved (C/S)X(A/P)XR motif, producing the aldehyde-containing amino acid formylglycine, which can serve as a conjugation handle for hydrazide or Hydrazino-iso-Pictet-Spengler (HIPS) conjugation.¹²⁵ First identified in 2003 as the posttranslational machinery responsible for activating type I sulfatases in eukaryotes,¹²⁶ FGE has since been recognized for its enzymatic diversity, with formylglycine-generating enzymes from various sources enabling dual site-specific protein conjugations.¹⁰⁹ One approach utilized human FGE (hFGE), which specifically targets proline-containing sequences (CTPSR), and FGE from bacteria such as Mycobacterium tuberculosis (MtFGE), which exhibits broader substrate specificity (CTPSR and CTAGR), in the presence of Cu2⁺. Homogeneous dual-payload conjugation was demonstrated by introducing formylglycine and two payloads, MMAE and fluorescein, alternately onto an EGFR-targeting scFv-Fc, which contained both CTPSR and CTAGR sequences at the C-terminus of the HC (Figure 6d). Cell killing and imaging assays confirmed the successful dual labeling of the scFv-Fc. Compared to other conjugation methods requiring the production of two different enzymes, this approach simplifies the process by requiring only a single FGE, provided that the initial aldehyde group is generated in vivo.

Tyrosinase is capable of oxidizing solvent-exposed tyrosine residues in proteins to o-quinone, which then undergoes bioconjugation reactions with thiols,¹²⁷ strain-promoted cycloaddition,¹²⁸ or light-induced photoaddition.¹²⁹ In IgG1, tyrosine residues Y296 and Y300 become accessible to tyrosinase oxidation once glycans are removed. Since both tyrosine residues undergo oxidation, the linker payload can conjugate to either site; however, steric hindrance restricts accessibility such that after conjugation at one site, the other becomes inaccessible. Consequently, only DAR2 conjugates have been observed.¹²⁷ Site-specificity might be improved by mutating either Y296 or Y300, though this modification remains to be experimentally validated. Alternatively, introducing a GGGY tag at the C-terminus of the HC or LC enhances tyrosinase accessibility.¹²⁸ ADCs have been demonstrated using both native antibodies and those modified with a GGGY tag. For example, a dual-payload conjugate containing MMAE and lissamine was created using glycoengineering in conjunction with tyrosinase-mediated oxidative coupling (Figure 6e).¹¹⁰ In principle, the combination of GGGY tag and native tyrosine in Fc region in a stepwise process may enable dual-payload conjugates.

Figure 6.

Homogeneous dual-payload conjugation mediated by enzymes with orthogonal activities of (e) glycoengineering and tyrosinase, (f) glycoengineering and affinity tag guided site-specific conjugation method, (g) engineered sortases, (i) glycoengineering and sortase. Reactive functional groups from amino acids in protein and linker payloads are highlighted in orange and blue. Stars in blue or orange represent payloads. Blue square: N-acetylgalactosamine; green cycle: mannose; yellow cycle: galactose; red triangle: fucose.

Affinity-based conjugation methods offer another promising strategy for site-specific conjugation of native antibodies. This approach leverages small proteins or peptides with high affinity toward antibodies to selectively attach bio-orthogonal groups or drugs at defined positions. Various methods have been developed to enable traceless conjugation in one or multiple steps.^130–132 Recent advances in affinity-based conjugation techniques have achieved specificity for selected amino acids, such as K248 in IgG1, a commonly targeted conjugation site. In combination with glycoengineering, employing engineered endoglycosidase endoS2 enables a one-pot, two-step dual-payload conjugation method (Figure 6f).^133,134 This approach enabled the production of a HER2-targeting dual-payload conjugate with MMAE and MMAF. Although the combination benefit of MMAE and MMAF was not observed, the conjugation method on its own remains promising. The superior efficacy of the single payload ADC compared to the 2  +  2 dual-payload ADC in the NCI-N87 xenograft model underscores the critical importance of careful selection of payload combinations and optimization of the ratio of the two payloads.

Engineered enzymes

A key challenge with sortase enzymes, particularly in bioconjugation applications, is their inherently slow conjugation kinetics. Using yeast display technology, researchers evolved a variant known as eSrtA, which exhibits a 140-fold increase in catalytic activity relative to the wild-type SrtA.¹³⁵ Building upon eSrtA, two orthogonal sortase A variants, eSrtA (2A–9) and eSrtA (4S–9), were engineered to specifically recognize altered substrates, LAXTG and LPXSG, respectively, achieving high activity and specificity. This was accomplished through a combination of selective substrate binding and the destabilization of the mischarged SrtA intermediate.¹¹² The first demonstration of homogeneous dual-payload conjugation using the two orthogonal variants, eSrtA(2A–9) and eSrtA(4S–9), involved conjugating a PEG-bearing polyglycine and an Alexa Fluor 750-linked LAETG peptide to fibroblast growth factor 1 (FGF1). This approach was further applied by conjugating two distinct payloads to the C-termini of the HC and LC of Fabs, showcasing its potential for developing dual-labeled tumor-targeting therapies (Figure 6g). Notably, the study found that close proximity of the two sortags did not compromise conjugation efficiency, while achieving near-quantitative conversion with rapid kinetics. However, this method has certain limitations. Achieving the desired conjugation efficiency required a high linker-to-protein ratio (50 equivalents), elevated SrtA concentration (0.75 equivalents), and additional purification steps between conjugations.¹¹³ Another major constraint of sortase-mediated conjugation is its reversibility. To drive the reaction forward, a large stoichiometric excess of the intended nucleophile is necessary, as demonstrated in the above study. Alternatively, using synthetic depsipeptide substrates can release less nucleophilic glycolic acid, reducing the likelihood of reaction reversal.¹³⁶

Combining sortase-mediated labeling with glycan remodeling has recently been demonstrated as an effective approach for producing dual-payload conjugates, specifically featuring a Topo1i and an immune agonist (Figure 6h).⁴⁹ The process achieved significant simplification through the use of an engineered endoglycosidase, which can cleave glycans and attach a linker payload in a single step. This innovation eliminates the need for additional adaptors commonly required in enzyme-mediated conjugation processes, thereby streamlining the workflow. Additionally, the technology leverages branched linker payloads, which further reduces procedural complexity and enables facile adjustments in DAR and the ratios of the two payloads. This adaptability enhances the versatility of conjugate design, allowing for the precise customization of payload ratios, which is especially beneficial in optimizing therapeutic efficacy and minimizing off-target toxicities of ADCs.

Process complexity, flexibility to adjust total DAR, and payload ratios

The production of dual-payload ADCs generally demands a more intricate process than single-payload ADCs, primarily due to the additional reaction steps required (Table 4). The dual conjugation process complexity is largely influenced by the introduction of orthogonal conjugation handles, with the post-protein synthesis steps directly affecting the overall complexity of the process. Balancing the potency of the two payloads with different MOAs is crucial for the success of dual-payload ADCs; thus, the DARs of the two payloads are important design parameters. Flexibility in adjusting the total DAR and the ratio of the two payloads is critical for optimization of therapeutic efficacy and safety profiles of different linker payload combinations.

Conjugation handles that use orthogonal amino acids or enzymatic tags can be incorporated during protein synthesis, which greatly simplifies the dual conjugation process. When comparing different strategies, dual-payload conjugation with DVD-IgG offers a relatively streamlined process, as it requires minimal modifications. However, increasing the DAR or adjusting the ratio of the two payloads through additional conjugation sites proves challenging with this approach. Non-canonical amino acids provide greater flexibility in modifying both total DAR and the ratio of two payloads while maintaining reasonable process complexity, but they require specialized protein expression systems, limiting their broader application. Dual-payload ADCs constructed through orthogonal enzymatic tags, such as sortase/butelase and sortase/engineered endoS combinations, can be produced through a relatively straightforward process. While they allow for precise conjugation, they suffer from slower conjugation kinetics and necessitate the removal of enzymes post-conjugation. Consequently, enzyme-mediated methods often involve adapters and require column purification to remove enzymes from the final product. Tyrosinase-mediated oxidative coupling significantly enhances reaction kinetics and reduces the required enzyme quantity. However, as with other enzyme-mediated conjugation methods, the limited number of accessible tyrosine residues restricts flexibility, particularly in optimizing the DAR and adjusting the ratio of the two payloads.

Most methods that use a multifunctional linker strategy leverage established conjugation chemistries from single-payload ADC development, alongside existing antibody expression systems. Typically, these approaches involve attaching an adapter to address steric hindrance at conjugation sites, particularly when interchain thiols serve as primary conjugation handles. By using adapters, the process can better accommodate the effects of large linker payloads on conjugation kinetics, especially when enzymes are the primary conjugation agents. The versatility of a library of multifunctional adapters, combined with an array of linker payloads, offers flexibility for developing varied ADC designs. This modular approach enables the creation of diverse total DAR and payload ratio profiles, catering to different research and therapeutic needs. As an alternative, direct chemical synthesis of multifunctional linker payloads reduces the number of conjugation steps, potentially streamlining the process. Depending on the drug developer’s resources, technical expertise, and specific objectives, both the multifunctional adapter and multifunctional linker payload methods offer adaptable paths for customized drug design. Each approach can contribute to optimizing drug efficacy and targeting precision through strategic adjustments in DAR and payload distribution.

The combination of one non-canonical amino acid with cysteine, the utilization of two non-canonical amino acids, as well as a branched multifunctional linker payload, has demonstrated the ease of manufacturability and the feasibility of achieving desirable DARs. Additionally, the combination of one non-canonical amino acid with cysteine and the combination of two non-canonical amino acids allow for robust fine-tuning the DAR and the ratio of the two payloads, which is critical for optimizing the synergy of the two MOAs. By carefully evaluating these factors, ADC developers can select the most suitable methods to achieve effective dual conjugation and to optimize the pharmacologic properties of dual-payload conjugates.

In summary, the selection of a precise dual-payload conjugation method involves careful consideration of multiple criteria, including process complexity and compatibility with existing single-payload conjugation processes, linker payload MOAs, and antibody production platforms. There is no single criterion for ranking of the various dual-payload conjugation methods as multiple disciplines need to be integrated, including PK, pharmacodynamics, safety, and tumor biology in addition to process complexity of ADC manufacturing and preferences may vary across different ADC developers.

Discussion

Combination therapy with compounds exerting different MOAs is often considered during clinical development of oncology drug candidates to improve therapeutic efficacy. Combination with traditional chemotherapy, which lacks tumor-targeted delivery, is often associated with increased toxicity and a narrower therapeutic window.^137,138 The promising data from the Phase I clinical trial (NCT04724018) investigating the safety and efficacy when combining two ADCs, sacituzumab govitecan and enfortumab vedotin, demonstrated the potential of co-administration of two ADCs with different classes of payloads.¹³⁹ These early clinical observations demonstrated added benefit when combining two different payload MOAs and suggested that development of dual-payload ADCs may offer additional therapeutic benefits compared to the combination of two separate ADCs, as outlined below.

Focused targeting: A single ADC with dual payloads can deliver both agents directly to the same cancer cell. This enhances efficacy by allowing both drugs to act within the same tumor microenvironment.

Synergistic mechanisms: The dual payloads can exploit synergistic MOAs, resulting in greater therapeutic efficacy compared to two independent single-payload ADCs by the simultaneous release of both payloads in tumors, which can lead to more effective tumor cell killing, particularly if the payloads block two different pathways of oncogenesis.

Improved drug delivery: Two single-payload ADCs targeting the same antigen compete for limited binding sites and limiting internalization of each ADC, whereas a single dual-payload ADC delivers more payloads per internalized antibody, leading to improved efficacy.

Streamlined clinical development and manufacturing: Developing a single ADC with dual payloads is less complex than developing two single-payload ADCs in parallel. This approach simplifies regulatory pathways, clinical development, and manufacturing. A single dual-payload ADC requires only one regulatory data package, standard monotherapy dose escalation design, and one CMC development campaign.

Simplified treatment regimens and regulatory requirements: A single dual-payload ADC streamlines the treatment logistics for patients and healthcare providers. Instead of coordinating and optimizing the co-administration of two different ADCs, a single agent simplifies logistics and enhances patient compliance.

Potential to combine with other therapeutic agents: A dual-payload ADC offers greater clinical potential for combination with therapies such as immune checkpoint inhibitors, compared to using two separate single-payload ADCs in conjunction with a third therapy.

Overall, a dual-payload ADC can provide a more effective and efficient approach to cancer treatment compared to a combination of separate ADCs and ultimately improve the therapeutic benefit for cancer patients.

Here, we reviewed homogenous dual conjugation methods that were reported in the literature and that were experimentally validated. While there is significant ongoing research into the development of dual-payload ADCs, only a fraction of successful methods have been reported, making it difficult to cite all relevant sources and references. However, in theory, combining any two orthogonal conjugation methods can enable precise dual conjugates. For example, this can include the combination of non-canonical amino acids with engineered cysteine, the pairing of non-canonical amino acids with enzyme-mediated conjugation, or enzymatic conjugation in combination with inter-chain cysteine. When developing dual-payload ADCs, it is crucial to assess not only pharmacological properties, such as cell killing, efficacy in tumor models, and safety, but also the physicochemical properties, PK, manufacturability, and cost of these complex, dual-payload therapeutics. Understanding the physicochemical properties ensures optimal stability and solubility, which are essential for ADC efficacy and safety.^5,8,140,141 Factors such as linker design, conjugation technology, conjugation sites, and DAR play critical roles in achieving favorable physicochemical properties. The linker-payload design directly affects the hydrophobicity of the conjugates, which is correlated to exposure and efficacy.¹⁴² The increased total DAR of dual-payload conjugates compared to single-payload conjugates presents additional challenges, necessitating further optimization to enhance hydrophilicity and mitigate aggregation propensity, both of which directly influence the tolerability of the conjugates.¹⁴³ Additionally, evaluating PK characteristics informs how the ADC will behave in vivo, influencing dosing strategies and therapeutic outcomes. Furthermore, manufacturability considerations are critical for scaling up production and ensuring consistent quality of clinical ADCs, which ultimately impacts clinical success. By integrating these multifaceted assessments into the development process, researchers can better predict the performance of dual-payload ADCs in clinical settings to enhance their potential as effective cancer therapies. Researchers have pushed the boundary and demonstrated feasibility of multi-payload conjugates by combining four orthogonal site-specific conjugation methods.¹⁴⁴ The application of the multiple-payload conjugates is yet to be explored, but the tool has become available for drug developers. The considerations discussed here will apply to these more complex modalities.

We would like to emphasize that the DAR and the ratios of the two payloads are critical for achieving optimal synergy between the MOAs in dual-payload ADCs. Ensuring the right DAR and payload ratios is essential for maximizing ADC efficacy and minimizing potential toxicity. ADC development must prioritize these key design parameters and should avoid compromises based on conjugation platform limitations in order to optimize the therapeutic index and to provide maximal therapeutic benefit to cancer patients. By maintaining emphasis on these critical design factors, and how to translate them into clinical response, it is possible to improve the overall effectiveness of ADCs and patient outcomes.

Here, we aimed to review the therapeutic potential, production methods, and key considerations for this novel product concept, rather than to validate the concept of dual-payload ADCs or specific payload combinations by clinical data. Dual-payload ADCs offer a promising strategy to enhance efficacy by combining two distinct MOA. However, this approach also faces potential challenges, including the complexity of managing multiple mechanisms of toxicity, the potential for increased toxicity, and the loss of flexibility in titrating two separate single-payload ADCs.

Therapeutic index is a critical factor in drug development, and a thorough assessment of the efficacy and toxicity of dual-payload ADCs in comparison to mono-payload ADCs is essential. Similar to other drug development efforts, there is a risk that preclinical data may not fully translate to clinical outcomes. However, there is increasing evidence that preclinical toxicity studies conducted in non-human primates are predictive for most, if not all platform and on-target toxicities of ADCs.¹⁴⁵ Similarly, testing ADCs at clinically relevant dose levels in patient-derived xenograft tumors grown in mice was shown to be highly predictive of clinical response rates, across multiple linker payload types.¹⁴⁶ Therefore, establishing an exposure-based safety vs. efficacy relationship for ADCs, i.e., therapeutic index,¹⁴¹ can guide the selection of the most promising linker payload combination as well as the ratio of the two payloads. Selecting clinically validated, distinct payloads with minimal overlapping toxicities and leveraging synergistic effects may facilitate optimization of the payload ratio preclinically, mitigating safety concerns, and improving the therapeutic index, particularly in drug-resistant tumors.

As dual-payload ADCs remain an emerging concept and have yet to be validated in the clinic, further preclinical and clinical studies are necessary to optimize their design and to demonstrate therapeutic superiority over single payload ADCs. Addressing toxicity concerns through strategic payload selection and rigorous preclinical evaluation to determine their therapeutic index will be key to maximizing therapeutic benefits. In addition to the rapid emergence of preclinical studies, the advancement of KH815, a Trop2-targeting Topo1i and RNA polymerase II inhibitor dual-payload ADC developed by Chengdu Kanghong Biotech, into clinical trials (NCT068856450) marks a major step forward in dual-payload ADC development.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author Contributions

Conceptualization: GY

Initial drafts of the manuscript: MW, GY

Investigation: MW, YP

Figures:MW, AY, DC

Tables: MW

Writing – original draft: MW, GY

Writing – review & editing: MW, GY and HP

All authors have read and agreed to the published version of the manuscript.

Data availability statement

The data presented in this study are available on request from the corresponding author.