DVD-IgG1 Antibody–Drug Conjugates: Expanding the Landscape of Targeted Cancer Therapy

Abstract

Antibody–drug conjugates (ADCs) are transforming targeted cancer therapy, yet conventional IgG-based formats are often limited by stochastic conjugation, yielding heterogeneous ADCs. Dual-variable domain IgG1 (DVD-IgG1) antibodies offer a modular solution by enabling precise, site-specific conjugation through catalytic lysine and engineered arginine or cysteine residues, affording homogeneous ADCs. This review summarizes recent advances in DVD-IgG1–based ADCs, highlighting site-specific conjugation, compatibility with diverse linker chemistries, and incorporation of both established and experimental payloads. Together, these developments position DVD-IgG1s as a versatile platform to expand the chemical and biological design space of next-generation ADCs.

Introduction

Antibody–drug conjugates (ADCs) have emerged as one of the most impactful therapeutic classes in modern oncology, bridging the specificity of monoclonal antibodies (mAbs) with the potency of cytotoxic small molecules that would be intolerable as systemic agents. [1] Since the approval of gemtuzumab ozogamicin (Mylotarg^®) with a calicheamicin (CAL)-type payload (Figure 1A) in 2000 for the treatment of CD33-positive acute myeloid leukemia, the ADC landscape has grown considerably, with 14 FDA-approved agents and a pipeline exceeding 100 clinical assets spanning both solid tumors and hematologic malignancies.[2] Despite these advances, significant challenges remain, particularly in conjugation technology.[3] Traditional stochastic methods—where cytotoxic payloads are attached to endogenous cysteine (Cys) or lysine (Lys) residues—dominate current practice and account for all approved ADCs, albeit next-generation platforms enabling site-specific and homogeneous conjugation, such as chemical or enzyme-mediated strategies, have emerged as powerful alternatives.[3] However, these traditional approaches yield heterogeneous mixtures with variable drug-to-antibody ratios (DARs, typically 0–8), complicating pharmacokinetics, quality control, and dose optimization. Each species within this distribution can display distinct efficacy, toxicity, and pharmacokinetic properties, collectively narrowing the therapeutic index of stochastically assembled ADCs. Moreover, linker instability further compresses this window, requiring a delicate balance between over- and under-dosing in order to avoid toxicity and maintain efficacy, respectively. These limitations highlight the need for antibody platforms that support site-specific conjugation strategies, thus overcoming the performance bottlenecks of current ADCs.[4,5]

Figure 1:

Schematic representation of conventional IgG1-based antibody-drug conjugates (ADCs) by stochastic conjugation and dual-variable domain (DVD)-IgG1-based ADCs by site-specific conjugation. (A) FDA-approved anti-CD33 ADC gemtuzumab ozogamicin with a calicheamicin (CAL)-type payload represents a heterogeneous ADC with an average drug-to-antibody ratio (DAR) of 2–3. (B) Investigational anti-HER2 DVD-IgG1 ADC with a monomethyl auristatin F (MMAF) payload displays a highly homogeneous ADC with DAR of 2.

All currently approved ADCs are derived from the immunoglobulin G (IgG) antibodies IgG1 and IgG4.[6] Breakthroughs in mAb engineering have positioned dual-variable domain IgG1s (DVD-IgG1s) as powerful bispecific immunotherapies for cancer treatment, however, this technology has yet to be effectively translated into a clinical ADC platform.[7–9] Herein we summarize recent developments that set the stage for DVD-IgG1-based ADCs as contenders to address the shortcomings with the current portfolio of clinically approved ADCs. These developments include site-specific conjugation strategies, functionalization of both validated payloads in FDA-approved ADCs and novel payload candidates, and systematic optimization of available linker chemistries (Figure 1B).[10,11] These preclinical successes have demonstrated the viability of DVD-IgG1-based ADCs for utilizing novel payloads, their modularity for targeting multiple antigens, and the ability to rapidly optimize linker chemistry.[12,13] Future iterations of this technology can be further developed to install two distinct payloads within a single ADC, further widening the therapeutic potential of the emerging landscape of DVD-IgG1 ADCs.

DVD-IgG1s—an emerging platform for precise and site-specific conjugation, and rapid ADC development

The unique architecture of DVD-IgG1s enables a relatively straightforward approach to the design of multi-specific mAbs, offering numerous advantages over combination therapies and contributing to clinical success.[8,9] DVD-IgG1s are engineered antibodies that assemble into tetravalent structures, typically in a homodimeric fashion with an inner and outer Fv, in contrast to the natural IgG1 architecture consisting of a single Fv domain (Figure 2A). Despite their increased molecular weight (~200 kDa versus ~150 kDa for IgG1), DVD-IgG1s exhibit pharmacokinetics and tissue penetration profiles comparable to IgGs. This functional similarity enables engagement with the neonatal fragment crystallizable receptor (FcRn), facilitating DVD-IgG1 recycling and prolonging serum half-life. The binding specificity of each Fv can be independently engineered by adopting Fvs from previously developed mAbs. This design enables dual antigen specificity, thereby addressing antigen heterogeneity and offering the potential for improved clinical efficacy and broader patient coverage compared with IgGs that target single antigen.[7,14–16] Two bispecific DVD-IgG1 that have advanced into late-stage clinical trials include the interleukin (IL)-1α/IL-1β-targeting lutikizumab, for immune-mediated diseases such as hidradenitis suppurativa [17] and the delta-like 4 protein (DLL4)/vascular endothelial growth factor (VEGF)-targeting dilpacimab in metastatic colorectal cancer and other advanced solid malignancies.[18] These examples highlight the utility of DVD-IgG1s for targeting either redundant antigens in a single pathway (IL-1α/IL-1β) or antigens involved in disparate biological pathways (DLL4/VEGF).

Figure 2:

General overview of the modular design of DVD-IgG1 molecules and the aldolase antibody h38C2-IgG1, serving as the basis for engineered DVD-IgG1 formats and their site-specific conjugation strategies. (A) The original modular design of DVD-IgG1 molecules enables flexible engineering for targeted applications. (B) The parental aldolase antibody h38C2 in IgG1 format lays the foundation for homo- and heterodimeric engineered DVD-IgG1 molecules that can be leveraged to prepare homogeneous ADCs with one or two distinct payloads via site-specific conjugation. The antibody binding site results from the convergence of six complementarity determining regions (CDRs), shown as ovals, three provided by each variable light (V_L) and heavy chain domains (V_H). Light chains are shown in white, heavy chains in gray. The variable fragment (Fv; V_L + V_H) of h38C2 contains a uniquely reactive Lys (shown in blue) residue (pKa ~6) at the bottom of an 11-Å deep hydrophobic pocket that constitutes the catalytic site. DVD-IgG1s graft an outer Fv that binds to a cell surface antigen (shown in yellow) on top of h38C2 IgG1 or h38C2 mutant Lys99Arg (shown in brown). Inner and outer variable domains of both light-chain and heavy-chain fragment are connected by a short peptide spacer. The heterodimeric DVD-IgG1 combines a parental h38C2 inner Fv in one arm with h38C2 mutant Lys99Arg in the other arm and utilizes knobs-into-holes mutations (brown triangle) for heavy-chain heterodimerization. (C) Site-specific and orthogonal conjugation approaches via Lys, Arg, and Cys are depicted in blue, brown, and purple ovals, respectively. The β-lactam moiety undergoes irreversible amide bond formation via the Lys residues, whereas methylsulfone phenyloxadiazoles (MS-PODA) engage the Lys residues to generate stable amine linkages. Phenylglyoxal derivatives covalently react with the guanidino group of the buried Arg residues, resulting in the formation of stable hydroxyimidazole adducts. Dibromomaleimide derivatives covalently react with the engineered Cys residues to afford stable C-S adducts. R1 and R2 are referring to respective amino acid residues.

The catalytic antibody 38C2, combined with DVD-IgG1s, enables the incorporation of nontraditional site-specific conjugation chemistries into a mAb with programmable antigen specificity—providing a robust and flexible platform for ADC development (Figure 2B). While the DVD architecture was originally designed for targeting multiple antigens, this modularity was later leveraged to incorporate elements of a catalytic antibody enabling site-specific conjugation. Specifically, mouse mAb 38C2 was originally developed to mimic aldolases with catalytic Lys residues and generated by immunization with a reactive β-diketone hapten attached to a carrier protein.[19,20] Humanization and incorporation of IgG1 elements within 38C2 resulted in mAb h38C2,[21] which preserves the IgG1-like structure and the aldolase activity conferred by 38C2 while attenuating immunogenicity, enabling conjugation of various peptide-based payloads for therapeutic applications.[11,22,23] Several clinical assets have utilized the h38C2 platform for targeting cancer, pain, and diabetes.[24] The most advanced example, CVX-060, which incorporates an angiopoietin-binding peptide payload, reached phase II clinical trials in second-line metastatic renal cell carcinoma prior to its discontinuation due to safety concerns, in particular an increased incidence of thrombotic events observed in combination therapy studies. [25,26] These early examples demonstrate the versatility of h38C2-based immunotherapies for various indications, however, the payload choices were limited to peptides, and the antibody-peptide conjugates suffered from safety, pharmacokinetic, and pharmacodynamic issues.

The programmable antigen-targeting specificity of DVD-IgG1s, combined with the catalytic flexibility of h38C2, provides a platform to target numerous antigens in cancer therapy with one or two distinct payloads in a single construct (Figure 2B). The architecture of these DVD-IgG1s mirrors that described above, except the inner Fv is designated to supporting site-specific conjugation, while the outer Fv can still be varied to bind the antigen of interest. The architecture of the DVD-IgG1s containing the h38C2 inner Fv enables the preparation of homodimeric ADCs, by virtue of a uniquely reactive Lys for site-specific conjugation, giving rise to homogeneous ADCs with a DAR of 2. Unlike surface-exposed Lys residues, this buried Lys—located at position 99 (position 93 by Kabat numbering) in the variable heavy chain domain at the base of an 11 Å–deep hydrophobic pocket—exhibits markedly enhanced nucleophilicity (pKa ~6) at physiological pH. Successful conjugation has been carried out between h38C2-derived DVD-IgG1s and various payload precursors containing a methylsulfone phenyloxadiazole (MS-PODA), dibromomaleimide, or β-lactam functional group (Figure 2C).[12,13,22,23,27] By substituting Lys with arginine (Arg) in heterodimeric DVD-IgG1 (Figure 2B), orthogonal conjugation strategies for assembling ADCs with two distinct payloads become feasible, due to the reactivity of the Arg residue with payload precursors containing a glyoxal functional group (Figure 2C). Because the Lys-to-Arg mutation is confined to a single residue in the heavy chain, heterodimeric constructs can be readily generated that feature a buried Lys in one arm, a buried Arg in the other arm, and identical light chains.[11,28] This design enables orthogonal conjugation via β-lactam- and phenylglyoxal-containing payload precursors in a single reaction, allowing efficient loading of heterodimeric DVD-IgG1s with two distinct payloads and thereby providing a convenient and versatile platform for dual-payload ADCs (Figure 2C). Orthogonality was also achieved with a Lys-to-Cys mutation that afforded a free thiol group for selective conjugation to maleimide derivatives.[27] In contrast to conventional cysteine engineering—where surface-exposed cysteines are often capped (i.e., oxidized by cysteine, homocysteine, or glutathione) and therefore require a reductive activation step—Cys99 remains uncapped. As a result, Cys99 can be directly conjugated to electrophiles without prior reduction (Figure 2C). Previous studies have further highlighted the inherent modularity of the DVD-IgG1-based ADC platform, whereby variation of the outer Fv facilitated the parallel generation of DVD-IgG1 ADCs that target CD138 in multiple myeloma,[22] CD79b in non-Hodgkin lymphoma,[13] FOLR1 in ovarian cancer,[9] and TrkB or HER2 in breast cancer, each with a defined DAR of 2.[22,29,30]

Benchmarking the DVD-IgG1–based ADC platform with FDA-approved payloads

The first successful combinations of cytotoxic small molecule payloads, h38C2 conjugation chemistry, and homodimeric DVD-IgG1 architecture featured HER2-targeting antibodies tethered to monomethyl auristatin F (MMAF)-derived payloads.[10,22] The constructs were benchmarked against ado-trastuzumab emtansine (Kadcyla^®), an FDA-approved ADC for HER2+ malignancies. A β-lactam–functionalized MMAF derivative (1) served as the first-generation ADC precursor (Figure 3A). Efficient and quantitative sitespecific conjugation to anti-HER2 DVD-IgG1, led to full modification of the catalytic Lys residue and a uniform DAR of 2 (Figure 3B). Importantly, all other Lys residues remained unaffected due to the unique hapten-driven specificity of the hydrophobic pocket for the β-lactam–modified payload. Conjugation of compound 1 to anti-HER2 DVD-IgG1 was conveniently monitored using a fluorescence-based retro-aldol assay that quantifies the conversion of methodol to its parent aldehyde.[22] Residual retro-aldol activity directly reflects the presence of unconjugated h38C2 Lys residues, enabling rapid and reliable assessment of conjugation efficiency. Owing to its simplicity, low cost, and compatibility with miniaturized formats, this assay is readily adaptable to high-throughput screening, representing a further advantage of h38C2-based site-specific conjugation over not only conventional stochastic but also other site-specific conjugation strategies. The resulting MMAF–anti-HER2 DVD-IgG1 ADC exhibited subnanomolar, strictly target-dependent cytotoxicity in vitro comparable to ado-trastuzumab emtansine. Ultimately, the conjugate demonstrated high in vivo efficacy, surpassing the FDA-approved ADC ado-trastuzumab emtansine in an orthotopic mouse xenograft model of human breast cancer. To elaborate on further conjugation techniques, a second-generation MMAF–anti-HER2 DVD-IgG1 ADC was developed, demonstrating the utility of MS-PODA MMAF derivative (2) (Figure 3A).[23,31] The resulting conjugate displayed high homogeneity and retained potent, selective cytotoxic activity against a panel of breast cancer cell lines (Figure 3B). Given the propensity of β-lactams to undergo hydrolysis, the MS-PODAs provide a convenient alternative.

Figure 3:

DVD-IgG1-based ADCs employing the FDA-approved payload MMAF. (A) Structures of β-lactam–functionalized MMAF derivative (1), methylsulfone phenyloxadiazole–functionalized MMAF derivative (2), and phenylglyoxal-functionalized MMAF derivative (3). (B) 1, 2, and 3 are site-specifically conjugated to homo- and heterodimeric DVD-IgG1s, yielding homogeneous MMAF-anti-HER2 DVD-IgG1 ADCs with drug-to-antibody ratios (DARs) of ~2 for homodimeric and ~1 (i.e., ~1 for each of the two distinct payloads or ~2 for both payloads) for heterodimeric constructs.

The utility of the DVD-IgG1 format is further expanded by a Lys99Arg mutation in the h38C2-based active site, featuring conjugation chemistry that is orthogonal to the original Lys-based strategy, enabling the construction of heterodimeric dual payload ADCs (Figure 2B).[11] The structural conservation of the deep pocket suggested that, analogous to the buried Lys residue in h38C2-IgG1, substitution with Arg might confer a similarly unique reactivity that could be harnessed for site-specific conjugation (Figure 2C). A successful proof-of-concept study on h38C2_Arg DVD-Fab highlighted Arg as a viable alternative conjugation site within engineered antibody scaffolds and prompted the construction of Arg-derived anti-HER2 DVD-IgG1 ADCs. After conjugation of phenylglyoxal-functionalized MMAF derivative (3), the Arg-based anti-HER2 DVD-IgG1 ADC, with a DAR of ~1.7, revealed similar potency and selectivity when compared with the Lys-based anti-HER2 DVD-IgG1 ADC. The orthogonality of the Lys-based and Arg-based conjugation chemistries further expands the utility of the h38C2 chemistries to prepare dual-payload ADCs. A heterodimeric anti-HER2 DVD-IgG1 pairs a parental h38C2-IgG1 inner Fv in one arm with the h38C2-IgG1 Lys99Arg mutant in the other, and employs knobs-into-holes mutations (brown triangle) to ensure heavy-chain heterodimerization (Figure 2B).[32] Conjugation of the heterodimeric anti-HER2 DVD-IgG1 with either a β-lactam- or phenylglyoxal-functionalized MMAF derivative (1 or 3) resulted in ADCs with a DAR of ~1 (Figure 3B). These conjugates retained high potency and selectivity, although the in vitro cytotoxicity was slightly lower than those of the corresponding homodimeric anti-HER2 DVD-IgG1 ADCs with a DAR of ~2. To assess the feasibility of orthogonal conjugation for one-pot labeling, the β-lactam–functionalized MMAF derivative (1) and a phenylglyoxal–functionalized TAMRA fluorophore were simultaneously attached to the heterodimeric anti-HER2 DVD-IgG1. The heterodimeric ADC maintained the cytotoxicity and fluorescence conferred by each of the small molecule precursors, enabling direct visualization of ADC internalization and trafficking by confocal microscopy.[11] This pioneering work has set the stage for ADCs with two distinct payloads by further leveraging the orthogonal Lys/Arg site-specific conjugation chemistries.

Expanding the DVD-IgG1–based ADCs platform with experimental payloads

Natural products (NPs) such as the enediyne CAL, the peptidic auristatins (MMAE and MMAF), and camptothecin derivatives have demonstrated exceptional utility as ADC payloads.[33] Nonetheless, the repertoire of payload scaffolds currently employed remains limited compared with the vast diversity of NPs known to possess potent anticancer activity.[34] At the same time, clinical resistance to established ADCs is increasingly reported, often driven by payload-specific mechanisms such as the upregulation of drug efflux transporters or alterations in intracellular targets.[35] These challenges highlight an urgent need for the development of novel payloads that can circumvent resistance while broadening the therapeutic spectrum of ADCs through tumor-selective, highly effective targeting. So far, one novel class of NPs has been applied to DVD-IgG1-based ADCs, and future efforts are sure to expand the repertoire of payloads.

Within this context, anthraquinone-fused enediynes (AFEs), including uncialamycin (UCM) and the tiancimycins (TNMs), have emerged as particularly promising payloads. The AFEs are a structurally novel class of ADC payloads with an established mechanism of action (MoA), extensive set of structure-activity relationship (SAR) data, ease of material access via fermentation of engineered microbial producers, and ample opportunities for precise functionalization and ADC linker installation (Figure 4A).[36,37] AFEs consist of an anthraquinone moiety, critical for DNA intercalation, fused to variably oxygenated ten-membered enediyne cores that mediate potent DNA-damaging activity.[38] The DNA-damaging MoA of AFEs mirrors that of CAL, the validated payload utilized in two FDA-approved ADCs. Extensive synthetic and biosynthetic studies have established a robust SAR for the AFE scaffold.[39–42] Advances in metabolic engineering have enabled Streptomyces strains to produce TNMs in sufficient titers (>10 mg/L) for further functionalization, with ample opportunities to utilize enzymes from the biosynthetic pathways that encode for the production of AFEs as biocatalysts to facilitate regio-specific linker installation.[43–45] This biotechnological accessibility has facilitated the generation of functionalized TNM analogues, which in turn have served as highly attractive starting points for the development of DVD-IgG1–based ADCs targeting B-cell malignancies.

Figure 4:

Anti-CD79b DVD-IgG1-based antibody–drug conjugates (ADCs) employing tiancimycin (TNM) experimental payloads. (A) General structures of the anthraquinone-fused enediynes uncialamycin (UCM) and various tiancimycins (TNMs). (B) β-Lactam-functionalized C7-TNM derivative (4) and β-lactam-functionalized C26-TNM derivative (5) are site-specifically conjugated to homodimeric DVD-IgG1s, yielding homogeneous TNM-anti-CD79b DVD-IgG1 ADCs with drug-to-antibody ratios (DARs) of ~1.6 and ~1.9 for the C7- and C26-TNM payloads, respectively.

The C7 position at the TNM scaffold was functionalized in a biocatalytic fashion utilizing the S-adenosyl methionine-dependent O-methyltransferase TnmH, a tailoring enzyme in the TNM biosynthetic pathway to provide the β-lactam containing C7-modified TNM (4),[12,42] whereas the C26-modified TNM (5) was made available via an efficient strategy combining strain engineering and fermentation optimization with semisynthesis (Figure 4B).[13] Conjugation of the β-lactam functionalized C7-TNM derivative (4) to homodimeric anti-CD79b DVD-IgG1 yielded an ADC with a DAR of ~1.6, which was shown to be CD79b-selective with nanomolar potency in vitro.[12,44] In the second generation of AFE-based DVD-IgG1 ADCs, optimized conjugation of the β-lactam functionalized C26- TNM derivative (5) to anti-CD79b DVD-IgG1 yielded an ADC with a DAR of ~2. The optimized ADC displayed potent and selective activity across multiple CD79b-expressing cell lines compared to the CD79b-targeting ADC polatuzumab vedotin (Polivy^®), and, most notably, showed activity in patient-derived primary chronic lymphocytic leukemia cells.[13] Collectively, integrating unique drug-payload properties, including engineered payload biosynthesis, biocatalysis, and protecting-group-free chemical modification and linker installation, with distinctive antibody-carrier features, alongside efficient site-specific conjugation under physiological conditions, positions this platform as a versatile approach for generating homogeneous AFE- and DVD-IgG1-based ADCs. In doing so, it enables the development of next-generation targeted chemotherapies against a broad range of antigens.

Conclusions and Future Outlook

The development of DVD-IgG1–based ADCs represents an important step forward in addressing the inherent limitations of conventional IgG-based conjugates. By combining the modularity of DVDs with the precision of Lys- and Arg-mediated site-specific conjugation chemistries, the DVD-IgG1 platform enables the generation of structurally homogeneous ADCs with defined DARs, enhanced stability, and versatile payload/antigen compatibility. Proof-of-concept studies with validated payloads of FDA-approved ADCs, such as MMAF, and experimental payload scaffolds, including AFEs, highlight the capacity of this format to expand the therapeutic repertoire beyond existing payload classes. Importantly, the ability to engineer homodimeric and heterodimeric DVD-IgG1 constructs further broadens the design space, allowing further diversification to dual-payload and dual antigen-targeting modalities.

The effectiveness of DVD-IgG1-based ADCs has been demonstrated in the pioneering examples laid out here, and the stage is set for expansion into more elaborate designs with heightened clinical relevance. Looking ahead, several directions are likely to shape the advancement of DVD-IgG1–based ADCs. Systematic evaluation of pharmacokinetics, safety, and efficacy in preclinical models will be essential to benchmark their translational value relative to existing ADC formats. The expansion of orthogonal conjugation strategies, particularly those enabling site-specific dual payload installation, may open avenues for combination payloads that simultaneously address resistance mechanisms and broaden cytotoxic coverage.[46] The integration of engineered production of designer NP payloads and biocatalysis for linker installation, exemplified by AFEs, underscores the potential of biotechnological and semisynthetic strategies to unlock underexplored chemical space for next-generation ADC development. Finally, the inherent modularity of the DVD-IgG1 architecture offers opportunities for rapid and modular adaptation to newly validated tumor antigens, paving the way for personalized chemotherapy strategies. Alternatively, bispecific antibodies that target multiple antigens on the same cell or different cells to overcome resistance are an attractive future direction,[15] which are similarly enabled by the heterodimeric DVD architecture. Taken together, DVD-IgG1–based ADCs stand at the intersection of antibody engineering, cancer biology, and NP chemistry. By uniting precise site-specific conjugation with payload innovation, design, and scalable production by synthetic biology and biotechnology, this emerging platform has the potential to redefine the next generation of targeted cancer therapies and extend the clinical impact of ADCs to a broader spectrum of malignancies.

Data availability

No data was used for the research described in the article.