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.

Keywords: Antibody drug conjugate; conjugation; drug resistance; dual-payload; homogenous; manufacturability; next generation cancer therapy; process complexity.

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.

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.

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.

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.

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.

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.

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.