Exploring the next generation of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising cancer treatment modality that enables the selective delivery of highly cytotoxic payloads to tumours. However, realizing the full potential of this platform necessitates innovative molecular designs to tackle several clinical challenges such as drug resistance, tumour heterogeneity and treatment-related adverse effects. Several emerging ADC formats exist, including bispecific ADCs, conditionally active ADCs (also known as probody-drug conjugates), immune-stimulating ADCs, protein-degrader ADCs and dual-drug ADCs, and each offers unique capabilities for tackling these various challenges. For example, probody-drug conjugates can enhance tumour specificity, whereas bispecific ADCs and dual-drug ADCs can address resistance and heterogeneity with enhanced activity. The incorporation of immune-stimulating and protein-degrader ADCs, which have distinct mechanisms of action, into existing treatment strategies could enable multimodal cancer treatment. Despite the promising outlook, the importance of patient stratification and biomarker identification cannot be overstated for these emerging ADCs, as these factors are crucial to identify patients who are most likely to derive benefit. As we continue to deepen our understanding of tumour biology and refine ADC design, we will edge closer to developing truly effective and safe ADCs for patients with treatment-refractory cancers. In this Review, we highlight advances in each ADC component (the monoclonal antibody, payload, linker and conjugation chemistry) and provide more-detailed discussions on selected examples of emerging novel ADCs of each format, enabled by engineering of one or more of these components.

An Enzymatically Cleavable Tripeptide Linker for Maximizing the Therapeutic Index of Antibody-Drug Conjugates.

Valine-citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody-drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid-glycine-citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease-mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg-1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

Glioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Homogeneous antibody-angiopep 2 conjugates for effective brain targeting.

Antibody-based therapy has shown great success in the treatment of many diseases, including cancers. While antibodies and antibody-drug conjugates (ADCs) have also been evaluated for central nervous system (CNS) disorders as well as brain tumors, their therapeutic efficacy can be substantially limited due to low permeability across the blood-brain barrier (BBB). Thus, improving BBB permeability of therapeutic antibodies is critical in establishing this drug class as a reliable clinical option for CNS diseases. Here, we report that, compared with a conventional heterogeneous conjugation, homogeneous conjugation of the synthetic BBB shuttle peptide angiopep-2 (Ang2) to a monoclonal antibody (mAb) provides improved binding affinity for brain microvascular endothelial cells in vitro and accumulation into normal brain tissues in vivo. In a mouse model, we also demonstrate that the homogeneous anti-EGFR mAb-Ang2 conjugate administered intravenously efficiently accumulates in intracranial tumors. These findings suggest that homogeneous conjugation of BBB shuttle peptides such as Ang2 is a promising approach to enhancing the therapeutic efficacy of antibody agents for CNS diseases.

Enhanced anti-angiogenetic effect of transferrin receptor-mediated delivery of VEGF-trap in a glioblastoma mouse model.

Glioblastoma (GBM) is a common and aggressive brain cancer that accounts for 60% of adult brain tumors. Anti-angiogenesis therapy is an attractive option due to the high vasculature density of GBM. However, the best-known anti-angiogenic therapeutics, bevacizumab, and aflibercept, have failed to show significant benefits in GBM patients. One of the reasons is the limited brain penetration of antibody-based therapies due to existence of the blood-brain barrier (BBB), which is further strengthened by the blood vessel normalization effects induced by anti-angiogenic therapies. To investigate if increased drug concentration in the brain by transferrin receptor (TfR)-mediated delivery across the BBB can enhance efficacy of anti-angiogenic antibody therapies, we first identified an antibody that binds to the apical domain of the mouse TfR and does not compete with the natural ligand transferrin (Tf) binding to TfR. Then, we engineered two bispecific antibodies fusing a vascular endothelial growth factor (VEGF)-Trap with the TfR-targeting antibody. Characterization of the two bispecific formats using multiple in vitro assays, which include endocytosis, cell surface and whole-cell TfR levels, human umbilical vein endothelial cell growth inhibition, and binding affinity, demonstrated that the VEGF-Trap fused with a monovalent αTfR (VEGF-Trap/moAb4) has desirable endocytosis without the induction of TfR degradation. Peripherally administered VEGF-Trap/moAb4 improved the brain concentration of VEGF-Trap by more than 10-fold in mice. The distribution of VEGF-Trap/moAb4 was validated to be in the brain parenchyma, indicating the molecule was not trapped inside the vasculature. Moreover, improved VEGF-Trap brain distribution significantly inhibited the angiogenesis of U-87 MG GBM tumors in a mouse model.

Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance.

Breast tumors generally consist of a diverse population of cells with varying gene expression profiles. Breast tumor heterogeneity is a major factor contributing to drug resistance, recurrence, and metastasis after chemotherapy. Antibody-drug conjugates (ADCs) are emerging chemotherapeutic agents with striking clinical success, including T-DM1 for HER2-positive breast cancer. However, these ADCs often suffer from issues associated with intratumor heterogeneity. Here, we show that homogeneous ADCs containing two distinct payloads are a promising drug class for addressing this clinical challenge. Our conjugates show HER2-specific cell killing potency, desirable pharmacokinetic profiles, minimal inflammatory response, and marginal toxicity at therapeutic doses. Notably, a dual-drug ADC exerts greater treatment effect and survival benefit than does co-administration of two single-drug variants in xenograft mouse models representing intratumor HER2 heterogeneity and elevated drug resistance. Our findings highlight the therapeutic potential of the dual-drug ADC format for treating refractory breast cancer and perhaps other cancers.

Chemical generation of small molecule-based bispecific antibody-drug conjugates for broadening the target scope.

Antibody-drug conjugates (ADCs) hold great therapeutic promise for cancer indications; however, treating tumors with intratumor heterogeneity remains challenging. We hypothesized that ADCs that can simultaneously target two different cancer antigens could address this issue. Here, we report controlled production and evaluation of bispecific ADCs chemically functionalized with tumor-targeting small molecules. Enzyme-mediated conjugation of bi-functional branched linkers and following sequential orthogonal click reactions with payload and tumor targeting modules (folic acid or RGD peptide) afforded homogeneous bispecific ADCs with defined ligand/drug-to-antibody ratios ranging from 4 + 4 to 16 + 4 (ligand/payload). Most bispecific ADCs were stable under physiological conditions for 14 days. Functionalization with the cancer-specific ligands did not impair cathepsin B-mediated payload release from ADCs. Bispecific ADCs targeting the folate receptor (FR)/human epidermal growth factor receptor 2 (HER2) demonstrated specific binding and high cell killing potency only in cells expressing either antigen (FR or HER2). Integrin/HER2 bispecific ADCs equipped with RGD peptides also showed target-specific binding and cytotoxicity in integrin- or HER2-positive cells. These findings suggest that our small-molecule based bispecific ADCs have the potential to effectively treat tumors with heterogeneous antigen expression.

Total Synthesis of the Monomeric Unit of Lomaiviticin A.

The architecturally symmetrical and synthetically challenging marine natural products lomaiviticins A and B present alluring synthetic targets due to their molecular complexity, potent antitumor properties, and natural scarcity. Herein, we report the total synthesis of the fully glycosylated monomeric unit of lomaiviticin A, monolomaiviticin A. The retrosynthetically derived synthetic strategy relied on an intramolecular palladium-catalyzed coupling reaction to complete the tetracyclic aglycon scaffold and gold-promoted glycosylations to install the synthetically challenging α- and ß-glycoside moieties of the target molecule. This accomplishment paves a path for the eventual total synthesis of lomaiviticins A and B and opens opportunities for biological investigations within this family of compounds.

LILRB4-targeting Antibody-Drug Conjugates for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.

Transglutaminase-Mediated Conjugations.

Microbial transglutaminase (MTGase) catalyzes site-specific transpeptidation between a primary amine within linkers and the side chain of glutamine 295 within deglycosylated chimeric, humanized, and human IgG1s, affording homogeneous antibody-drug conjugates (ADCs). This method can be empowered by mutation of asparagine 297, insertion of a glutamine-containing peptide tag, and the use of branched linkers. Such modifications facilitate the conjugation process and provide flexibility in adjusting the conjugation site and drug-to-antibody ratio (DAR). Here, we present a protocol optimized in our group for MTGase-mediated linker incorporation and subsequent click chemistry-based payload installation. Both small linear linkers and bulky branched linkers can be incorporated into the Fc moiety within various antibodies, affording homogeneous ADCs with defined DARs. Thanks to the high homogeneity, ADCs constructed using this method can be analyzed using a single-quadrupole electrospray ionization (ESI) mass spectrometer, which many laboratories own for regular analysis of small molecules and peptides. The approach presented here allows for facile and cost-effective production of various homogeneous ADCs and other antibody conjugates for research and clinical purposes.

Disrupting LILRB4/APOE Interaction by an Efficacious Humanized Antibody Reverses T-cell Suppression and Blocks AML Development.

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

Glutamic acid-valine-citrulline linkers ensure stability and efficacy of antibody-drug conjugates in mice.

Valine-citrulline linkers are commonly used as enzymatically cleavable linkers for antibody-drug conjugates. While stable in human plasma, these linkers are unstable in mouse plasma due to susceptibility to an extracellular carboxylesterase. This instability often triggers premature release of drugs in mouse circulation, presenting a molecular design challenge. Here, we report that an antibody-drug conjugate with glutamic acid-valine-citrulline linkers is responsive to enzymatic drug release but undergoes almost no premature cleavage in mice. We demonstrate that this construct exhibits greater treatment efficacy in mouse tumor models than does a valine-citrulline-based variant. Notably, our antibody-drug conjugate contains long spacers facilitating the protease access to the linker moiety, indicating that our linker assures high in vivo stability despite a high degree of exposure. This technology could add flexibility to antibody-drug conjugate design and help minimize failure rates in pre-clinical studies caused by linker instability.

Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands.

We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues 1-4 have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1-4 with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1-4 increased with incubation time. Enone compounds 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compounds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogues 1-4. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD.

Enzymatic conjugation using branched linkers for constructing homogeneous antibody-drug conjugates with high potency.

Antibody-drug conjugates (ADCs) are emerging therapeutic agents in the treatment of cancer, and various conjugation strategies and chemical linkers have been developed to efficiently construct ADCs. Despite previous extensive efforts for improving conjugation efficiency and ADC homogeneity, most ADC linkers developed to date load only single payloads. Branched linkers that can load multiple payload molecules have yet to be fully explored. It is logical to envisage that a multi-loading strategy allows for increase in drug-to-antibody ratio (DAR) with less chemical or enzymatic modification to the antibody structure compared to traditional linear linkers, leading to efficient ADC construction, minimal destabilization of the antibody structure, and enhanced ADC efficacy. Herein, we report that the branched linkers we designed can be quantitatively installed on an anti-HER2 monoclonal antibody by microbial transglutaminase (MTGase)-mediated conjugation without impairing its antigen binding affinity, enabling modular installation of payload molecules and construction of homogeneous ADCs with increased DARs (up to 8). An anti-HER2 antibody-monomethyl auristatin F conjugate constructed using our branched linkers showed greater in vitro cytotoxicity against HER2-expressing breast cancer cell lines than that consisting of linear linkers, demonstrating the effectiveness of the branched linker-based payload delivery. Our finding demonstrates that enzymatic ADC construction using branched linkers is a promising strategy, which may lead to innovative cancer therapeutics.

Truncated Autoinducing Peptide Conjugates Selectively Recognize and Kill Staphylococcus aureus.

The accessory gene regulator (agr) of Staphylococcus aureus coordinates various pathogenic events and is recognized as a promising therapeutic target for virulence control. S. aureus utilizes autoinducing peptides (AIPs), cyclic-peptide signaling molecules, to mediate the agr system. Despite the high potency of synthetic AIP analogues in agr inhibition, the potential of AIP molecules as a delivery vehicle for antibacterial agents remains unexplored. Herein, we report that truncated AIP scaffolds can be fused with fluorophore and cytotoxic photosensitizer molecules without compromising their high agr inhibitory activity, binding affinity to the receptor AgrC, or cell specificity. Strikingly, a photosensitizer-AIP conjugate exhibited 16-fold greater efficacy in a S. aureus cell-killing assay than a nontargeting analogue. These findings highlight the potential of truncated AIP conjugates as useful chemical tools for in-depth biological studies and as effective anti-S. aureus agents.

SRC2-3 binds to vitamin D receptor with high sensitivity and strong affinity.

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates the expression of target genes through ligand binding. To express the target gene, coactivator binding to the VDR/ligand complex is essential. Although there are many coactivators in living cells, precise interactions between coactivators and VDR have not been clarified. Here, we synthesized two coactivator peptides, DRIP205-2 and SRC2-3, evaluated their affinity for the ligand-binding domain (LBD) of VDR using 1α,25-dihydroxyvitamin D3, partial agonist 1, and antagonist 2 by surface plasmon resonance (SPR), and assessed their interaction modes with VDR-LBD using X-ray crystallographic analysis. This study showed that the SRC2-3 peptide is more sensitive to the ligands (agonist, partial agonist, and antagonist) and shows more intimate interactions with VDR-LBD than DRIP205-2 peptide.

Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by Hybrid Approach Combining Small-Angle X-ray Scattering and Molecular Dynamics.

Vitamin D receptor (VDR) controls the expression of numerous genes through the conformational change caused by binding 1α,25-dihydroxyvitamin D3. Helix 12 in the ligand-binding domain (LBD) is key to regulating VDR activation. The structures of apo VDR-LBD and the VDR-LBD/antagonist complex are unclear. Here, we reveal their unprecedented structures in solution using a hybrid method combining small-angle X-ray scattering and molecular dynamics simulations. In apo rat VDR-LBD, helix 12 is partially unraveled, and it is positioned around the canonical active position and fluctuates. Helix 11 greatly bends toward the outside at Q396, creating a kink. In the rat VDR-LBD/antagonist complex, helix 12 does not generate the activation function 2 surface, and loop 11-12 is remarkably flexible compared to that in the apo rat VDR-LBD. On the basis of these structural insights, we propose a "folding-door model" to describe the mechanism of agonism/antagonism of VDR-LBD.

Helix12-Stabilization Antagonist of Vitamin D Receptor.

To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and synthesized vitamin D analogues with bulky side chains based on the "active antagonist" concept in which antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds 3a and 3b showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and static X-ray crystal structure analyses indicated that compound 3a stabilizes H11-H12 but displaces H6-H7 so that 3a is a novel rather than "active" or "passive" type of antagonist. We classified 3a as a third type of antagonist and called it "H11-H12 stabilization antagonist". HDX-MS analysis indicated that antagonist 3b is an "active" antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse depending on each antagonist structure. Additionally, HDX-MS was proven to be very useful for investigations of protein structure alterations resulting from ligand binding.

Fine tuning of agonistic/antagonistic activity for vitamin D receptor by 22-alkyl chain length of ligands: 22S-Hexyl compound unexpectedly restored agonistic activity.

1α,25-Dihydroxyvitamin D3 exerts its actions by binding to vitamin D receptor (VDR). We are continuing the study related to the alteration of pocket structure of VDR by 22-alkyl substituent of ligands and the relationships between the alteration and agonistic/antagonistic activity. Previously we reported that compounds 2 (22-H), 3 (22S-Et), and 4 (22S-Bu) are VDR agonist, partial agonist and antagonist, respectively. Here, we describe the synthesis and biological evaluation of 22S-hexyl analog 5 (22S-Hex), which was designed to be a stronger VDR antagonist than 4. Unexpectedly, 5 showed partial agonistic but not antagonistic activity when bound to VDR, indicating that it is not necessarily true that the bulkier the side chain is, the stronger the antagonistic activity will be. X-ray crystallographic analysis of the VDR-ligand-binding domain (VDR-LBD) accommodating compound 5 indicated that the partial agonist activity of 5 is dependent on the mixed population of the agonistic and antagonistic conformations. Binding of compound 5 may not bring the complex into the only antagonistic conformation due to the large conformational change of the VDR-LBD. From this study it was found that fine tuning of agonistic/antagonistic activity for VDR is possible by 22-alkyl chain length of ligands.

Development of vitamin D analogs modulating the pocket structure of vitamin D receptor.

The first determination of the X-ray crystal structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) complexed with 1α,25-dihydroxyvitamin D3 was reported in 2000. Since then several dozen crystal structures of VDR accommodating various ligands have been presented. Almost all of these complexes display the canonical active conformation observed in the VDR-LBD/1α,25- dihydroxyvitamin D3 complex, and all have quite similar ligand binding pocket (LBP) architectures. To develop new VDR ligands as therapeutic agents, it is important to separate the various biological activities of 1α,25- dihydroxyvitamin D3, such as calcium regulation, cell differentiation and anti-proliferation, and immune modulation. We focused on the structure of the LBP and discovered that vitamin D analogs with a branched side chain induce structural rearrangement of the amino acid residues lining the LBP. These analogs formed an additional cavity in the LBP for accommodation of the side chain and thus altered the structure of the LBP. Interestingly, the ligands showed agonistic, partial agonistic, or antagonistic activity depending upon the structure of the side chain. These results indicate that ligands which alter the pocket structure open a new perspective for the development of VDR ligands exhibiting a specific biological activity.