Receptor-mediated endocytosis plays a crucial role in the success of numerous therapies and remains central to advancing drug development. This process begins with ligand binding to specific receptors, triggering the internalization and intracellular trafficking of receptor-ligand complexes. These complexes are subsequently directed into distinct routes, either toward lysosomal degradation or recycling to the cell surface, with implications for therapeutic outcomes. This review examines receptor-ligand interactions as key modulators of endocytosis, emphasizing their role in shaping therapeutic design and efficacy. Advances in selecting receptor-ligand pairs and engineering ligands with optimized properties have enabled precise control over internalization, endosomal sorting, and trafficking, providing tailored solutions for diverse therapeutic applications. Leveraging these insights, strategies such as RNA-based therapies, antibody-drug conjugates (ADCs), and targeted protein degradation (TPD) platforms have been refined to selectively avoid or promote lysosomal degradation, thereby enhancing therapeutic efficacy. By bridging fundamental mechanisms of receptor-mediated endocytosis with innovative therapeutic approaches, this review offers a framework for advancing precision medicine.

Although a considerable number of disease-related biomolecular interactions occur in the cytosol, therapeutic and diagnostic application of target-specific binding proteins is largely confined to surface-exposed or extracellular targets. Therefore, protein-cargo delivery approaches, including cell-penetrating peptides and cytosol-penetrating antibodies, are being explored to overcome this limitation. In this context, we have developed a modular approach for cytosolic penetration of tumor cells based on bispecific antibodies containing a masked cytosol-penetrating Fab on one arm and a tumor-targeting scFv linked via an endosomal cleavable linker on the other arm. The relevance of the antigen-specific binding, internalization, and cytosolic cargo delivery was demonstrated in several in vitro assays using different cell lines with anti-B7-H3 scFv, the well-characterized trastuzumab (HER2), and inotuzumab (CD22) as examples. In addition, presence of the masking moiety to prevent non-specific surface binding, as well as the activation of cytosol-penetrating capabilities in the tumor microenvironment upon release by tumor-specific proteases was confirmed using the catalytic domain of Pseudomonas exotoxin as model cargo for cytosol delivery. Tumor microenvironment-dependent as well as tumor-associated antigen-specific cytosol-penetrating antibodies of the type developed here have the potential to serve as a modular platform to deliver macromolecular cargoes for addressing intracellular targets in tumor cells.

Understanding the mechanisms driving specific and nonspecific tissue uptake of antibodies can inform protein engineering strategies that maximize therapeutic efficacy in target tissues while minimizing off-target tissue toxicities. While in vitro cell assays are typically used to study these internalization mechanisms, there are few methods readily available to evaluate these pathways in vivo. Single photon emission computed tomography (SPECT) imaging with a non-residualizing radiohalogen probe can measure total levels of intact antibody, and a residualizing radiometal-chelate probe, in combination with a non-residualizing probe, can measure catabolized antibody associated with receptor-mediated and nonspecific internalization processes. Here, we describe a SPECT imaging study in human epidermal growth factor receptor 2 (HER2)-expressing tumor-bearing mice aimed at measuring whole body disposition kinetics of tumor-targeting trastuzumab (anti-HER2) and non-targeting (anti-gD) antibodies. Mice received these molecules labeled with either a non-residualizing prosthetic group ([125I]SIB) or with a residualizing radiometal-chelate (111In-DOTA).

SPECT imaging data confirmed significant HER2-mediated tumor uptake and catabolism of anti-HER2, evidenced by the high 111In-DOTA-anti-HER2 signal over time relative to 111In-DOTA-anti-gD and the respective [125I]SIB-labeled molecules. [125I]SIB-anti-HER2 still showed noticeably higher tumor signal than [125I]SIB-anti-gD, demonstrating a meaningful pool of intact anti-HER2 in the interstitial tumor compartment. Spleen showed the greatest catabolism of both mAbs amongst all non-tumor tissues. Compartmental modeling of the SPECT data demonstrated that cell-associated anti-HER2 was primarily receptor-bound, with a peak receptor occupancy of 35% at 13 h post administration of a 10 mg/kg dose, with minimal free and pinocytosed mAb.

Here, we successfully developed an imaging and modeling approach to capture anti-HER2 antibody receptor binding as well as specific and nonspecific internalization over time in vivo. These data and analyses demonstrate the power of SPECT imaging using both non-residualizing and residualizing radioisotopes to better characterize the different biological states (free, bound, and catabolized) of antibodies within interstitial and intracellular compartments. Understanding these distinct antibody internalization mechanisms in tumor and non-tumor tissues enables more informed decisions on dose selection to optimize treatment of tumors with heterogeneous antigen expression while minimizing nonspecific toxicities.

Natural compound-derived chemotherapies remain central to cancer treatment, however, they often cause off-target side effects that negatively impact patients' quality of life. In contrast, antibody-drug conjugates (ADCs) combine cytotoxic payloads with antibodies to specifically target cancer cells. Most approved and clinically investigated ADCs utilize naturally derived payloads, while those with conventional synthetic molecular payloads remain limited. This review focuses on approved ADCs that enhance the efficacy of naturally derived payloads by linking them with antibodies. We provide an overview of the core components of ADCs, their working mechanisms, and FDA-approved ADCs featuring naturally derived payloads, such as calicheamicin, camptothecin, dolastatin 10, maytansine, pyrrolbenzodiazepine (PBD), and the immunotoxin Pseudomonas exotoxin A. This review also explores recent clinical advancements aimed at broadening the therapeutic potential of ADCs, their applicability in treating heterogeneously composed tumors and their potential use beyond oncology. Additionally, this review highlights naturally derived payloads that are currently being clinically investigated but have not yet received approval. By summarizing the current landscape, this review provides insights into promising avenues for exploration and contributes to the refinement of treatment protocols for improved patient outcomes.

Antibody-drug conjugates (ADCs) are antitumor drugs composed of monoclonal antibodies and cytotoxic payload covalently coupled by a linker. Currently, 15 ADCs have been clinically approved worldwide. More than 100 clinical trials at different phases are underway to investigate the newly developed ADCs. ADCs represent one of the fastest growing classes of targeted antitumor drugs in oncology drug development. It takes advantage of the specific targeting of tumor-specific antigen by antibodies to deliver cytotoxic chemotherapeutic drugs precisely to tumor cells, thereby producing promising antitumor efficacy and favorable adverse effect profiles. However, emergence of drug resistance has severely hindered the clinical efficacy of ADCs. In this review, we introduce the structure and mechanism of ADCs, describe the development of ADCs, summarized the latest research about the mechanisms of ADC resistance, discussed the strategies to overcome ADCs resistance, and predicted biomarkers for treatment response to ADC, aiming to contribute to the development of ADCs in the future.

HER2 and αVβ6 integrin are independent predictors of breast cancer survival and metastasis. We identify an αVβ6/HER2 cross-talk mechanism driving invasion, which is dysregulated in drug-resistant HER2+ breast cancer cells. Proteomic analyses reveal ligand-bound αVβ6 recruits HER2 and a trafficking subnetwork, comprising guanosine triphosphatases RAB5 and RAB7A and the Rab regulator guanine nucleotide dissociation inhibitor 2 (GDI2). The RAB5/RAB7A/GDI2 functional module mediates direct cross-talk between αVβ6 and HER2, affecting receptor trafficking and signaling. Acute exposure to trastuzumab increases recruitment of the subnetwork to αVβ6, but trastuzumab resistance decouples GDI2 recruitment. GDI2, RAB5, and RAB7A cooperate to regulate migration and transforming growth factor-β activation to promote invasion. However, these mechanisms are dysregulated in trastuzumab-resistant cells. In patients, RAB5A, RAB7A, and GDI2 expression correlates with patient survival and αVβ6 expression predicts relapse following trastuzumab treatment. Thus, the RAB5/RAB7A/GDI2 subnetwork regulates αVβ6-HER2 cross-talk to drive breast cancer invasion but is subverted in trastuzumab-resistant cells to drive αVβ6-independent and HER2-independent tumor progression.

Trastuzumab deruxtecan (T-DXd) is a human epidermal growth factor receptor 2 (HER2)-targeting antibody drug conjugate that has remarkable activity in HER2-positive cancers. However, the degree of benefit of T-DXd is not uniform among solid tumors even with high levels of HER2. Despite high HER2 expression, the HER2/T-DXd complex may not always undergo internalization and payload release dependent on the receptor's conformation and context. We hypothesize that epidermal growth factor receptor (EGFR), a dimerization partner of HER2, can modulate HER2 trafficking through endocytic pathways and affect T-DXd uptake. We demonstrate that elevated EGFR expression levels can promote EGFR/HER2 heterodimer formation and suppress T-DXd internalization and efficacy. Knockdown of EGFR expression or pharmacologic stimulation of EGFR endocytosis with EGFR monoclonal antibodies restores T-DXd trafficking and antitumor activity in EGFR-overexpressing cancers in vivo. Our results reveal EGFR overexpression to be a potential mechanism of resistance to T-DXd, which can be overcome by combination therapy strategies targeting EGFR.

In this work, we conceived and developed antibody-drug conjugates (ADCs) that could efficiently release the drug after enzymatic cleavage of the linker moiety by tumoral proteases. The antibody-drug linkers we used are the result of a rational optimization of a previously reported PEGylated linker, PUREBRIGHT® MA-P12-PS, which showed excellent drug loading capacities but lacked an inbuilt drug discharge mechanism, thus limiting the potency of the resulting ADCs. To address this limitation, we chose to incorporate a protease-sensitive trigger into the linker to favor the release of a "PEGless" drug inside the tumor cells and, therefore, obtain potent ADCs. Currently, most marketed ADCs are based on the Val-Cit dipeptide followed by a self-immolative spacer for releasing the drug in its unmodified form. Here, we selected two untraditional peptide sequences, a Phe-Gly dipeptide and a Val-Ala-Gly tripeptide and placed one or the other in between the drug on one side (N-terminus) and the rest of the linker, including the PEG moiety, on the other side (C-terminus), without a self-immolative group. We found that both linkers responded to cathepsin B, a reference lysosomal enzyme, and liberated a PEG-free drug catabolite, as desired. We then used the two linkers to generate ADCs based on trastuzumab (a HER2-targeting antibody) and DM1 (a microtubule-targeted cytotoxic agent) with an average drug-to-antibody ratio (DAR) of 4 or 8. The ADCs showed restored cytotoxicity in vitro, which was proportional to the DM1 loading and generally higher for the ADCs bearing Val-Ala-Gly in their structure. In an ovarian cancer mouse model, the DAR 8 ADC based on Val-Ala-Gly behaved better than Kadcyla® (an approved ADC of DAR 3.5 used as control throughout this study), leading to a higher tumor volume reduction and more prolonged median survival. Taken together, our results depict a successful linker optimization process and encourage the application of the Val-Ala-Gly tripeptide as an alternative to other existing protease-sensitive triggers for ADCs.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Lysosome-targeting degradation technologies have emerged as a promising therapeutic strategy for the selective depletion of target extracellular and cell-surface proteins by harnessing a cell-surface effector protein such as lysosome-targeting receptors (LTRs) or transmembrane E3 ligases that direct lysosomal degradation. We recently developed a lysosome-targeting degradation platform termed signal-mediated lysosome-targeting chimeras (SignalTACs) that functions independently of an LTR or E3 ligase; these are engineered fusion proteins comprising a target binder, a cell-penetrating peptide (CPP), and a lysosomal sorting signal motif (P1). Herein, we present the next-generation SignalTACs containing a single endocytic signal that bypasses the need for a CPP. We demonstrate that the fusion with a 10-amino acid endocytic signaling peptide (P3) derived from the cation-independent mannose-6-phosphate receptor (CI-M6PR) induces robust internalization and lysosomal degradation of the target protein. The P3-based SignalTAC exhibited enhanced antitumor efficacy compared to the parent antibody. We envision that the fusion of the endocytic signaling peptide P3 to a target binder may allow the construction of an effective degrader for membrane-associated targets. Furthermore, mechanistic studies identified different drivers for the activities of the P3- and P1-based SignalTACs, which is expected to provide crucial insights toward the harnessing of the intrinsic signaling pathways to direct protein trafficking and degradation.

A next generation multiscale quantitative systems pharmacology (QSP) model for antibody drug conjugates (ADCs) is presented, for preclinical to clinical translation of ADC efficacy. Two HER2 ADCs (trastuzumab-DM1 and trastuzumab-DXd) were used for model development, calibration, and validation. The model integrates drug specific experimental data including in vitro cellular disposition data, pharmacokinetic (PK) and tumor growth inhibition (TGI) data for T-DM1 and T-DXd, as well as system specific data such as properties of HER2, tumor growth rates, and volumes. The model incorporates mechanistic detail at the intracellular level, to account for different mechanisms of ADC processing and payload release. It describes the disposition of the ADC, antibody, and payload inside and outside of the tumor, including binding to off-tumor, on-target sinks. The resulting multiscale PK model predicts plasma and tumor concentrations of ADC and payload. Tumor payload concentrations predicted by the model were linked to a TGI model and used to describe responses following ADC administration to xenograft mice. The model was translated to humans and virtual clinical trial simulations were performed that successfully predicted progression free survival response for T-DM1 and T-DXd for the treatment of HER2+ metastatic breast cancer, including differential efficacy based upon HER2 expression status. In conclusion, the presented model is a step toward a platform QSP model and strategy for ADCs, integrating multiple types of data and knowledge to predict ADC efficacy. The model has potential application to facilitate ADC design, lead candidate selection, and clinical dosing schedule optimization.

Antibody-drug conjugates (ADCs) consist of antibodies, linkers and payloads. They offer targeted delivery of potent cytotoxic drugs to tumor cells, minimizing off-target effects. However, the therapeutic efficacy of ADCs is compromised by heterogeneity in the drug-to-antibody ratio (DAR), which impacts both cytotoxicity and pharmacokinetics (PK). Additionally, the emergence of drug resistance poses significant challenges to the clinical advancement of ADCs. To overcome these limitations, a variety of strategies have been developed, including the design of multi-specific drugs with accurate DAR. This review critically summarizes the current challenges faced by ADCs, categorizing key issues and evaluating various innovative solutions. We provide an in-depth analysis of the latest methodologies for achieving homogeneous DAR and explore design strategies for multi-specific drugs aimed at combating drug resistance. Our discussion offers a current perspective on the advancements made in refining ADC technologies, with an emphasis on enhancing therapeutic outcomes.

Overexpression of HER2 occurs in 25% of breast cancer. Targeting HER2 has proven to be an effective therapeutic strategy for HER2-positive breast cancer. While trastuzumab is the most commonly used HER2 targeting agent, which has significantly improved outcomes, the overall response rate is low. To develop novel therapies to boost trastuzumab efficacy, it is critical to identify the mechanisms underlying trastuzumab action and resistance. We recently showed that the inhibition of breast cancer cell growth by trastuzumab is not through the inhibition of HER2 canonical signaling. Here we report the identification of a novel non-canonical HER2 signaling pathway and its interference by trastuzumab. We showed that HER2 signaled through a non-canonical pathway, regulated intramembrane proteolysis (RIP). In this pathway, HER2 is first cleaved by metalloprotease ADAM10 to produce an extracellular domain (ECD) that is released and the p95HER2 that contains the transmembrane domain (TM) and intracellular domain (ICD). p95HER2, if further cleaved by an intramembrane protease, γ-secretase, produced a soluble ICD p75HER2 with nuclear localization signal (NLS). p75HER2 is phosphorylated and translocated to the nucleus. Nuclear p75HER2 promotes cell proliferation. Trastuzumab targets this non-canonical HER2 pathway via inhibition of the proteolytic cleavage of HER2 by both ADAM10 and γ-secretase. However, p75HER2 pathway also confers resistance to trastuzumab once aberrantly activated. Combination of trastuzumab with ADAM10 and γ-secretase inhibitors completely blocks p75HER2 production in both BT474 and SKBR3 cells. We concluded that HER2 signals through the RIP signaling pathway that promotes cell proliferation and is targeted by trastuzumab. The aberrant HER2 RIP signaling confers resistance to trastuzumab that could be overcome by the application of inhibitors to ADAM10 and γ-secretase.

Antibody-drug conjugates (ADCs) represent potent cancer therapies that deliver highly toxic drugs to tumor cells precisely, thus allowing for targeted treatment and significantly reducing off-target effects. Despite their effectiveness, ADCs can face limitations due to acquired resistance and potential side effects.

This study focuses on advances in various ADC components to improve both the efficacy and safety of these agents, and includes the analysis of several novel ADC formats. This work assesses whether the unique features of VHHs-such as their small size, enhanced tissue penetration, stability, and cost-effectiveness-make them a viable alternative to conventional antibodies for ADCs and reviews their current status in ADC development.

Following PRISMA guidelines, this study focused on VHHs as components of ADCs, examining advancements and prospects from 1 January 2014 to 30 June 2024. Searches were conducted in PubMed, Cochrane Library, ScienceDirect and LILACS using specific terms related to ADCs and single-domain antibodies. Retrieved articles were rigorously evaluated, excluding duplicates and non-qualifying studies. The selected peer-reviewed articles were analyzed for quality and synthesized to highlight advancements, methods, payloads, and future directions in ADC research.

VHHs offer significant advantages for drug conjugation over conventional antibodies due to their smaller size and structure, which enhance tissue penetration and enable access to previously inaccessible epitopes. Their superior stability, solubility, and manufacturability facilitate cost-effective production and expand the range of targetable antigens. Additionally, some VHHs can naturally cross the blood-brain barrier or be easily modified to favor their penetration, making them promising for targeting brain tumors and metastases. Although no VHH-drug conjugates (nADC or nanoADC) are currently in the clinical arena, preclinical studies have explored various conjugation methods and linkers.

While ADCs are transforming cancer treatment, their unique mechanisms and associated toxicities challenge traditional views on bioavailability and vary with different tumor types. Severe toxicities, often linked to compound instability, off-target effects, and nonspecific blood cell interactions, highlight the need for better understanding. Conversely, the rapid distribution, tumor penetration, and clearance of VHHs could be advantageous, potentially reducing toxicity by minimizing prolonged exposure. These attributes make single-domain antibodies strong candidates for the next generation of ADCs, potentially enhancing both efficacy and safety.

Trastuzumab and pertuzumab combination has been approved for the treatment of patients with HER2-positive metastatic breast cancer. However, trastuzumab and pertuzumab combination did not show improvement in overall survival in patients with HER2-positive metastatic gastric cancer.

We developed a new HER2-targeted monoclonal antibody, HLX22, targeting HER2 subdomain IV as trastuzumab but with non-overlapping epitopes. We examined the antitumor effects of this novel HER2-antibody in gastric cell lines and cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

HLX22 in combination with HLX02 (trastuzumab biosimilar) induced enhancement of HER2/HER2 homodimers and HER2/EGFR heterodimers internalization, which ultimately led to the reduction in signal transductions involving STAT3, P70 S6, and AKT; gene expressions of FGF-FGFR-PI3K-MTOR, EGF-EGFR-RAS, TGF-β-SMAD, PLCG and cell cycle progression related pathways that favor tumor development, proliferation, progression, migration and survival in gastric cancer cell line NCI-N87 were also reduced. These differing but complementary actions contributed to the synergistic antitumor efficacy of the HLX22 and HLX02 combination in gastric cancer cell lines, CDX and PDX. In addition, HLX22 in combination with HLX02 demonstrated stronger antitumor efficacy than HLX02 and HLX11 (a potential pertuzumab biosimilar) combination treatment both in vitro and in vivo.

These results suggested that the application of non-competing antibodies HLX22 and HLX02 targeting HER2 subdomain IV together may be of substantial benefit to gastric cancer patients who currently respond suboptimal to trastuzumab therapy.

Breast cancer remains one of the predominant malignancies worldwide. In the context of inoperable advanced or metastatic human epidermal growth factor receptor 2 (HER2)-positive breast cancer, systemic management primarily relies on HER2-targeting monoclonal antibodies. With the successful development of anti-HER2 antibody-drug conjugates (ADCs), these agents have been increasingly integrated into therapeutic regimens for metastatic breast cancer. Here, we present the case of a 42-year-old female patient with HER2-positive pulmonary metastatic breast cancer who underwent an extensive treatment protocol. This protocol included chemotherapy, radiation therapy, hormonal therapy, surgical intervention on the breast, and anti-HER2 therapies. The anti-HER2 therapies involved both singular and dual targeting strategies using trastuzumab and the ADC disitamab vedotin (RC48) over an 8-year period. After experiencing disease progression following HER2-targeted therapy with RC48, the patient achieved noticeable partial remission through a therapeutic regimen that combined trastuzumab deruxtecan (DS8201) and tislelizumab. The data suggest a promising role for DS8201 in managing advanced stages of HER2-amplified metastatic breast cancer, especially in cases that demonstrate progression after initial HER2-directed therapies using ADCs. Furthermore, its combination with anti-PD-1 agents enhances therapeutic efficacy by augmenting the anti-tumoral immune response.

Urothelial carcinoma (UC) is the most common malignancy of the urinary tract in dogs and has aggressive behaviour. Although human epidermal growth factor receptor 2 (HER2) is a known therapeutic target with evidence in canine UC, the efficacy of anti-HER2 antibody drugs remains unknown. This study aimed to investigate the effects of anti-HER2 antibody drugs including trastuzumab and trastuzumab emtansine (T-DM1) on canine UC cell lines in vitro and in vivo. Four canine UC cell lines (Nene, TCCUB, Love, and Sora) were used. In western blotting, HER2 protein expression was observed in all the cell lines. Although both trastuzumab and T-DM1 showed dose-dependent growth inhibitory activity in the cell lines, T-DM1 showed much stronger activity than that of trastuzumab. In flow cytometry analyses with the canine UC cell line (Sora), T-DM1 but not trastuzumab significantly increased the percentages of early and late apoptotic cells in annexin V apoptotic assays and the sub-G1 phase fraction in cell cycle analyses. For the in vivo experiment, the canine UC cells (Sora) were subcutaneously injected into nude mice. Four days after inoculation, trastuzumab, T-DM1, or vehicle was administered intraperitoneally once a week for three times. Tumour volumes were significantly smaller in the T-DM1 group compared to the trastuzumab and vehicle control groups. These findings indicate that T-DM1 exerts a stronger antitumour effect than that of trastuzumab on canine UC cells in vitro and in vivo, possibly by inducing apoptosis due to DM1.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Cadherin-6 (CDH6) is expressed in several cancer types, but no CDH6-targeted therapy is currently clinically available. Here, we generated raludotatug deruxtecan (R-DXd; DS-6000), a novel CDH6-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and evaluated its properties, pharmacologic activities, and safety profile. In vitro pharmacologic activities and the mechanisms of action of R-DXd were assessed in serous-type ovarian cancer and renal cell carcinoma cell lines. In vivo pharmacologic activities were evaluated with several human cancer cell lines and patient-derived xenograft mouse models. The safety profile in cynomolgus monkeys was also assessed. R-DXd exhibited CDH6 expression-dependent cell growth-inhibitory activity and induced tumor regression in xenograft models. In this process, R-DXd specifically bound to CDH6, was internalized into cancer cells, and then translocated to the lysosome. The DXd released from R-DXd induced the phosphorylation of Chk1, a DNA damage marker, and cleaved caspase-3, an apoptosis marker, in cancer cells. It was also confirmed that the DXd payload had a bystander effect, passing through the cell membrane and impacting surrounding cells. The safety profile of R-DXd was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. R-DXd demonstrated potent antitumor activity against CDH6-expressing tumors in mice and an acceptable safety profile in monkeys. These findings indicate the potential of R-DXd as a new treatment option for patients with CDH6-expressing serous-type ovarian cancer and renal cell carcinoma in a clinical setting.

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.

Proteins are multifunctional large organic compounds that constitute an essential component of a living system. Hence, control over their bioconjugation impacts science at the chemistry-biology-medicine interface. A chemical toolbox for their precision engineering can boost healthcare and open a gateway for directed or precision therapeutics. Such a chemical toolbox remained elusive for a long time due to the complexity presented by the large pool of functional groups. The precise single-site modification of a protein requires a method to address a combination of selectivity attributes. This review focuses on guiding principles that can segregate them to simplify the task for a chemical method. Such a disintegration systematically employs a multi-step chemical transformation to deconvolute the selectivity challenges. It constitutes a disintegrate (DIN) theory that offers additional control parameters for tuning precision in protein bioconjugation. This review outlines the selectivity hurdles faced by chemical methods. It elaborates on the developments in the perspective of DIN theory to demonstrate simultaneous regulation of reactivity, chemoselectivity, site-selectivity, modularity, residue specificity, and protein specificity. It discusses the progress of such methods to construct protein and antibody conjugates for biologics, including antibody-fluorophore and antibody-drug conjugates (AFCs and ADCs). It also briefs how this knowledge can assist in developing small molecule-based covalent inhibitors. In the process, it highlights an opportunity for hypothesis-driven routes to accelerate discoveries of selective methods and establish new targetome in the precision engineering of proteins and antibodies.

Model-informed drug discovery advocates the use of mathematical modeling and simulation for improved efficacy in drug discovery. In the case of monoclonal antibodies (mAbs) against cell membrane antigens, this requires quantitative insight into the target tissue concentration levels. Protein mass spectrometry data are often available but the values are expressed in relative, rather than in molar concentration units that are easier to incorporate into pharmacokinetic models. Here, we present an empirical correlation that converts the parts per million (ppm) concentrations in the PaxDb database to their molar equivalents that are more suitable for pharmacokinetic modeling. We evaluate the insight afforded to target tissue distribution by analyzing the likely tumor-targeting accuracy of mAbs recognizing either epidermal growth factor receptor or its homolog HER2. Surprisingly, the predicted tissue concentrations of both these targets exceed the Kd values of their respective therapeutic mAbs. Physiologically based pharmacokinetic (PBPK) modeling indicates that in these conditions only about 0.05% of the dosed mAb is likely to reach the solid tumor target cells. The rest of the dose is eliminated in healthy tissues via both nonspecific and target-mediated processes. The presented approach allows evaluation of the interplay between the target expression level in different tissues that determines the overall pharmacokinetic properties of the drug and the fraction that reaches the cells of interest. This methodology can help to evaluate the efficacy and safety properties of novel drugs, especially if the off-target cell degradation has cytotoxic outcomes, as in the case of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) have greatly improved the outcomes of advanced breast tumors. However, the treatment of breast tumors with existing ADCs is still hindered by many issues, such as tumor antigen heterogeneity and drug resistance. Therefore, ADCs against new targets would provide options for the treatment of these challenges. Sortilin-1 (SORT1) may be a promising target for ADC as it is upregulated in breast cancer. To evaluate the possibility of SORT1 as an ADC target, a humanized antibody_8D302 with high affinity against SORT1 was generated. Additionally, 8D302 was conjugated with MMAE and DXd to generate two ADCs_8D302-MMAE and 8D302-DXd, respectively. Both 8D302-MMAE and 8D302-DXd showed effective cytotoxicity against SORT1 positive breast tumor cell lines and induced bystander killing. Consequently, 8D302-MMAE showed relatively better anti-tumor activity than 8D302-DXd both in vitro and in vivo, but 8D302-DXd had superior safety profile and pharmacokinetics profile over 8D302-MMAE. Furthermore, SORT1 induced faster internalization and lysosomal trafficking of antibodies and had a higher turnover compared with HER2. Also, 8D302-DXd exhibited superior cell cytotoxicity and tumor suppression over trastuzumab-DXd, a HER2-targeted ADC. We hypothesize that the high turnover of SORT1 enables SORT1-targeted ADC to be a powerful agent for the treatment of SORT1-positive breast tumor.

Trastuzumab, a therapeutic monoclonal antibody directed against HER2, is routinely used to treat HER2-positive breast cancer with a good response rate. However, concerns have arisen in the clinical practice due to adverse side effects. One way to overcome these limitations is to encapsulate trastuzumab in nanoparticles to improve cytotoxic activity, increase intracellular drug concentrations, escape the immune system and avoid systemic degradation of the drug in vivo.

A double emulsion method was used to encapsulate trastuzumab into poly(lactic-co-glycolic) nanoparticles, effective for their biocompatibility and biodegradability. These nanocarriers, hereafter referred to as TZPs, were characterised in terms of size, homogeneity, zeta potential and tested for their stability and drug release kinetics. Finally, the TZPs cytotoxicity was assessed in vitro on the HER2 positive SKBR3 breast cancer cell line and compared to free trastuzumab.

The TZPs were stable, homogeneous in size, with a reduced zeta potential. They showed higher encapsulation efficiency and drug loading, a prolonged trastuzumab release kinetics that retained its physicochemical properties and functionality. TZPs showed a stronger cytotoxicity and increased apoptosis than similar doses of free trastuzumab in the cell line analysed. Confocal microscopy and flow cytometry assessed TZPs and trastuzumab cellular uptake while Western blot evaluated downstream signalling, overall HER2 content and shedding.

TZPs exert more robust effects than free trastuzumab via a dual mode of action: TZPs are taken up by cells through an endocytosis mechanism and release the drug intracellularly for longer time. Additionally, the TZPs that remain in the extracellular space release trastuzumab which binds to the cognate receptor and impairs downstream signalling. This is the sole modality used by free trastuzumab. Remarkably, half dose of TZPs is as efficacious as the highest dose of free drug supporting their possible use for drug delivery in vivo.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

With almost 20 million new cases per year, cancer constitutes one of the most important challenges for public health systems. Unlike traditional chemotherapy, targeted anti-cancer strategies employ sophisticated therapeutics to precisely identify and attack cancer cells, limiting the impact of drugs on healthy cells and thereby minimizing the unwanted side effects of therapy. Protein drug conjugates (PDCs) are a rapidly growing group of targeted therapeutics, composed of a cancer-recognition factor covalently coupled to a cytotoxic drug. Several PDCs, mainly in the form of antibody-drug conjugates (ADCs) that employ monoclonal antibodies as cancer-recognition molecules, are used in the clinic and many PDCs are currently in clinical trials. Highly selective, strong and stable interaction of the PDC with the tumor marker, combined with efficient, rapid endocytosis of the receptor/PDC complex and its subsequent effective delivery to lysosomes, is critical for the efficacy of targeted cancer therapy with PDCs. However, the bivalent architecture of contemporary clinical PDCs is not optimal for tumor receptor recognition or PDCs internalization. In this review, we focus on multivalent PDCs, which represent a rapidly evolving and highly promising therapeutics that overcome most of the limitations of current bivalent PDCs, enhancing the precision and efficiency of drug delivery to cancer cells. We present an expanding set of protein scaffolds used to generate multivalent PDCs that, in addition to folding into well-defined multivalent molecular structures, enable site-specific conjugation of the cytotoxic drug to ensure PDC homogeneity. We provide an overview of the architectures of multivalent PDCs developed to date, emphasizing their efficacy in the targeted treatment of various cancers.

Antibody-drug conjugates (ADCs) have emerged as a revolutionary therapeutic class, combining the precise targeting ability of monoclonal antibodies with the potent cytotoxic effects of chemotherapeutics. Notably, ADCs have rapidly advanced in the field of breast cancer treatment. This innovative approach holds promise for strengthening the immune system through antibody-mediated cellular toxicity, tumor-specific immunity, and adaptive immune responses. However, the development of upfront and acquired resistance poses substantial challenges in maximizing the effectiveness of these therapeutics, necessitating a deeper understanding of the underlying mechanisms. These mechanisms of resistance include antigen loss, derangements in ADC internalization and recycling, drug clearance, and alterations in signaling pathways and the payload target. To overcome resistance, ongoing research and development efforts are focused on urgently identifying biomarkers, integrating immune therapy approaches, and designing novel cytotoxic payloads. This Review provides an overview of the mechanisms and clinical effectiveness of ADCs, and explores their unique immune-boosting function, while also highlighting the complex resistance mechanisms and safety challenges that must be addressed. A continued focus on how ADCs impact the tumor microenvironment will help to identify new payloads that can improve patient outcomes.

Spontaneous dimerization of EGF receptors (EGFR) and dysregulation of EGFR signaling has been associated with the development of different cancers. Under normal physiological conditions and to maintain homeostatic cell growth, once EGFR signaling occurs, it needs to be attenuated. Activated EGFRs are rapidly internalized, sorted through early endosomes, and ultimately degraded in lysosomes by a process generally known as receptor down-regulation. Through alterations to EGFR trafficking, tumors develop resistance to current treatment strategies, thus highlighting the necessity for combination treatment strategies that target EGFR trafficking. This review covers EGFR structure, trafficking, and altered surface expression of EGFR receptors in cancer, with a focus on how therapy targeting EGFR trafficking may aid tyrosine kinase inhibitor treatment of cancer.

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

Human epidermal growth factor receptor 2-targeted (HER2-targeted) therapy is the mainstay of treatment for HER2+ breast cancer. However, the proteolytic cleavage of HER2, or HER2 shedding, induces the release of the target epitope at the ectodomain (ECD) and the generation of a constitutively active intracellular fragment (p95HER2), impeding the effectiveness of anti-HER2 therapy. Therefore, identifying key regulators in HER2 shedding might provide promising targetable vulnerabilities against resistance. In the current study, we found that upregulation of dolichyl-phosphate N-acetylglucosaminyltransferase (DPAGT1) sustained high-level HER2 shedding to confer trastuzumab resistance, which was associated with poor clinical outcomes. Upon trastuzumab treatment, the membrane-bound DPAGT1 protein was endocytosed via the caveolae pathway and retrogradely transported to the ER, where DPAGT1 induced N-glycosylation of the sheddase - ADAM metallopeptidase domain 10 (ADAM10) - to ensure its expression, maturation, and activation. N-glycosylation of ADAM10 at N267 protected itself from ER-associated protein degradation and was essential for DPAGT1-mediated HER2 shedding and trastuzumab resistance. Importantly, inhibition of DPAGT1 with tunicamycin acted synergistically with trastuzumab treatment to block HER2 signaling and reverse resistance. These findings reveal a prominent mechanism for HER2 shedding and suggest that targeting DPAGT1 might be a promising strategy against trastuzumab-resistant breast cancer.

One of the main challenges of PET imaging with 89Zr-labeled monoclonal antibodies (mAbs) remains the long blood circulation of the radiolabeled mAbs, leading to high background signals, decreasing image quality. To overcome this limitation, here we report the use of a bioorthogonal linker cleavage approach (click-to-release chemistry) to selectively liberate [89Zr]Zr-DFO from trans-cyclooctene-functionalized trastuzumab (TCO-Tmab) in blood, following the administration of a tetrazine compound (trigger) in BT-474 tumor-bearing mice. Methods: We created a series of TCO-DFO constructs and evaluated their performance in [89Zr]Zr-DFO release from Tmab in vitro using different trigger compounds. The in vivo behavior of the best performing [89Zr]Zr-TCO-Tmab was studied in healthy mice first to determine the optimal dose of the trigger. To find the optimal time for the trigger administration, the rate of [89Zr]Zr-TCO-Tmab internalization was studied in BT-474 cancer cells. Finally, the trigger was administered 6 h or 24 h after [89Zr]Zr-TCO-Tmab- administration in tumor-bearing mice to liberate the [89Zr]Zr-DFO fragment. PET scans were obtained of tumor-bearing mice that received the trigger 6 h post-[89Zr]Zr-TCO-Tmab administration. Results: The [89Zr]Zr-TCO-Tmab and trigger pair with the best in vivo properties exhibited 83% release in 50% mouse plasma. In tumor-bearing mice the tumor-blood ratios were markedly increased from 1.0 ± 0.4 to 2.3 ± 0.6 (p = 0.0057) and from 2.5 ± 0.7 to 6.6 ± 0.9 (p < 0.0001) when the trigger was administered at 6 h and 24 h post-mAb, respectively. Same day PET imaging clearly showed uptake in the tumor combined with a strongly reduced background due to the fast clearance of the released [89Zr]Zr-DFO-containing fragment from the circulation through the kidneys. Conclusions: This is the first demonstration of the use of trans-cyclooctene-tetrazine click-to-release chemistry to release a radioactive chelator from a mAb in mice to increase tumor-to-blood ratios. Our results suggest that click-cleavable radioimmunoimaging may allow for substantially shorter intervals in PET imaging with full mAbs, reducing radiation doses and potentially even enabling same day imaging.

Antibody-drug conjugates (ADCs) are a promising class of cancer biopharmaceuticals that exploit the specificity of a monoclonal antibody (mAb) to selectively deliver highly cytotoxic small molecules to targeted cancer cells, leading to an enhanced therapeutic index through increased antitumor activity and decreased off-target toxicity. ADCs hold great promise for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer after the approval and tremendous success of trastuzumab emtansine and trastuzumab deruxtecan, representing a turning point in both HER2-positive breast cancer treatment and ADC technology. Additionally and importantly, a total of 29 ADC candidates are now being investigated in different stages of clinical development for the treatment of HER2-positive breast cancer. The purpose of this review is to provide an insight into the ADC field in cancer treatment and present a comprehensive overview of ADCs approved or under clinical investigation for the treatment of HER2-positive breast cancer.

Radiolabeled drug nanocarriers that can be easily imaged via positron emission tomography (PET) are highly significant as their in vivo outcome can be quantitatively PET-traced with high sensitivity. However, typical radiolabeling of most PET-guided theranostic vehicles utilizes modification with chelator ligands, which presents various challenges. In addition, unlike passive tumor targeting, specific targeting of drug delivery vehicles via binding affinity to overexpressed cancer cell receptors is crucial to improve the theranostic delivery to tumors. Herein, we developed 89Zr-labeled triblock copolymer polymersomes of 60 nm size through chelator-free radiolabeling. The polymersomes are assembled from poly(N-vinylpyrrolidone)5-b-poly(dimethylsiloxane)30-b-poly(N-vinylpyrrolidone)5 (PVPON5-PDMS30-PVPON5) triblock copolymers followed by adsorption of a degradable tannin, tannic acid (TA), on the polymersome surface through hydrogen bonding. TA serves as an anchoring layer for both 89Zr radionuclide and targeting recombinant humanized monoclonal antibody, trastuzumab (Tmab). Unlike bare PVPON5-PDMS30-PVPON5 polymersomes, TA- and Tmab-modified polymersomes demonstrated a high radiochemical yield of more than 95%. Excellent retention of 89Zr by the vesicle membrane for up to 7 days was confirmed by PET in vivo imaging. Animal biodistribution using healthy BALB/c mice confirmed the clearance of 89Zr-labeled polymersomes through the spleen and liver without their accumulation in bone, unlike the free nonbound 89Zr radiotracer. The 89Zr-radiolabeled polymersomes were found to specifically target BT474 HER2-positive breast cancer cells via the Tmab-TA complex on the vesicle surface. The noncovalent Tmab anchoring to the polymersome membrane can be highly advantageous for nanoparticle modification compared to currently developed covalent methods, as it allows easy and quick integration of a broad range of targeting proteins. Given the ability of these polymersomes to encapsulate and release anticancer therapeutics, they can be further expanded as precision-targeted therapeutic carriers for advancing human health through highly effective drug delivery strategies.

Antibody drug conjugates (ADCs) are powerful anti-cancer therapies comprising an antibody joined to a cytotoxic payload through a chemical linker. ADCs exploit the specificity of antibodies for their target antigens, combined with the potency of cytotoxic drugs, to selectively kill target antigen-expressing tumour cells. The recent rapid advancement of the ADC field has so far yielded twelve and eight ADCs approved by the US and EU regulatory bodies, respectively. These serve as effective targeted treatments for several haematological and solid tumour types. In the development of an ADC, the judicious choice of an antibody target antigen with high expression on malignant cells but restricted expression on normal tissues and immune cells is considered crucial to achieve selectivity and potency while minimising on-target off-tumour toxicities. Aside from this paradigm, the selection of an antigen for an ADC requires consideration of several factors relating to the expression pattern and biological features of the target antigen. In this review, we discuss the attributes of antigens selected as targets for antibodies used in clinically approved ADCs for the treatment of haematological and solid malignancies. We discuss target expression, functions, and cellular kinetics, and we consider how these factors might contribute to ADC efficacy.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Over the past two decades, we have seen an increase in the complexity and diversity of biotherapeutic modalities pursued by biopharmaceutical companies. These biologics are multifaceted and susceptible to post-translational modifications and in vivo biotransformation that could impose challenges for bioanalysis. It is vital to characterize the functionality, stability and biotransformation products of these molecules to enable screening, identify potential liabilities at an early stage and devise a bioanalytical strategy. This article highlights our perspective on characterization and bioanalysis of biologics using hybrid LC-MS in our global nonregulated bioanalytical laboratories. AbbVie's suite of versatile, stage-appropriate characterization assays and quantitative bioanalytical approaches are discussed, along with guidance on their utility in answering project-specific questions to aid in decision-making.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Comprehensive analyses of intracellular disposition and in vivo pharmacokinetics were performed for small interfering RNA (siRNA) conjugated with the Fab fragment of panitumumab, a fully humanized monoclonal antibody against epidermal growth factor receptor (EGFR). The Fab-siRNA conjugate was internalized into EGFR-expressing cancer cells in an antigen-dependent manner. Intracellular disposition was quantitatively evaluated using fluorescent-labeled panitumumab and confocal microscopy. The majority of internalized panitumumab was suggested to be transferred into lysosomes. In vivo pharmacokinetics were evaluated in EGFR-expressing tumor-bearing mice. Intact Fab-siRNA was measured by immunoprecipitation using anti-Fab antibody followed by quantitative polymerase chain reaction. The Fab portion was measured by a ligand binding assay. Intact Fab-siRNA concentrations rapidly decreased in the plasma and tumor, although the Fab portion concentration remained high, suggesting extensive degradation in the linker-siRNA portion. After incubation of Fab-siRNA in mouse plasma, samples were digested with proteinase K, and extracted siRNA tagged with Fab-derived peptide was subjected to an ion-pair reversed-phase liquid chromatography with mass spectrometry analysis. Results suggested that hydrolysis from the 3' end of the antisense strand of siRNA is the major metabolizing pathway. Based on these findings, endosomal escape and stability in lysosomes, blood, and tumor are key factors to improve to achieve efficient target gene knockdown in tumors, and stabilizing the 3' end of the antisense strand was suggested to be most efficient. Our approaches clearly identified the key issues of Fab-siRNA from a pharmacokinetics aspect, which will be useful for improving the in vivo activity of siRNA conjugated with not only Fab but also other immunoproteins. SIGNIFICANCE STATEMENT: The intracellular and in vivo disposition of Fab-small interfering RNA (siRNA) conjugate was comprehensively investigated using various approaches, including newly developed analytical methods. This study clearly shows that improvements in siRNA stability in lysosomes, blood, and tumor are needed for target gene knockdown in tumors. The major metabolic pathway of Fab-siRNA is 3' exonuclease degradation, suggesting that optimization of the conjugation site to Fab might help improve stability.

The dysregulated endocytic traffic of oncogenic receptors, such as the EGFR family especially HER2, contributes to the uncontrolled activation of the downstream oncogenic signaling and progression of various carcinomas, including 90% of ovarian carcinoma. However, the key regulators in the intracellular trafficking of HER2 and their impacts for cancer progression remain largely unknown. In this study, through a genome-wide CRISPR/Cas9 screening for key genes affecting the peritoneal disseminated metastasis of ovarian carcinoma, we identified a member of COMMD family, that is, COMMD3, as a key regulator in the endosomal trafficking of HER2. In the patients with high-grade serous ovarian carcinoma (HGSOC), the expression of COMMD3 is dramatically decreased in the peritoneal disseminated ovarian carcinoma cells comparing with that in the primary ovarian carcinoma cells. COMMD3 greatly inhibits the proliferation, migration, and epithelial-mesenchymal transition (EMT) of HGSOC cells, and dramatically suppresses the tumor growth, the formation of malignant ascites, and the peritoneal dissemination of cancer cells in the orthotopic murine model of HGSOC. Further transcriptome analysis reveals that silencing COMMD3 boosts the activation of HER2 downstream signaling. As a component in the Retriever-associated COMMD/CCDC22/CCDC93 complex responsible for the recognition and recycling of membrane receptors, COMMD3 physically interacts with HER2 for directing it to the slow recycling pathway, leading to the attenuated downstream tumor-promoting signaling.

Collectively, this study reveals a novel HER2 inactivation mechanism with a high value for the clinic diagnosis of new ovarian carcinoma types and the design of new therapeutic strategy.

Antibody-drug conjugates (ADCs) are a rapidly developing therapeutic approach in cancer treatment that has shown remarkable activity in breast cancer. Currently, there are two ADCs approved for the treatment of human epidermal growth factor receptor 2-positive breast cancer, one for triple-negative breast cancer, and multiple investigational ADCs in clinical trials. However, drug resistance has been noticed in clinical use, especially in trastuzumab emtansine. Here, the mechanisms of ADC resistance are summarized into four categories: antibody-mediated resistance, impaired drug trafficking, disrupted lysosomal function, and payload-related resistance. To overcome or prevent resistance to ADCs, innovative development strategies and combination therapy options are being investigated. Analyzing predictive biomarkers for optimal therapy selection may also help to prevent drug resistance.