Protocol for designing and evaluating antibody-drug conjugates to promote endocytosis and lysosomal trafficking

Summary

Receptor endocytosis and lysosomal delivery are critical for efficient intracellular delivery of antibody-drug conjugates (ADCs), yet many cancer targets exhibit limited internalization. Here, we present a protocol for engineering and evaluating ADCs to induce receptor ubiquitination, thereby promoting receptor uptake and transport toward lysosomal compartments. We describe steps for designing antibodies fused with E3 ligase-binding modules, drug conjugation, and cellular assays to quantify ubiquitination-dependent internalization and lysosomal accumulation in cancer cell models.

For complete details on the use and execution of this protocol, please refer to Zhuang et al.¹

Graphical abstract

Highlights

• •Protocol for constructing receptor-ubiquitination-targeting ADCs
• •Workflow for site-specific conjugation and quality characterization
• •Techniques for evaluating ADC trafficking and lysosomal delivery
• •Approaches to validate E3 ligase-dependent ADC mechanisms

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Receptor endocytosis and lysosomal delivery are critical for efficient intracellular delivery of antibody-drug conjugates (ADCs), yet many cancer targets exhibit limited internalization. Here, we present a protocol for engineering and evaluating ADCs to induce receptor ubiquitination, thereby promoting receptor uptake and transport toward lysosomal compartments. We describe steps for designing antibodies fused with E3 ligase-binding modules, drug conjugation, and cellular assays to quantify ubiquitination-dependent internalization and lysosomal accumulation in cancer cell models.

Before you begin

Antibody-drug conjugates (ADCs) have emerged as a promising class of targeted therapeutics that combine the specificity of monoclonal antibodies with the potency of cytotoxic payloads.^2,3 However, their efficacy remains limited by inefficient receptor uptake and delivery to lysosomal compartments, which restrict payload liberation inside cells and consequently attenuate antitumor activity.^4,5 Many tumor-associated antigen, including EGFR and HER2, exhibit slow endocytosis or recycling to the plasma membrane, resulting in suboptimal payload delivery and reduced therapeutic index.^6,7

Recently, our group reported a receptor-ubiquitination-targeting ADCs (ubitaADCs) platform designed to utilize membrane E3 ligase–dependent ubiquitination for promoting receptor uptake and trafficking toward lysosomes (Figure 1A).¹ By fusing a modified RSPO2 FU module capable of interacting with the transmembrane E3 ligases RNF43 and ZNRF3 to a receptor-targeting antibody (Figure 1B), ubitaADCs promote rapid ubiquitination of the target receptor. This ubiquitination serves as the initiating signal for receptor endocytosis, thereby accelerating intracellular trafficking toward lysosomes and enhancing intracellular payload delivery. Using EGFR-ubitaADC as a prototype, we demonstrated that ubiquitination-driven receptor trafficking results in enhanced internalization, lysosomal delivery, receptor degradation, and improved tumor suppression compared with conventional ADCs. As with conventional EGFR-targeting antibodies and ADCs, the primary tumor selectivity of EGFR-ubitaADC is determined by EGFR expression. In addition, the ubitaADC platform requires recruitment of the membrane-associated E3 ubiquitin ligases RNF43 and ZNRF3, which are highly expressed in many colorectal cancers. Thus, efficient receptor ubiquitination and lysosomal trafficking depend on the presence of both EGFR and RNF43/ZNRF3, providing an additional level of tumor selectivity. When applying this protocol to other tumor models, the expression of both the target receptor and the relevant E3 ligases should be verified before experimentation.

Figure 1.

Receptor-ubiquitin-targeting antibody-drug conjugates (ubitaADCs) overview

(A) Schematic illustration of ubitaADC mechanism. By simultaneously engaging membrane E3 ligases (e.g., RNF43/ZNRF3) and tumor-associated receptors (e.g., EGFR, HER2, etc), ubitaADCs promote ubiquitin-mediated receptor internalization and lysosomal trafficking, thereby enhancing cytotoxin delivery and receptor degradation.

(B) Structural design of ubitaADCs comprising a bivalent anti-RNF43/ZNRF3 binder fused to an anti-receptor antibody conjugated with the cytotoxic payload MMAE via a cleavable Val-Cit-PAB linker. Inset shows a representative structure of glycan-engineered N297 site for site-specific drug conjugation.

In this protocol, we outline the detailed procedures for designing, producing, and evaluating EGFR-ubitaADC, including the construction of the EGFR-ubiquitination-targeting antibody (EGFR-ubitaAb), site-specific chemoenzymatic conjugation, and functional assays for EGFR-ubitaADC-induced EGFR ubiquitination and degradation, EGFR-ubitaADC internalization, lysosomal trafficking, as well as in vitro and in vivo efficacy. This protocol provides a generalizable framework for applying ubiquitination-driven trafficking control to optimize ADC performance and other receptor-targeted biologics.

Innovation

The ubitaADCs introduce a mechanistic and design innovation that rewires receptor fate through engineered ubiquitination. By co-targeting tumor-associated receptors and membrane-tethered E3 ligases, these conjugates actively trigger receptor ubiquitination and redirect receptor trafficking toward lysosomal degradation. This built-in mechanism enhances payload delivery into lysosome-rich degradative compartments, even for receptors such as EGFR that otherwise favor recycling, thereby providing a generalizable and directional strategy to potentiate cytotoxic payload release within tumor cells. Beyond a single target, ubitaADCs establish a modular and broadly applicable platform that can be readily adapted to different receptor-ligase pairs, enabling precise control of receptor internalization and degradation. We demonstrate this flexibility by constructing ubitaADCs targeting both EGFR and HER2 in combination with distinct E3 ligases (RNF43 and ZNRF3), achieving consistent improvements in lysosomal trafficking and cytotoxic efficacy across multiple cancer cell lines and xenograft models. Importantly, ubitaADCs integrate a dual-function mechanism that synergistically combines targeted receptor ubiquitination-mediated degradation with efficient cytotoxic payload delivery, allowing simultaneous silencing of oncogenic surface receptors and selective elimination of tumor cells. This unified design provides a mechanistically informed framework for optimizing ADC performance and expanding their therapeutic potential against refractory solid tumors.

Institutional permissions

All animal experiments included in this protocol were performed following institutional policies governing the care and use of laboratory animals and were authorized by the Animal Ethics Committee of Zhejiang University. Experimental procedures complied with national requirements for animal welfare and ethical conduct in biomedical research. Investigators seeking to implement or modify this protocol should secure approval from their local Institutional Animal Care and Use Committee (IACUC) or corresponding oversight authority before initiating studies involving live vertebrate animals.

Generation of stable EGFR-eGFP-expressing HEK293T cells

Timing: 2–3 weeks

• 1.
  Prepare lentiviral plasmids.

  • a.
    Use pLVX-EGFR-eGFP as the transfer vector encoding full-length human EGFR fused to eGFP at the C terminus.
  • b.
    Use psPAX2 as the lentiviral packaging construct together with pMD2.G as the plasmid encoding the VSV-G envelope protein.
  • c.
    Verify plasmid integrity by sequencing before use.
• 2.
  Seed HEK-293T cells for lentivirus production.

  • a.
    Seed 2.5–3.0 × 10⁶ HEK-293T cells (passages 5–20 are recommended) in complete DMEM in a 10 cm culture dish approximately 24 h before transfection, allowing the cells to reach approximately 60%–70% confluency at the time of transfection.
  • b.
    Maintain the cells overnight in a humidified incubator at 37°C supplemented with 5% CO₂.
• 3.
  Produce lentiviral particles encoding EGFR-eGFP.

  • a.
    Prepare a transfection mixture containing a total of 20 μg plasmid DNA, consisting of 8 μg pLVX-EGFR-eGFP, 6 μg psPAX2, and 2 μg pMD2.G (mass ratio 4:3:1).
  • b.
    Perform transfection using Lipofectamine™ 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions (https://www.thermofisher.cn/cn/zh/home/life-science/cell-culture/transfection/transfection-reagents/lipofectamine-3000-reagent.html?CID=bid_clb_tfx_r04_cn_0se_bng_nt_pur_CBTFX_gene_tra_aBID_pc_mkt_031326_NC0K0BID00156550&msclkid=18e995b39be81034ac5aff3a2ae). For a 10 cm culture dish, use 20 μg total plasmid DNA together with 40 μL P3000 reagent and 30 μL Lipofectamine 3000 as a recommended starting condition. Optimization may be required depending on cell status and culture conditions.
  • c.
    Remove the transfection medium and add freshly prepared complete DMEM approximately 6–8 h following transfection.
  • d.
    Collect viral supernatants at 48 h and 72 h post-transfection.

CRITICAL: Handle lentiviral supernatants under appropriate biosafety conditions according to institutional guidelines.

• 4.
  Clarify and prepare viral supernatants.

  • a.
    Centrifuge the collected supernatants at 500 × g for 10 min at 4°C to remove cell debris.
  • b.
    Pass the collected supernatants through a 0.45 μm low-protein-retention membrane filter before further use.
  • c.
    Either use the viral preparation immediately after collection or divide it into aliquots and preserve at −80°C until needed.

Note: In this protocol, freshly prepared lentiviral supernatants were used directly for cell transduction without prior viral titer determination. For applications requiring precise control of transduction efficiency or comparison among virus preparations, viral titers may be estimated using standard methods.

• 5.
  Transduce HEK-293T cells with EGFR-eGFP lentivirus.

  • a.
    Seed naïve HEK-293T cells at 30%–40% confluency one day before transduction.
  • b.
    Approximately 24 h after cell seeding, when the naïve HEK-293T cells reach approximately 30%–40% confluency, replace the culture medium with lentiviral supernatant supplemented with polybrene (e.g., 8 μg/mL) to enhance transduction efficiency.
  • c.
    Incubate cells for 12–24 h, then replace with fresh complete medium.
• 6.
  Select stably transduced cells.

  • a.
    Begin antibiotic selection 48–72 h after transduction using 1 μg/mL puromycin to select cells carrying the pLVX-EGFR-eGFP vector.
  • b.
    Replace the selection medium every 2–3 days. In parallel, maintain a separate dish of non-transduced HEK-293T cells under the same puromycin selection conditions as a negative control. Continue selection until all cells in the non-transduced control dish are eliminated, indicating successful selection of the transduced population.
• 7.
  Enrich and expand EGFR-eGFP-positive cells.

  • a.
    Monitor eGFP expression by fluorescence microscopy.
  • b.
    Isolate EGFR-eGFP–positive cells using fluorescence-activated cell sorting (FACS), or alternatively maintain and expand the bulk population under ongoing selection pressure.
  • c.
    Expand the stable EGFR-eGFP-positive cell population until sufficient cells are available for downstream experiments (typically enough to seed two to three 10 cm culture dishes). It is recommended to cryopreserve one aliquot of the validated cell population before initiating subsequent experiments.
• 8.
  Validate stable EGFR-eGFP expression and localization.

  • a.
    Confirm EGFR-eGFP expression by flow cytometry (Figure S1A).
  • b.
    Verify predominant plasma membrane localization of EGFR-eGFP under basal conditions using fluorescence microscopy.

CRITICAL: Excessive EGFR-eGFP expression may alter receptor trafficking dynamics and confound the interpretation of ubiquitination-dependent endocytosis and lysosomal trafficking. After puromycin selection, evaluate EGFR-eGFP expression by flow cytometry and/or fluorescence microscopy, and select cell populations or single-cell–derived clones exhibiting moderate and relatively homogeneous EGFR-eGFP fluorescence for downstream experiments. Avoid populations containing a high proportion of extremely bright EGFR-eGFP-positive cells.

Pause point: Confirmed EGFR-eGFP-expressing HEK-293T stable cell lines may be cryopreserved in liquid nitrogen for long-term storage and subsequently thawed for downstream experiments.

Transiently express ZNRF3 or the ZNRF3 ΔRING mutant in EGFR-eGFP-HEK293T stable cell lines

Timing: 2–3 days

• 9.
  Seed EGFR-eGFP-expressing stable HEK-293T cells for transient transfection.

  • a.
    Plate EGFR-eGFP-expressing stable HEK-293T cells in complete DMEM at 40%–50% confluency in 6-well plates one day before transfection.
  • b.
    Maintain the cells overnight at 37°C in a humidified incubator supplemented with 5% CO₂.
• 10.
  Prepare expression plasmids.

  • a.
    Prepare high-quality, endotoxin-free pCDNA3.1(+)-ZNRF3 and pCDNA3.1(+)-ZNRF3 ΔRING plasmid DNA using a commercial endotoxin-free plasmid purification kit. Adjust the DNA concentration to approximately 1 μg/μL (or another concentration suitable for the planned transfection) and store aliquots at −20°C to avoid repeated freeze–thaw cycles.
  • b.
    Use Use pCDNA3.1(+)-ZNRF3 to express full-length human ZNRF3. Use pCDNA3.1(+)-ZNRF3 ΔRING to express a RING-domain-deleted ZNRF3 variant.
  • c.
    Confirm plasmid sequences before transfection.
• 11.
  Prepare Lipofectamine 3000 transfection complexes (per well of a 6-well plate).

  • a.
    Dilute 2.5 μg plasmid DNA in 125 μL Opti-MEM, then add 5 μL P3000 reagent and mix gently.
  • b.
    In a separate tube, dilute 7.5 μL Lipofectamine 3000 reagent in 125 μL Opti-MEM.
  • c.
    Combine the diluted DNA and Lipofectamine 3000 solutions, gently mix by pipetting, and incubate for 10–15 min at room temperature before adding the complexes dropwise to the cells.
  • d.
    Mix the diluted DNA/P3000 solution with the diluted Lipofectamine 3000 reagent, and allow the mixture to incubate at room temperature for 10–15 min.
• 12.
  Transiently transfect EGFR-eGFP-HEK-293T cells.

  • a.
    Add the transfection complexes dropwise onto the cells, then gently rock the plate to ensure uniform distribution.
  • b.
    Maintain the cells at 37°C in a humidified incubator with 5% CO₂ for 24 h, which is generally sufficient for robust expression of ZNRF3 or ZNRF3 ΔRING and is recommended for most downstream assays. If transgene expression is insufficient, continue incubation for up to 48 h. During this period, monitor cell morphology, confluency, and viability, as prolonged incubation may lead to overconfluent cultures or compromised cell health, potentially affecting receptor trafficking analyses.

CRITICAL: Quantify purified ZNRF3 and ZNRF3 ΔRING plasmids using a NanoDrop spectrophotometer and adjust them to the same working concentration before transfection. Use identical DNA masses and transfection conditions for both constructs. Confirm comparable protein expression by flow cytometry (Step 14) before performing downstream experiments.

• 13.
  Define experimental cell populations.

  • a.
    Designate cells transfected with pCDNA3.1(+)-ZNRF3 as ZNRF3/EGFR-eGFP-HEK-293T cells.
  • b.
    Designate cells transfected with pCDNA3.1(+)-ZNRF3 ΔRING as ZNRF3 ΔRING/EGFR-eGFP-HEK-293T cells.
• 14.
  Validate transient expression of ZNRF3 constructs.

  • a.
    Assess ZNRF3 or ZNRF3 ΔRING expression by flow cytometry (Figures S1B and S1C).Verify successful transfection by comparing the percentage of positive cells and the fluorescence intensity distribution with the corresponding negative control.
  • b.
    Confirm that EGFR-eGFP expression levels remain comparable across all experimental groups by comparing the median fluorescence intensity (MFI) of the EGFR-eGFP-positive population. Large shifts in EGFR-eGFP MFI may indicate unequal receptor expression and could affect downstream trafficking analyses.

Note: The ZNRF3 ΔRING mutant is used as a ubiquitination-inactive control to differentiate E3 ligase–mediated effects on EGFR internalization and intracellular trafficking.

Pause point: Transfected cells can be used directly for endocytosis and lysosomal trafficking assays 24–48 h post-transfection.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Cy5-conjugated anti-human IgG (H+L) (1:100 dilution)	Abcame	Cat# ab97172
Cy3-labeled anti-rabbit IgG (H+L) (1:100 dilution)	Beyotime	Cat# A0516
Anti-human LAMP-1 (1:1000 dilution)	Abcame	Cat# ab24170
FITC-conjugated anti-human IgG (H+L) (1:500 dilution)	Beyotime	Cat# A0556
HRP-conjugated anti-mouse IgG (H+L) (1:5000 dilution)	Beyotime	Cat# A0216
HRP-conjugated anti-rabbit IgG (H+L) (1:5000 dilution)	Beyotime	Cat# A0208
Anti-human β-Actin (1:1000 dilution)	Beyotime	Cat# AF0003
Anti-human EGFR (1:1000 dilution)	Abcame	Cat# ab52894
Chemicals, peptides, and recombinant proteins
Penicillin-Streptomycin Liquid	Solarbio	Cat# P1400
DAPI	Beyotime	Cat# C1002
Puromycin	Solarbio	Cat# P8231
Protease and phosphatase inhibitor cocktail	Beyotime	Cat# P1046
Cell lysis buffer for Western and IP	Beyotime	Cat# P0013J
PMSF	Beyotime	Cat# ST506
endo-BCN-PEG4-Val-Cit-PAB-MMAE	MedChemExpress	Cat# HY-141155
RSPO2-FU domain-Fc	This paper	N/A
EGFR-ubitaAb-v1	This paper	N/A
EGFR-ubitaAb-v2	This paper	N/A
EGFR-Ab (Cetuximab)	This paper	N/A
ZNRF3-extracellular domain (ECD)-Fc	This paper	N/A
RNF43-ECD-Fc	This paper	N/A
HER2-ECD-Fc	This paper	N/A
EGFR-ECD-Fc	This paper	N/A
Critical commercial assays
BeyoECL Plus	Beyotime	Cat# CK04
Cell Counting Kit-8	Dojindo	Cat# P0018S
Experimental models: Cell lines
HEK-293F	Laboratory of Dr. Linqi Zhan	N/A
HEK-293T	ATCC	Cat# CRL-3216
LS-513	Cell Bank, China Academy of Sciences	Cat# CRL-2134
HCT8	ATCC	Cat# CCL-244
RKO	Cell Bank, China Academy of Sciences	Cat# TCHu116
DLD-1	Cell Bank, China Academy of Sciences	Cat# TCHu134
SW480	Cell Bank, China Academy of Sciences	Cat# TCHu172
HT29	Cell Bank, China Academy of Sciences	Cat# TCHu103
Experimental models: Organisms/strains
Nude mice (BALB/cNj-Foxn1nu/Gpt, 6 to 8 weeks old, female, body weight 20-30g)	Hangzhou Medical College	BALB/c-nu mice
Recombinant DNA
pCDNA3.1(+)-RSPO2-FU domain-Fc	Tsingke Biotech	N/A
pCDNA3.1(+)-EGFR-ubitaAb-v2-light chain	Tsingke Biotech	N/A
pCDNA3.1(+)-EGFR-ubitaAb-v1-heavy chain	Tsingke Biotech	N/A
pCDNA3.1(+)-EGFR-Ab (Cetuximab)-light chain	Tsingke Biotech	N/A
pCDNA3.1(+)-EGFR-Ab (Cetuximab)-heavy chain	Tsingke Biotech	N/A
pCDNA3.1(+)-ZNRF3-ECD-Fc	Tsingke Biotech	N/A
pCDNA3.1(+)-RNF43-ECD-Fc	Tsingke Biotech	N/A
pCDNA3.1(+)-EGFR-ECD-Fc	Tsingke Biotech	N/A
Software and algorithms
Prism software	GraphPad	https://www.graphpad.com/scientific-software/prism/
Other
SMM 293-TI Expression Medium	SinoBiological	Cat# M293TI
FBS	Gibco	Cat# A5670701
RPMI-1640	Gibco	Cat# 11875093
DMEM	Gibco	Cat# 11965092

Materials and equipment

PEI stock solution (pH 6.8)

Reagent	Final concentration	Amount
PEI	1 mg/mL	50 mg
ddH2O	N/A	50 mL
Total	1 mg/mL	50 mL

Note: Store at 4°C for up to 1 month, but it is recommended to prepare fresh before use.

PBS (pH 7.4)

Reagent	Final concentration	Amount
NaCl	137 mM	8.0 g
KCl	2.7 mM	0.2 g
Na2HPO4	10 mM	1.42 g
KH2PO4	1.8 mM	0.24 g
ddH2O	N/A	1 L
Total	N/A	1 L

Note: Prepare fresh before use.

PBST (pH 7.4)

Reagent	Final concentration	Amount
NaCl	137 mM	8.0 g
KCl	2.7 mM	0.2 g
Na2HPO4	10 mM	1.42 g
KH2PO4	1.8 mM	0.24 g
Tween-20	0.05% (v/v)	0.5 mL
ddH2O	N/A	1 L
Total	N/A	1 L

Note: Prepare fresh before use.

Citrate buffer (pH 3.0)

Reagent	Final concentration	Amount
Citric acid monohydrate	0.1 M	14.71 g
Trisodium citrate dihydrate	0.1M	6.04 g
ddH2O	N/A	1 L
Total	N/A	1 L

Note: Store at 4°C for up to 1 month, but it is recommended to prepare fresh before use.

Tris-HCl

Reagent	Final concentration	Amount
Tris base	1 M	1.21 g
HCl	N/A	420 μL (36%–38%)
ddH2O	N/A	10 mL
Total	N/A	10 mL

Note: Store at 4°C for up to 1 month, but it is recommended to prepare fresh before use.

EDC buffer

Reagent	Final concentration	Amount
EDC	0.4 M	76.7 mg
ddH2O	N/A	1 mL
Total	N/A	1 mL

Note: It is recommended to prepare fresh before use.

NHS buffer

Reagent	Final concentration	Amount
NHS	0.1 M	11.5 mg
ddH2O	N/A	1 mL
Total	N/A	1 mL

Note: It is recommended to prepare fresh before use.

MES buffer

Reagent	Final concentration	Amount
MES monohydrate	10 mM	2.133 g
ddH2O	N/A	1 L
Total	N/A	1 L

Note: It is recommended to prepare fresh before use.

TBST (pH 7.6)

Reagent	Final concentration	Amount
Tris base	20 mM	2.42 g
NaCl	150 mM	8.77 g
Tween-20	0.1% (v/v)	1 mL
ddH2O	N/A	1 L
Total	N/A	1 L

Note: Store at 4°C for up to 1 month, but it is recommended to prepare fresh before use.

HPLC-HIC Mobile phase A (pH 7.0)

Reagent	Final concentration	Amount
Sodium phosphate	25 mM	3.55 g
Ammonium sulfate	2 M	264.28 g
ddH2O	N/A	1 L
Total	N/A	1 L

Note: It is recommended to prepare fresh before use.

HPLC-HIC Mobile phase B (pH 7.0)

Reagent	Final concentration	Amount
Sodium phosphate	25 mM	3.55 g
Isopropanol	20% (v/v)	200 mL
ddH2O	N/A	800 mL
Total	N/A	1 L

Note: It is recommended to prepare fresh before use.

Step-by-step method details

Design and functional screening of EGFR-ubitaAbs with improved cellular uptake and receptor ubiquitination-dependent degradation capabilities

Timing: 7–10 days (depending on transient transfection efficiency, antibody expression yield, purification, and completion of downstream functional assays)

This section outlines the design, production, and functional evaluation of EGFR-targeting receptor-ubiquitination antibodies (EGFR-ubitaAbs) developed to engage membrane-associated E3 ubiquitin ligases and promote EGFR uptake together with ubiquitination-driven receptor degradation in colorectal cancer cells.

• 1.
  Design and generation of EGFR-ubitaAbs.

  • a.
    Design and cloning of EGFR-ubitaAb expression constructs (Figure 2A).

    • i.
      Use cetuximab heavy-chain (HC) and light-chain (LC) sequences as the parental antibody framework.
    • ii.
      For EGFR-ubitaAb-v1, attach the RSPO2 FU domain carrying the F109A mutation to the N-terminus of the HC using a flexible (Gly₄Ser)₃ linker.
    • iii.
      For EGFR-ubitaAb-v2, connect the RSPO2 FU domain containing the F109A mutation to the N-terminus of the LC through the same flexible linker.
    • iv.
      Clone all HC and LC constructs into pcDNA3.1(+) mammalian expression vectors. The amino acid sequences of the parental cetuximab heavy and light chains, the RSPO2 FU domain carrying the F109A mutation, and the engineered EGFR-ubitaAb fusion constructs are provided in the supplemental information (Table S1).

      Note: The RSPO2 FU domain carrying the F109A mutation preserves its ability to interact with RNF43/ZNRF3 while eliminating binding to LGR4-6 receptors.

  • b.
    Transient transfection for antibody expression.

    • i.
      Prepare HEK-293F or Expi293 cells in suspension culture using SMM 293-TI Expression Medium.
    • ii.
      Adjust cell density to 5×10⁶ cells/mL prior to transfection.
    • iii.
      For each transfection, mix a total of 600 μg plasmid DNA (HC and LC plasmids at a 1:1 mass ratio) with SMM 293-TI Expression Medium.
    • iv.
      Add 2 mL of PEI (1 mg/mL stock solution) to the DNA mixture, vortex briefly, and incubate at room temperature for 15 min to allow complex formation.
    • v.
      Add the DNA-PEI complexes dropwise to the cell culture while gently swirling.
    • vi.
      Culture transfected cells at 37°C with 5% CO₂, shaking at 120 rpm.
    • vii.
      Maintain cells in SMM 293-TI Expression Medium without medium exchange.
    • viii.
      Harvest culture supernatants 3–4 days post-transfection by centrifugation at 3,000×g for 10 min to remove cells and debris.

      Note: HEK-293F and Expi293 cells are suspension-adapted mammalian expression cell lines that are widely used for transient antibody production because they combine high transfection efficiency with high recombinant protein yield while supporting appropriate protein folding and mammalian post-translational modifications, including glycosylation.

      CRITICAL: Maintain a DNA:PEI mass ratio of approximately 1:3 to ensure efficient transfection and antibody expression.

  • c.
    Antibody purification and buffer exchange.

    • i.
      Clarify supernatants by filtration through a 0.45 μm filter.
    • ii.
      Load supernatants onto a Protein A affinity chromatography column pre-equilibrated with PBS.
    • iii.
      Wash the column thoroughly with PBS to remove unbound proteins.
    • iv.
      Elute bound antibodies using 0.1 M citrate buffer (pH 3.0)Elute bound antibodies in a single batch using 0.1 M citrate buffer (pH 3.0). Collect the eluate over approximately 2–3 column volumes into a single collection tube.
    • v.
      Immediately neutralize the acidic eluate by gradually adding small aliquots of 1 M Tris-HCl while gently mixing. Monitor the pH using pH indicator paper during neutralization and continue adding Tris-HCl until the eluate reaches approximately pH 7.0, then proceed with buffer exchange.
    • vi.
      Pool antibody-containing fractions and exchange into PBS using desalting columns or ultrafiltration.
    • vii.
      Concentrate the antibodies to approximately 1–5 mg/mL using ultrafiltration as needed. Measure the antibody concentration using a NanoDrop spectrophotometer. If necessary, continue ultrafiltration until a final concentration of 1–5 mg/mL is achieve.
  • d.
    Quality control of purified antibodies.

    • i.
      Determine the concentration of purified antibodies by measuring the absorbance at 280 nm using a NanoDrop spectrophotometer. Calculate the total antibody yield from the measured concentration and the final sample volume.
    • ii.
      Load 2–5 μg of purified antibody per lane and evaluate antibody purity and structural integrity by SDS-PAGE using a 4%–20% gradient gel under both reducing and non-reducing conditions. (Figure S2A).
    • iii.
      Use the same gel composition for both reducing and non-reducing analyses to facilitate comparison of antibody integrity and confirm correct heavy- and light-chain assembly prior to downstream functional assays.
• 2.
  Evaluation of EGFR binding and E3 ligase recruitment.

  • a.
    Surface plasmon resonance (SPR) instrument setup and sensor chip activation.

    • i.
      Conduct SPR measurements on a WeSPR One Auto system at 25°C. Alternative SPR instruments, such as Biacore platforms, may also be used provided that comparable experimental conditions are maintained, including ligand immobilization strategy, analyte concentration series, experimental temperature, and kinetic analysis using the same 1:1 Langmuir binding model.
    • ii.
      Perform all binding analyses using carboxylate-functionalized sensor chips provided by WeSPR. Equivalent carboxymethylated sensor chips compatible with other SPR platforms (e.g., CM5 sensor chips) may also be used, provided that comparable ligand immobilization chemistry and immobilization levels are maintained.
    • iii.
      Initiate sensor chip activation by flowing a 1:1 solution of 0.4 M EDC and 0.1 M NHS (100 μL each) across the surface at 10 μL/min.
  • b.
    Ligand immobilization.

    • i.
      Prepare recombinant EGFR-ECD-Fc, RNF43-ECD-Fc, or ZNRF3-ECD-Fc at a final concentration of 50 μg/mL using 10 mM MES buffer (pH 5.0).
    • ii.
      Apply 100 μL of each ligand to the activated sensor chip surface to obtain an immobilization level of approximately 770 response units (RU).
    • iii.
      Set up reference flow channels using the same activation and blocking procedures but without immobilizing ligand proteins.
    • iv.
      Quench residual active ester groups with a casein-containing blocking solution supplied by WeSPR.

      CRITICAL: Maintain comparable immobilization levels across ligand surfaces to ensure accurate affinity comparison.

  • c.
    Antibody binding measurements.

    • i.
      Generate serially diluted solutions of EGFR-Ab (cetuximab), EGFR-ubitaAb-v1, and EGFR-ubitaAb-v2 using PBST buffer.
    • ii.
      Flow antibody analytes across both ligand-coated and reference sensor surfaces at 10 μL/min.
    • iii.
      Monitor and record both the association and dissociation kinetics during each analyte injection.
    • iv.
      In our experiments, sensor surface regeneration was generally not required because fresh ligand-coated sensor surfaces were used when necessary. If regeneration is required, the regeneration solution should be empirically optimized to completely dissociate bound analytes while preserving ligand activity.
  • d.
    Data processing and kinetic analysis (Figures S2B–S2D).

    • i.
      Subtract reference channel and buffer responses from all sensorgrams.
    • ii.
      Process the binding results using WeSPR Analysis Software v2.0.
    • iii.
      Apply a 1:1 Langmuir interaction model to fit the binding kinetics curves.
    • iv.
      Calculate equilibrium dissociation constant (KD) values based on steady-state affinity analysis.

      Note: Similar EGFR-binding affinities observed for EGFR-ubitaAbs and the parental cetuximab antibody suggest that addition of the E3 ligase–binding modules does not impair EGFR recognition, whereas concurrent interaction with RNF43-ECD-Fc and ZNRF3-ECD-Fc demonstrates effective recruitment of E3 ubiquitin ligases.

• 3.
  Flow cytometry–based analysis of antibody internalization in HT29 cells.

  • a.
    Cell seeding and preparation.

    • i.
      Plate HT29 cells in standard tissue culture-treated 12-well plates at a density of 5×10⁵ cells per well using complete growth medium. No additional coating is required because HT29 cells exhibit sufficient adherence under standard culture conditions.
    • ii.
      Incubate the cells overnight at 37°C in a humidified incubator containing 5% CO₂ to facilitate cell attachment.
  • b.
    Antibody binding at the cell surface.

    • i.
      Rinse the cells once with ice-cold PBS.
    • ii.
      Treat cells with 10 nM EGFR-Ab, EGFR-ubitaAb-v1, or EGFR-ubitaAb-v2 in serum-free DMEM and incubate at 4°C for 30 min to permit antibody binding to cell-surface EGFR.

      CRITICAL: Perform all binding steps at 4°C to prevent antibody internalization during surface labeling.

  • c.
    Initiation of antibody internalization.

    • i.
      Remove unbound antibodies by washing cells twice with PBS.
    • ii.
      Add pre-warmed complete culture medium and maintain the cells at 37°C for 0, 3, 6, 12, and 48 h to characterize antibody internalization kinetics. For routine comparison of antibody internalization, one or more representative time points (e.g., 24 h or 48 h) may be selected according to the experimental objective.
  • d.
    Termination of internalization and surface antibody labeling.

    • i.
      At each time point, immediately transfer the plates onto ice to halt further internalization.
    • ii.
      Rinse cells with ice-cold PBS containing 1% BSA.
    • iii.
      Treat cells with FITC-labeled goat anti-human IgG (H+L) secondary antibody at a 1:500 dilution and incubate at 4°C for 30 min to detect surface-bound primary antibodies.
  • e.
    Acquire samples by flow cytometry and analyze the resulting data (Figure 2B; Figure S3).

    • i.
      Wash cells twice with cold PBS containing 1% BSA.
    • ii.
      Harvest cells by gentle pipetting or enzymatic detachment if required.
    • iii.
      Acquire samples using a NovoCyte flow cytometer and collect at least 10,000 live single-cell events for each sample.
    • iv.
      Analyze the data using FlowJo software. Gate sequentially on the cell population based on forward scatter (FSC) and side scatter (SSC), exclude cell doublets if necessary, and determine the median fluorescence intensity (MFI) of the FITC signal corresponding to surface-bound antibodies.
    • v.
      Calculate antibody internalization rates using the following equation: Internalization (%) = [1 − (MFI at 37°C for each time point/MFI at 4°C for 30 min)] × 100% where the 0 h sample represents 100% surface-bound antibody before internalization.

      Note: Higher internalization rates indicate enhanced endocytic trafficking mediated by recruitment of membrane E3 ligases.

• 4.
  Western blot–based assessment of EGFR degradation in HT29 cells.

  • a.
    Seeding and preparation.

    • i.
      Plate HT29 cells in 6-well plates at a density of 6×10⁵ cells per well using complete growth medium.
    • ii.
      Maintain the cells overnight at 37°C in a humidified incubator containing 5% CO₂.
    • iii.
      Expose cells to EGFR-Ab, EGFR-ubitaAb-v1, or EGFR-ubitaAb-v2 at final concentrations of 1 nM, 10 nM, or 100 nM in complete medium for 48 h.
  • b.
    Cell lysis and protein extraction.

    • i.
      After treatment, immediately place the plates on ice and rinse cells twice with ice-cold PBS.
    • ii.
      Lyse cells using RIPA buffer supplemented with protease and phosphatase inhibitor cocktails.
    • iii.
      Keep lysates on ice for 30 min, mixing gently from time to time.
    • iv.
      Centrifuge at 12,000 × g for 15 min at 4°C.
    • v.
      Collect the clarified supernatant carefully, and discard the remaining insoluble debris.
  • c.
    Protein quantification and SDS-PAGE.

    • i.
      Determine protein concentration using a BCA Protein Assay Kit following the manufacturer’s protocol (https://www.beyotime.com/product/P0011.htm).
    • ii.
      Equalize all samples to the same protein concentration by dilution with RIPA buffer.
    • iii.
      Load 20–30 μg total protein per lane and separate samples by SDS-PAGE under reducing conditions.
  • d.
    Protein transfer and membrane blocking.

    • i.
      Transfer the resolved proteins onto PVDF membranes using either a wet or semi-dry transfer apparatus.
    • ii.
      Block the membranes in 5% non-fat milk prepared in TBST for 1 h at room temperature.
  • e.
    Incubation with primary and secondary antibodies.

    • i.
      Incubate the membranes overnight at 4°C with primary antibodies diluted in blocking buffer, including anti-EGFR antibody (1:1,000) and anti-β-actin antibody (1:1,000).
    • ii.
      Wash the membranes three times with TBST, 5–10 min per wash.
    • iii.
      Probe the membranes with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG secondary antibodies (1:5,000) for 1 h at room temperature.
  • f.
    Signal detection and data acquisition (Figure 2C).

    • i.
      Rinse the membranes three times with TBST.
    • ii.
      Develop protein signals using enhanced chemiluminescence (ECL) reagents.
    • iii.
      Acquire images using a ChemiDoc imaging system.
    • iv.
      Perform densitometric analysis of band intensities using Image Lab or ImageJ software, and normalize EGFR expression levels to β-actin.

Note: Reduced EGFR protein levels indicate antibody-induced receptor degradation mediated by enhanced endocytosis and ubiquitination-dependent lysosomal trafficking.

Figure 2.

Engineering and screening of EGFR-ubitaAbs with enhanced internalization and ubiquitination-mediated degradation

(A) Schematic illustration of the EGFR-ubitaAb constructs and their corresponding expression cassettes. EGFR-ubitaAb-v1 was generated by fusing the RSPO2 FU1/FU2 domain variant (F109A) to the N-terminus of the cetuximab heavy chain through a flexible (G₄S)₃ linker, whereas EGFR-ubitaAb-v2 was generated by fusing the same module to the N-terminus of the cetuximab light chain. The lower panel illustrates the corresponding expression constructs cloned into the pcDNA3.1(+) vector under the control of the CMV promoter, including the signal peptide, RSPO2 FU1/FU2 domain variant, linker, and antibody heavy- or light-chain coding sequences.

(B) Flow cytometry analysis of EGFR internalization in HT29 cells treated with 10 nM of the indicated antibodies for different durations at 37°C. EGFR-ubitaAb-v1 and -v2 showed enhanced internalization compared to cetuximab. Data are mean ± s.d. of n = 3 biologically independent experiments. Statistical significance was assessed using unpaired two-tailed t-tests. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(C) Quantification of EGFR levels by western blot after 48 h treatment with increasing concentrations (1–100 nM) of EGFR-Ab (cetuximab), EGFR-ubitaAb-v1, or -v2 in HT29 cells. EGFR-ubitaAb-v2 induced the most pronounced EGFR degradation.

Engineering an EGFR-ubitaADC to promote receptor ubiquitination-mediated endocytosis, lysosomal trafficking, and tumor-cell killing

Timing: 7–10 days (depending on antibody conjugation efficiency, purification, quality control, and completion of in vitro characterization assays)

This step describes the rational conversion of the lead receptor-ubiquitination antibody identified in Major Step 1 (EGFR-ubitaAb-v2) into a site-specifically conjugated antibody-drug conjugate (EGFR-ubitaADC), and the systematic evaluation of its biochemical integrity, receptor and E3 ligase engagement, intracellular trafficking, and cytotoxic activity. Based on the superior internalization efficiency and ubiquitination-dependent EGFR degradation observed for EGFR-ubitaAb-v2, this antibody was selected as the scaffold for ADC development. Employing a two-enzyme, three-step chemoenzymatic glycoengineering approach, a cleavable MMAE payload was selectively conjugated to the conserved Fc N-glycan, generating EGFR-ubitaADC with a defined drug-to-antibody ratio. This step further includes the generation of ligase-binding-deficient control ADCs to dissect the functional contribution of RNF43/ZNRF3 recruitment, as well as comprehensive in vitro assays to assess antigen recognition, E3 ligase binding, endocytosis, lysosomal delivery, receptor degradation, ubiquitination, ADC recycling, and tumor cell killing. Together, this step establishes receptor ubiquitination as a modular and translatable design principle for enhancing ADC intracellular delivery and therapeutic efficacy.

• 5.
  Site-specific chemoenzymatic conjugation of MMAE to EGFR-ubitaAb (Figure 3A).

  • a.
    Trimming of Fc N-glycans using Endo S.

    • i.
      Prepare EGFR-Ab, EGFR-ubitaAb at 10 mg/mL in PBS (pH 7.4).
    • ii.
      Add Endo S from Streptococcus pyogenes at a ratio of 200 U per mg IgG.
    • iii.
      Maintain the reaction at room temperature for 2 h under gentle agitation.
  • b.
    Purification of GlcNAc-trimmed antibodies.

    • i.
      Load the reaction mixture onto Protein A agarose resin equilibrated with PBS.
    • ii.
      Wash the resin thoroughly with PBS to remove Endo S.
    • iii.
      Elute the antibodies with 0.1 M citrate buffer (pH 3.0) and promptly neutralize the eluate using 1 M Tris-HCl.
    • iv.
      Concentrate the antibodies and perform buffer exchange into reaction buffer (50 mM Tris-HCl, 125 mM NaCl, pH 7.4) using 50 kDa Amicon Ultra centrifugal filtration devices.
  • c.
    Installation of azido-modified GalNAc (GalNAz).

    • i.
      Supplement the reaction with β(1,4)-galactosyltransferase GalT(Y289L) at a ratio of 12.5 μg per nmol of antibody.
    • ii.
      Add UDP-GalNAz at 25 nmol per nmol antibody.
    • iii.
      Maintain the reaction at 30°C for 16 h.
    • iv.
      Purify GalNAz-labeled antibodies with Protein A agarose resin and subsequently exchange the buffer into PBS.
  • d.
    Payload conjugation via SPAAC.

    • i.
      Supplement the reaction with endo-BCN-PEG₄-Val-Cit-PAB-MMAE at a ratio of 20 nmol per nmol of antibody.
    • ii.
      Maintain the reaction at 30°C for 16 h to facilitate strain-promoted azide–alkyne cycloaddition.
    • iii.
      Remove excess unconjugated payload by repeated ultrafiltration using 50 kDa Amicon Ultra centrifugal filters. Perform 3–5 rounds of dilution with PBS followed by centrifugation at 4,000 × g for 10–15 min (or according to the filter manufacturer’s recommendations (https://www.sigmaaldrich.cn/CN/zh/product/mm/ufc9050) until the desired buffer exchange is achieved. Finally, concentrate the purified ADC to the desired volume.
    • iv.
      Determine final ADC concentration by UV absorbance at 280 nm.
• 6.
  Characterization of EGFR-ubitaADC conjugation quality.

  • a.
    SDS-PAGE analysis (Figure S4A).

    • i.
      Analyze unconjugated antibodies, glycoengineered intermediates, and final ADCs under reducing and non-reducing conditions.
    • ii.
      Visualize protein bands by Coomassie Brilliant Blue staining to confirm integrity and homogeneity.
  • b.
    HIC-HPLC analysis of DAR distribution (Figure S4B).

    • i.
      Dilute each ADC to 1 mg/mL in PBS and inject 20 μL per run.
    • ii.
      Conduct the analysis using an Agilent 1260 HPLC system equipped with a TSKgel Butyl-NPR HIC column.
    • iii.
      Perform the analysis under the following conditions: column temperature set to 25°C; mobile phase A consisting of 25 mM sodium phosphate with 2 M ammonium sulfate (pH 7.0); mobile phase B consisting of 25 mM sodium phosphate with 20% isopropanol (pH 7.0); gradient program from 80:20 to 20:80 (A:B) over 30 min; flow rate of 0.8 mL/min. Calculate the average DAR values based on peak area distributions.
• 7.
  Evaluation of EGFR-ubitaADC internalization and lysosomal trafficking.

  • a.
    Flow cytometry-based internalization assay (Figure S4C).

    • i.
      Plate HT29 cells in 12-well plates at a density of 5×10⁵ cells per well and incubate overnight.
    • ii.
      Bind ADCs at final concentrations of 0.1, 1, or 10 nM in separate wells containing serum-free DMEM and incubate at 4°C for 30 min. Perform each condition in at least three biological replicates and include an untreated control well to establish background fluorescence for subsequent analysis.
    • iii.
      Initiate internalization at 37°C for indicated times (24 h).
    • iv.
      Label surface-retained ADCs with goat anti-human IgG secondary antibody conjugated with FITC (1:500) at 4°C.
    • v.
      Analyze samples using a NovoCyte flow cytometer and calculate internalization rates.
  • b.
    Lysosomal trafficking assay using Incucyte FabFluor (Figure S4D).

    • i.
      Plate HT29 cells in black 96-well plates at a density of 8 × 10³ cells per well.
    • ii.
      Treat with ADCs (10 nM) plus FabFluor-pH anti-human IgG reagent (1:200).
    • iii.
      Capture live-cell images at 2 h intervals using the Incucyte S3 system.
    • iv.
      Quantify red fluorescence normalized to cell confluence.
  • c.
    Confocal microscopy for lysosomal colocalization (Figure 3B).

    • i.
      Plate HT29 cells in 35 mm glass-bottom culture dishes and incubate overnight at 37°C in a humidified atmosphere containing 5% CO₂.
    • ii.
      Place cells on ice, then incubate with EGFR-ADC or EGFR-ubitaADC (10 nM) at 4°C for 30 min to permit surface association while minimizing endocytosis.
    • iii.
      Rinse cells twice with ice-cold PBS to eliminate unbound ADCs, and subsequently transfer the cells to 37°C for 6 h to enable internalization and intracellular transport.
    • iv.
      Wash cells with PBS and fix them in 4% paraformaldehyde for 15 min at room temperature.
    • v.
      Permeabilize the cells using 0.1% Triton X-100 prepared in PBS for 10 min.
    • vi.
      Block nonspecific interactions with 1% BSA in PBS for 30 min at room temperature.
    • vii.
      Label lysosomes using an anti-LAMP-1 primary antibody followed by incubation with a Cy3-labeled goat anti-rabbit IgG secondary antibody.
    • viii.
      Visualize ADC localization using a Cy5-conjugated goat anti-human IgG (H+L) secondary antibody.
    • ix.
      Stain nuclei with DAPI and mount the samples prior to imaging.
    • x.
      Acquire confocal fluorescence images using an Olympus FV3000 confocal microscope. As a starting point, use excitation/emission settings appropriate for DAPI (Ex 405 nm/Em 425–475 nm), Cy3 (Ex 561 nm/Em 570–620 nm), and Cy5 (Ex 640 nm/Em 650–750 nm). Optimize laser power and detector gain according to signal intensity, but maintain identical acquisition settings for all samples within the same experiment. Acquire images from at least 5–10 randomly selected fields of view per condition for quantitative colocalization analysis.
    • xi.
      Analyze colocalization between ADCs and LAMP-1-positive lysosomes using ImageJ (Fiji). Briefly, subtract background fluorescence from each image, define regions of interest corresponding to individual cells, and perform colocalization analysis using the Coloc 2 plugin. Quantify the degree of colocalization using Pearson’s correlation coefficient. Apply identical thresholding and analysis parameters to all images within the same experiment to ensure valid comparisons across experimental groups.
• 8.
  Analysis of EGFR-ubitaADC–mediated EGFR ubiquitination, degradation, and cytotoxic activity.

  • a.
    Confocal microscopy for EGFR ubiquitination (Figure 3C).

    • i.
      Plate ZNRF3/EGFR-eGFP–HEK-293T cells in 35 mm glass-bottom dishes and incubate overnight.
    • ii.
      For inhibition of ubiquitination, pretreat cells with the E1 ubiquitin-activating enzyme inhibitor MLN7243 (1 μM) for 2 h before ADC exposure.
    • iii.
      Incubate the cells with EGFR-ADC or EGFR-ubitaADC (10 nM) at 4°C for 30 min to enable surface binding.
    • iv.
      Remove unbound ADCs by washing with PBS, then transfer the cells to 37°C for 6 h to initiate internalization.
    • v.
      Rinse cells twice with PBS, fix them using 4% paraformaldehyde for 15 min at room temperature, and permeabilize with 0.1% Triton X-100 for 10 min.
    • vi.
      Block nonspecific staining with 1% BSA prepared in PBS for 30 min.
    • vii.
      Detect ubiquitinated proteins using a rabbit anti-ubiquitin monoclonal antibody followed by a Cy3-conjugated goat anti-rabbit IgG secondary antibody.
    • viii.
      Visualize ADCs with a Cy5-labeled goat anti-human IgG (H+L) secondary antibody.
    • ix.
      Counterstain cell nuclei using DAPI.
    • x.
      Capture confocal fluorescence images using an Olympus FV3000 confocal microscope while applying identical image acquisition settings across all experimental groups.
    • xi.
      Analyze colocalization between EGFR-eGFP, ubiquitin, and ADC signals using ImageJ.
  • b.
    Immunoblot analysis of EGFR degradation (Figure S4E).

    • i.
      Plate HT29 cells in 6-well plates at a density of 6 × 10⁵ cells per well using complete DMEM, and incubate overnight at 37°C in a humidified incubator containing 5% CO₂ until cell confluence reaches approximately 70%–80%.
    • ii.
      Exchange the medium with freshly prepared complete DMEM containing the indicated concentrations (typically 1 nM, 10 nM, or 100 nM) of EGFR-ADC or EGFR-ubitaADC.
    • iii.
      Maintain the cells at 37°C for 48 h to permit antibody-mediated EGFR internalization and receptor degradation.
    • iv.
      Following treatment, place the culture plates on ice and rinse cells twice with ice-cold PBS to eliminate residual culture medium.
    • v.
      Lyse the cells using RIPA buffer supplemented with protease and phosphatase inhibitor cocktails, adding 150–200 μL lysis buffer to each well.
    • vi.
      Allow lysates to incubate on ice for 30 min, briefly vortexing every 5 min to facilitate complete cell disruption.
    • vii.
      Centrifuge the lysates at 12,000 × g for 15 min at 4°C to sediment insoluble material.
    • viii.
      Transfer the clarified supernatant into fresh tubes and quantify protein concentration using a BCA Protein Assay Kit.
    • ix.
      Adjust all samples to equivalent protein concentrations, combine with 4× SDS loading buffer, and heat at 95°C for 5 min before electrophoresis.
    • x.
      Separate 20–30 μg total protein per sample on 8%–10% SDS-PAGE gels, followed by transfer of proteins onto PVDF membranes.
    • xi.
      Block the membranes in 5% non-fat milk prepared in TBST for 1 h at room temperature with gentle agitation.
    • xii.
      Incubate membranes overnight at 4°C with primary antibodies diluted in blocking solution, including anti-EGFR antibody (1:1,000) and anti-β-actin antibody (1:1,000) as the loading control.
    • xiii.
      Rinse membranes three times with TBST for 10 min each wash.
    • xiv.
      Incubate membranes with HRP-conjugated secondary antibodies (1:5,000 dilution) for 1 h at room temperature.
    • xv.
      Wash the membranes three additional times using TBST.
    • xvi.
      Detect protein signals using enhanced chemiluminescence (ECL) reagents.
    • xvii.
      Capture images using a ChemiDoc imaging system.

      CRITICAL: MLN7243 pretreatment should be sufficient to inhibit ubiquitination but not compromise overall cell viability during the imaging window.

  • c.
    Cell viability assays (Figure 3D).

    • i.
      Plate EGFR-expressing colorectal cancer cells in 96-well plates at a density of 5,000 cells per well in 100 μL complete growth medium, and incubate overnight at 37°C in a humidified atmosphere with 5% CO₂ to permit cell attachment.
    • ii.
      Prepare serial dilutions of EGFR-ADC or EGFR-ubitaADC in complete medium, then replace the existing culture medium with 100 μL antibody-containing medium at the indicated concentrations. Perform each concentration in at least three replicate wells. Include untreated control wells containing cells cultured in complete medium without ADCs, as well as medium-only and CCK-8-only blank wells for background correction.
    • iii.
      Maintain cells with ADC treatment for 72 h at 37°C in a humidified incubator containing 5% CO₂.
    • iv.
      Add 10 μL CCK-8 reagent directly to each well and incubate for an additional 1–2 h at 37°C while protecting from light exposure.
    • v.
      Determine absorbance at 450 nm using a microplate reader.
    • vi.
      Subtract the background absorbance determined from the medium-only and CCK-8-only blank wells. Normalize the absorbance of each treatment group to the mean value of the untreated control wells, which are defined as 100% cell viability.
    • vii.
      Generate dose–response curves and calculate IC₅₀ values using nonlinear regression analysis in GraphPad Prism.

      CRITICAL: Cell density at seeding must be uniform across plates, as variations strongly influence ADC sensitivity and IC₅₀ estimation.

Figure 3.

Design and functional evaluation of EGFR-ubitaADC

(A) Schematic of EGFR-ubitaADC generated by site-specific glycan-directed conjugation of MMAE to the N297 site of EGFR-ubitaAb-v2.

(B) Confocal microscopy of HT29 cells treated with 10 nM EGFR-ADC or EGFR-ubitaADC for 6 h at 37°C. EGFR-ubitaADC showed greater internalization and lysosomal colocalization. Scale bars, 10 μm.

(C) Confocal imaging of ZNRF3 and EGFR-eGFP overexpressed HEK-293T cells immunostained for EGFR-ubitaADC or EGFR-ADC (magenta), EGFR-eGFP (green), ubiquitin (red), and nucleus (blue) after 6 h treatment. EGFR-ubitaADC induced stronger EGFR ubiquitination.

(D) Cell viability of HT29, SW480, RKO, LS-513, HCT8, DLD-1 cells treated with the indicated antibodies or ADCs for 72 h. EGFR-ubitaADC exhibited greater cytotoxicity than EGFR-ADC. Unconjugated antibodies showed minimal activity. Dose-response curves represent mean ± s.d. from n = 3 independent experiments.

EGFR-ubitaADC-induced endocytosis and lysosomal trafficking are dependent on E3 ubiquitin ligase activity

Timing: 7–10 days (depending on scheduling of imaging-based trafficking assays)

This step evaluates whether the increased endocytosis and lysosomal trafficking of EGFR-ubitaADC is mechanistically reliant on recruitment and catalytic activity of E3 ubiquitin ligases. To specifically dissect ubiquitination-driven trafficking events while minimizing interference from endogenous EGFR internalization pathways, a reductionist and inducible EGFR-eGFP system was generated in HEK-293T cells. HEK-293T cells were selected because of their inherently low basal EGFR endocytic and ubiquitination activity, providing a controlled cellular background to interrogate E3 ligase–dependent mechanisms rather than to recapitulate tumor-specific EGFR trafficking phenotypes. Using this system, the effects of ZNRF3 overexpression, pharmacological inhibition of ubiquitin activation, and genetic ablation of E3 ligase catalytic activity were systematically evaluated to define the requirement of ubiquitination for EGFR-ubitaADC-mediated intracellular routing.

• 9.
  Baseline trafficking analysis of EGFR-ADC and EGFR-ubitaADC in EGFR-eGFP–expressing HEK-293T cells (Figure 4A).

  • a.
    Cell seeding and preparation.

    • i.
      Plate EGFR-eGFP–HEK-293T cells in 35 mm glass-bottom dishes at a density of 2–3 × 10⁵ cells per dish using complete DMEM.
    • ii.
      Incubate the cells overnight at 37°C in a humidified atmosphere containing 5% CO₂.

      Note: This baseline model provides a low endocytic and low ubiquitination background, enabling isolation of ligand binding from ubiquitination-driven trafficking.

  • b.
    Surface binding and internalization assay.

    • i.
      Place cells on ice for 10 min, then incubate with EGFR-ADC or EGFR-ubitaADC (10 nM) in ice-cold serum-free DMEM at 4°C for 30 min to enable surface binding.
    • ii.
      Rinse the cells three times with ice-cold PBS to eliminate unbound ADCs.
    • iii.
      Trigger internalization by transferring cells to 37°C in complete medium and incubating for 6 h.
  • c.
    Fixation, staining, and imaging.

    • i.
      Rinse cells twice with PBS, then fix with 4% paraformaldehyde for 15 min at room temperature.
    • ii.
      Permeabilize cells using 0.1% Triton X-100 in PBS for 10 min.
    • iii.
      Block nonspecific binding sites with 5% BSA in PBS for 1 h at room temperature.
    • iv.
      Label lysosomes by incubating with anti-LAMP-1 rabbit monoclonal antibody overnight at 4°C.
    • v.
      Apply Cy3-conjugated goat anti-rabbit IgG secondary antibody and incubate for 1 h.
    • vi.
      Visualize ADCs using Cy5-conjugated goat anti-human IgG (H+L), and counterstain nuclei with DAPI.
    • vii.
      Capture fluorescence images using an Olympus FV3000 confocal microscope. Acquire images using the following fluorescence settings: DAPI (excitation 405 nm, emission 425–475 nm), Cy3 (excitation 561 nm, emission 570–620 nm), Cy5 (excitation 640 nm, emission 650–750 nm), and eGFP (excitation 488 nm, emission 500–550 nm). Maintain identical laser power, detector gain, pinhole size, and scanning parameters for all samples. Acquire at least 5 randomly selected fields of view per condition for subsequent quantitative analysis.
• 10.
  ZNRF3-dependent enhancement of EGFR-ubitaADC internalization in ZNRF3/EGFR-eGFP-HEK-293T cells (Figure 4B).

  • a.
    Seed ZNRF3/EGFR-eGFP–HEK-293T cells on poly-D-lysine–coated glass-bottom dishes and culture overnight until reaching approximately 50%–60% confluence.
  • b.
    Place cells on ice for 10 min, then incubate with EGFR-ADC or EGFR-ubitaADC (10 nM) in serum-free medium at 4°C for 30 min to enable surface binding.
  • c.
    Wash cells three times with ice-cold PBS to remove any unbound ADCs.
  • d.
    Add pre-warmed complete medium and incubate at 37°C for 6 h to permit internalization and intracellular trafficking.
  • e.
    Perform lysosomal staining, fixation, and confocal imaging as described in Step 9c.

Note: This assay directly evaluates ADC trafficking behavior in a ZNRF3-competent context without further manipulation of ligase expression levels.

• 11.
  Inhibition of EGFR-ubitaADC trafficking using MLN7243, an inhibitor of the E1 ubiquitin-activating enzyme (Figure 4C).

  • a.
    Plate ZNRF3/EGFR-eGFP–HEK-293T cells as described above.
  • b.
    Treat cells with MLN7243 (1 μM) or DMSO vehicle control for 1 h at 37°C prior to further manipulation.
  • c.
    While maintaining inhibitor exposure, incubate cells with EGFR-ubitaADC at 4°C for 30 min to permit surface binding.
  • d.
    After washing, culture cells at 37°C for 6 h in complete medium supplemented with MLN7243.
  • e.
    Proceed with lysosomal staining, fixation, and confocal imaging following the procedure described in Step 9c.

CRITICAL: MLN7243 treatment must be maintained throughout the internalization period to ensure sustained inhibition of ubiquitin conjugation.

• 12.
  Requirement of E3 ligase catalytic activity in ZNRF3 ΔRING/EGFR-eGFP-HEK-293T cells (Figure 4D).

  • a.
    Culture ZNRF3 ΔRING/EGFR-eGFP-HEK-293T cells in glass-bottom dishes overnight until reaching ∼50%–60% confluence.
  • b.
    b.Expose cells to EGFR-ADC or EGFR-ubitaADC at 4°C for 30 min to permit surface binding.
  • c.
    c.Rinse cells extensively with ice-cold PBS to eliminate unbound ADCs.
  • d.
    d.Replace with pre-warmed complete medium and incubate at 37°C for 6 h to enable internalization and trafficking.
  • e.
    Perform lysosomal staining, fixation, and confocal imaging as described in Step 9c.

CRITICAL: Loss of the RING domain abolishes E3 ligase catalytic activity while preserving membrane localization, enabling specific assessment of ubiquitination-dependent trafficking.

Figure 4.

EGFR-ubitaADC trafficking is dependent on E3 ligase–mediated ubiquitination

(A) Confocal microscopy of HEK-293T cells expressing EGFR-eGFP and treated with 10 nM EGFR-ADC or EGFR-ubitaADC for 6 h at 37°C. Both ADCs remained predominantly membrane-localized, with limited internalization or lysosomal delivery in the absence of E3 ligase expression.

(B) Co-expression of ZNRF3 and EGFR-eGFP in HEK-293T cells promoted robust internalization and lysosomal accumulation of EGFR-ubitaADC, as shown by co-localization of EGFR-eGFP (green), ADC (red), and lysosomes (blue) after 6 h incubation.

(C) Treatment with the E1 ubiquitin-activating enzyme inhibitor MLN7243 (1 μM, 2 h pretreatment) abolished the lysosomal trafficking of EGFR-ubitaADC in ZNRF3/EGFR-eGFP co-expressing HEK-293T cells, supporting the requirement of ubiquitination for internalization and endolysosomal routing.

(D) Replacement of wild-type ZNRF3 with a catalytically inactive mutant lacking the RING domain (ZNRF3-ΔRING) failed to promote lysosomal delivery of EGFR-ubitaADC, confirming that E3 ligase catalytic activity is required for receptor trafficking enhancement. Scale bars, 10 μm.

Receptor ubiquitination is essential for EGFR-ubitaADC activity and enables its antitumor efficacy even in the context of partial target depletion

Timing: 5–6 weeks (depending primarily on the duration of in vivo tumor studies)

This step provides functional validation that the increased cytotoxic and antitumor effects of EGFR-ubitaADC are strictly dependent on receptor ubiquitination and engagement of E3 ubiquitin ligases. Through pharmacological inhibition, competitive blocking strategies, receptor pre-depletion experiments, and in vivo xenograft models, it demonstrates that EGFR-ubitaADC retains strong antitumor activity even under conditions of partial target depletion, and achieves enhanced tumor growth suppression via ubiquitin–lysosome–dependent pathways.

• 13.
  Requirement of ubiquitin activation for EGFR-ubitaADC cytotoxicity (Figure 5A).

  • a.
    Plate HT29 cells in 96-well plates at a density of 5,000 cells per well and incubate overnight.
  • b.
    Treat cells with MLN7243, an E1 ubiquitin-activating enzyme inhibitor (1 μM), or vehicle control for 1 h at 37°C prior to subsequent experiments.
  • c.
    Add serial dilutions of EGFR-ubitaADC or EGFR-ADC directly to the wells while maintaining MLN7243 or vehicle in the culture medium.
  • d.
    Incubate cells for 72 h at 37°C.
  • e.
    Measure cell viability using CCK-8 reagent and calculate relative viability compared to untreated controls.

CRITICAL: MLN7243 must be present throughout the entire ADC treatment period to ensure sustained inhibition of ubiquitin activation.

• 14.
  Requirement of E3 ligase engagement for EGFR-ubitaADC activity (Figure 5B).

  • a.
    Plate HT29 cells in 96-well plates and incubate overnight.
  • b.
    Pre-incubate EGFR-ubitaADC with soluble RNF43-ECD-Fc and ZNRF3-ECD-Fc proteins at 37°C for 30 min.
  • c.
    Apply the resulting ADC–decoy protein mixtures to cells at the indicated concentrations.
  • d.
    Incubate cells for 72 h.
  • e.
    Quantify cell viability using CCK-8 assays and normalize to control conditions.

Note: Soluble RNF43/ZNRF3-ECD-Fc proteins act as competitive decoys to block EGFR-ubitaADC interaction with endogenous membrane-bound E3 ligases.

• 15.
  EGFR-ubitaADC activity despite partial receptor depletion (Figures 5C and 5D).

  • a.
    Plate HT29 cells in 6-well plates and incubate overnight.
  • b.
    Treat cells with EGFR-ubitaAb at a fixed concentration for 0, 24, 48, or 72 h to achieve graded levels of EGFR depletion.
  • c.
    At each time point, collect a subset of cells for flow cytometry analysis to quantify surface EGFR expression.
  • d.
    In parallel, reseed the pretreated cells into 96-well plates for subsequent assays.
  • e.
    Treat cells with serial dilutions of EGFR-ubitaADC for 72 h.
  • f.
    Assess cell viability using CCK-8 assays.

CRITICAL: Care should be taken to maintain comparable cell densities across different pretreatment durations to ensure accurate comparison of cytotoxic responses.

• 16.
  Evaluation of the in vivo antitumor efficacy of EGFR-ubitaADC in HT29 xenograft models (Figures 5E–5G).

  • a.
    Generate subcutaneous HT29 xenograft tumors in female BALB/c nude mice.
  • b.
    Once tumors reach approximately 100–150 mm³, randomly assign animals to different treatment groups.
  • c.
    Provide intravenous injections of EGFR-ubitaADC, EGFR-ADC, or PBS at a dose of 10 mg/kg every three days for a total of six administrations (q3d × 6).
  • d.
    Record tumor volume and body weight at 2–3 days intervals throughout the study.
  • e.
    At the experimental endpoint, euthanize the mice and harvest tumor tissues for analysis.
  • f.
    Prepare tumor lysates and analyze EGFR and RNF43 protein levels by immunoblotting.

CRITICAL: All animal studies should be conducted in compliance with relevant institutional and national ethical regulations.

Figure 5.

Receptor ubiquitination is required for EGFR-ubitaADC activity and supports efficacy in vivo despite partial target depletion

(A) HT29 cells were treated with EGFR-ubitaADC in the presence or absence of the E1 inhibitor MLN7243 for 72 h. Inhibition of ubiquitin activation impaired EGFR-ubitaADC cytotoxicity.

(B) Co-incubation with recombinant RNF43-ECD-Fc and ZNRF3-ECD-Fc (each at 1μM) competitively blocked the cytotoxic effect of EGFR-ubitaADC in HT29 cells.

(C) Flow cytometry analysis of EGFR surface levels after pretreatment with EGFR-ubitaAb (10 nM) for 0–72 h.

(D) Cytotoxicity of EGFR-ubitaADC after different durations of EGFR-ubitaAb pretreatment. EGFR-ubitaADC retained efficacy despite partial EGFR depletion.

(E) Dosing scheme for in vivo efficacy study. BALB/c nude mice bearing subcutaneous HT29 xenografts were intravenously administered PBS, EGFR-ADC, or EGFR-ubitaADC (10 mg/kg, every 3 days for 6 doses, q3d × 6, n = 5 per group).

(F) Tumor growth curves of HT29 xenografts in nude mice treated with EGFR-ubitaADC, EGFR-ADC, or PBS. Average tumour growth curves showing EGFR-ubitaADC led to significant tumour inhibition compared to control groups. Data are mean ± s.e.m.; statistical analysis by unpaired two-tailed t-tests. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(G) Mouse body weights remained stable throughout treatment.

Expected outcomes

This protocol enables the reproducible generation, characterization, and functional evaluation of EGFR receptor-ubiquitination-targeting antibodies (EGFR-ubitaAbs) and their corresponding antibody-drug conjugates (EGFR-ubitaADCs) (Figure 1). Successful completion of this protocol generates biochemical, cellular, and in vivo evidence showing that engagement of membrane-associated E3 ubiquitin ligases promotes EGFR internalization, trafficking to lysosomes, and antitumor efficacy.

The design and functional evaluation of EGFR receptor-ubiquitination antibodies generate EGFR-ubitaAbs that retain strong binding affinity toward EGFR while gaining the capacity to recruit the membrane-associated E3 ubiquitin ligases RNF43 and/or ZNRF3. Surface plasmon resonance analyses typically show preserved EGFR binding together with newly introduced interactions with RNF43-ECD-Fc and ZNRF3-ECD-Fc, whereas the parental EGFR antibody binds EGFR alone. In EGFR-expressing colorectal cancer cells, EGFR-ubitaAbs display accelerated internalization kinetics and enhanced EGFR downregulation relative to conventional EGFR antibodies. Immunoblot analysis further reveals ubiquitination-dependent receptor degradation, enabling identification of a lead EGFR-ubitaAb variant with superior intracellular trafficking properties (Figures 2 and S2).

Engineering EGFR-ubitaADCs to promote receptor ubiquitination–mediated internalization, lysosomal transport, and tumor cell cytotoxicity enables the generation of homogeneous EGFR-ubitaADC products with defined drug-to-antibody ratios through site-specific chemoenzymatic conjugation. SDS-PAGE and HIC-HPLC analyses confirm conjugation integrity and narrow DAR distributions. Compared with conventional EGFR-ADCs, EGFR-ubitaADC shows increased cellular uptake and more efficient lysosomal delivery in colorectal cancer cells. Confocal microscopy demonstrates stronger colocalization of EGFR-ubitaADC with lysosomal markers and elevated ubiquitin signals associated with EGFR-positive compartments. These trafficking differences are accompanied by enhanced cytotoxic activity in cell viability assays (Figures 3 and S3).

The dependence of EGFR-ubitaADC–induced endocytosis and lysosomal trafficking on E3 ubiquitin ligase activity is investigated using a simplified HEK-293T model stably expressing EGFR-eGFP. Without ectopic E3 ligase expression, both EGFR-ADC and EGFR-ubitaADC are predominantly localized at the cell surface. Following ZNRF3 overexpression, EGFR-ubitaADC exhibits pronounced cellular uptake and enrichment within LAMP-1–positive lysosomal compartments, whereas the conventional EGFR-ADC displays only limited intracellular trafficking. Inhibition of ubiquitin activation using pharmacological agents or disruption of the ZNRF3 RING domain through genetic modification eliminates this phenotype, indicating that the catalytic activity of the E3 ligase is essential for intracellular trafficking of EGFR-ubitaADC (Figure 4).

The requirement of receptor ubiquitination for EGFR-ubitaADC activity, as well as its ability to maintain antitumor efficacy despite partial target depletion, is confirmed through complementary in vitro and in vivo experiments. Inhibition of ubiquitin activation or competitive blockade of RNF43/ZNRF3 engagement markedly attenuates EGFR-ubitaADC cytotoxicity in colorectal cancer cells. Despite inducing partial depletion of surface EGFR upon prolonged exposure, EGFR-ubitaADC retains robust killing activity, indicating resilience to target self-consumption. In HT29 xenograft models, EGFR-ubitaADC treatment results in superior tumor growth inhibition compared with conventional EGFR-ADCs, without significant body weight loss. Analysis of tumor lysates reveals enhanced EGFR downregulation without changes in E3 ligase expression, supporting ubiquitin-mediated receptor degradation as the dominant mechanism in vivo (Figure 5).

Quantification and statistical analysis

Statistical analyses were conducted using GraphPad Prism 8.4. Data are expressed as mean ± s.d. or mean ± s.e.m., as indicated in the corresponding figure legends. Analyses of EGFR internalization in HT29 cells, including time-dependent studies and comparisons among distinct EGFR-ubitaAb constructs, were performed using unpaired two-tailed Student’s t-tests based on data collected from three independent biological experiments. For animal experiments, analyses of tumor growth and body weight were carried out as described in the relevant figure legends. Body weight measurements are presented as mean ± s.e.m. and were statistically evaluated using unpaired two-tailed Student’s t-tests. Tumor volume data from xenograft models (n = 5 mice per treatment group) are shown as mean ± s.e.m., and differences between groups at the indicated time points were assessed using unpaired two-tailed Student’s t-tests. Unless otherwise noted, all experiments were independently reproduced at least three times with consistent outcomes. Statistical significance was defined as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, whereas P > 0.05 was considered not significant (ns). Detailed information regarding exact P values, sample numbers, and statistical methods is provided in the corresponding figure legends.

Limitations

This protocol is designed to interrogate receptor ubiquitination-driven endocytosis and lysosomal trafficking using EGFR and the membrane-bound E3 ligases RNF43/ZNRF3 as a defined model system. Its robustness therefore depends on adequate expression, membrane localization, and catalytic activity of these ligases, and the magnitude of EGFR-ubitaADC-mediated effects may vary across cell types or tumor models with distinct endocytic landscapes. In addition, mechanistic dissection relies in part on engineered HEK-293T systems and pharmacological inhibition of ubiquitin activation, which enable controlled analysis but may not fully capture the complexity of endogenous receptor regulation or avoid broader effects on cellular ubiquitin homeostasis. Finally, in vivo validation is performed in immunodeficient xenograft models, and factors such as tumor microenvironment, vascularization, and immune interactions may influence ADC trafficking and efficacy beyond what is observed in vitro, warranting further evaluation in additional disease-relevant settings.

Troubleshooting

Problem 1

Low yield or poor integrity of EGFR-ubitaAbs after transient expression. Related to: Step 1 (Engineering and production of EGFR-ubitaAbs).

Potential solution

Low antibody yield is most commonly caused by suboptimal transfection efficiency or incorrect heavy- and light-chain stoichiometry. Ensure that HEK-293F or Expi293 cells are in logarithmic growth phase and that cell viability exceeds 95% prior to transfection. Strictly maintain the recommended DNA:PEI mass ratio and confirm that heavy- and light-chain plasmids are mixed at an equimolar ratio. If antibody degradation is observed, reduce the culture duration to 72 h and include protease inhibitors during purification. Alternative mammalian expression systems (e.g., ExpiCHO) may be used, provided that comparable antibody folding and glycosylation are achieved.

Problem 2

EGFR-ubitaAbs show reduced EGFR binding affinity compared to parental cetuximab. Related to: Step 2 (Evaluation of EGFR binding and E3 ligase recruitment).

Potential solution

Reduced EGFR binding may arise from steric interference caused by improper positioning of the RSPO2 FU domain. Verify correct fusion orientation and linker length by sequencing and SDS-PAGE. In SPR experiments, confirm that EGFR-ECD-Fc is immobilized at comparable response units across channels and that ligand integrity is preserved during regeneration. If necessary, reduce immobilization density or perform single-cycle kinetics to minimize avidity-related artifacts.

Problem 3

Minimal differences in internalization rates between EGFR-Ab and EGFR-ubitaAbs in flow cytometry assays. Related to: Step 3 (Flow cytometry-based antibody internalization assay).

Potential solution

Insufficient temperature control during surface binding is a frequent cause of high background internalization. Ensure that all binding and labeling steps are performed strictly at 4°C and that pre-chilled buffers are used. Confirm that secondary antibody labeling is restricted to surface-retained antibodies and that fluorescence signals are normalized to the 0 h time point. If internalization remains weak, extend the internalization window or increase antibody concentration within the non-saturating range.

Problem 4

EGFR degradation is not detectable by immunoblotting despite prolonged antibody treatment. Related to: Step 4 (Immunoblot-based analysis of EGFR degradation).

Potential solution

Failure to detect EGFR degradation may result from insufficient antibody concentration, incomplete receptor internalization, or suboptimal lysis conditions. Verify that cells reach appropriate confluency and that treatment duration is sufficient to allow lysosomal degradation. Use freshly prepared RIPA buffer supplemented with protease inhibitors and ensure equal protein loading across samples. Increasing gel resolution or extending exposure times during chemiluminescence detection may improve sensitivity.

Problem 5

Low or heterogeneous drug-to-antibody ratio (DAR) after chemoenzymatic conjugation. Related to: Step 5 and Step 6 (Site-specific conjugation and ADC characterization).

Potential solution

Incomplete glycan trimming or inefficient GalNAz installation can compromise subsequent payload conjugation. Confirm Endo S activity using SDS-PAGE mobility shifts and ensure that GalT(Y289L) and UDP-GalNAz are freshly prepared. Maintain reaction temperatures and incubation times precisely as specified. While alternative payloads or click chemistries may be compatible, modification of the Fc glycan site or reaction sequence is not recommended, as this may alter ADC homogeneity.

Problem 6

EGFR-ubitaADC fails to show enhanced lysosomal trafficking in E3 ligase-dependent models. Related to: Steps 9–12 (E3 ligase-dependent trafficking analyses).

Potential solution

Insufficient E3 ligase activity or loss of inhibitor efficacy may obscure ubiquitination-dependent effects. Confirm expression and membrane localization of ZNRF3 or ZNRF3 ΔRING constructs prior to imaging. For MLN7243 experiments, ensure continuous presence of the inhibitor throughout the internalization period and verify that cell viability is not compromised. Imaging parameters should be kept constant across conditions to allow accurate comparison of lysosomal colocalization.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Liqiang Pan (panliqiang@zju.edu.cn).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Xinlei Zhuang (zhuangxinlei@zju.edu.cn).

Materials availability

UbitaAb and ubitaADC are available from the lead contact with a completed materials transfer agreement.

Data and code availability

This study did not generate new datasets or code.

Acknowledgments

Funding for this work was provided by the National Natural Science Foundation of China (grant no. 82473824, 82504649), the Zhejiang Province Pioneer Plan (2025C01189 and 2025C01123), the Zhejiang Provincial Natural Science Foundation of China under grant no. LR26H300001, and the China Postdoctoral Science Foundation (certificate no. 2024M752837).

Author contributions

Conceptualization, L.P. and X.Z.; methodology, X.Z. and L.L.; manuscript writing – original draft, X.L.Z.; manuscript review and revision, L.P. and X.Z.; acquisition of funding support, L.P. and X.Z.; provision of research resources, L.P.; project supervision, L.P.

Declaration of interests

L.P. and X.Z. are inventors on Chinese patent applications (application nos. 2025119878035 and 2025119878001). These patent applications cover aspects of the receptor-ubiquitination-targeting antibody and antibody-drug conjugate technologies described in this study.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT in order to assist with writing. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.