In the era of precision oncology, antibody-drug conjugates (ADCs) have emerged as one of the most promising targeted cancer therapies. By combining monoclonal antibodies (mAbs) with potent cytotoxic payloads, ADCs deliver drugs directly to tumor cells while minimizing systemic toxicity.
However, traditional ADCs still face critical challenges:
● Limited tumor penetration due to large molecular size
● Off-target toxicity and heterogeneous drug distribution
● Acquired drug resistance
To overcome these limitations, single-domain antibodies (sdAbs), especially VHH (nanobodies), are gaining attention as next-generation ADC carriers. Their small size, high stability, and excellent engineering flexibility make nanobody-based ADCs (nADCs) a powerful alternative.
ADC is composed of monoclonal antibodies targeting specific antigens and small molecule cytotoxic drugs linked through linkers, combining the powerful killing effects of traditional small molecule chemotherapies with the tumor-targeting properties of antibody drugs. However, the large size of conventional antibodies (mAb) hinders their ability to extravasate and effectively penetrate tissues, and the drug attached to the mAb tends to be unevenly distributed within the tumor. Therefore, ADC development must consider improving tissue permeability and minimizing off-target effects. Single-domain antibody (sdAb), as antibodies with unique properties, have shown great potential in the development of new-generation ADC.
Fig1. The general structure of an ADC consists of three parts: the mAb, the payload, and a chemical linker between the payload and the antibody.
HCAbs are found in camelidae (Bactrians and camels, alpacas, and llamas) as well as cartilaginous fish (such as sharks). The VHH derived from Camelidae is a unique functional single domain of HCAb (Figure 2a). Although the molecular weight of the variable domain of the heavy chain in HCAbs is only one-tenth that of traditional IgG (12-17 kDa compared to 150 kDa), they retain high antigen-binding affinity and are the smallest naturally occurring antigen-binding fragments.
Fig2. Schematic diagrams of VHH and nADC structures.
(a) Schematic diagrams of HCAb, VHH, and nADC.
(b) Schematic diagram of the VHH structure and molecular 3D structure, showing the complementarity-determining regions (CDR) known to be responsible for antigen recognition.
VHH share many properties with mAb, allowing them to be conjugated to payloads. However, their simpler structure facilitates genetic modification without loss of affinity. Compared to mAb, VHH has a significant advantage in the generation of targeted ADC (Table 1).
mAb | VHH | |
Size | -14.5nm | 2.5~4nm |
Molecular weight | 150 kDa | 12~17kDa |
Antibody production | Requires mammalian cell post-translational modification | Mammalian or microbial, naked and without post-translational modification |
Immunogenicity and complexity | Highly glycosylated and interacts with immune cells via Fc/FcR | Low, no Fc/FcR interaction |
Stability | Highly dependent on pH and temperature. Aggregates with other proteins | Broad pH range, extremely high chemical andthermal stability. Low aggregation |
Clearance rate | Long liver half-life | Renal, relatively short half-life |
Tissue penetration | Low | High tissue permeability, crosses the BBB |
Epitope recognition | Difficult to recognize hidden epitopes | Potent, recognizes epitopes inaccessible tonormal antibodies |
Production cost and standardization | High | Relatively low |
Humanization and structural engineering | Potential for loss of function or stability | Easy to modify |
Affinity | nM-uM | pM-nM |
Table 1. Main differences between mAb and VHH
VHH can also be conjugated using lysine, cysteine, or site-specific conjugation. Compared to mAb, VHH is rich in amino acids such as lysine, aspartic acid, and glutamic acid, allowing for higher DARs through conjugation of these residues. The VHH structure and lack of complex post-translational modifications offer advantages over IgG antibodies in introducing non-natural amino acids or cysteines for site-specific conjugation. Site-specific conjugation is preferred because the presence of lysines in the CDR binding site can reduce affinity if conjugated via these sites. Introducing additional cysteines at positions distal to the antigen-binding site, preferably at the C-terminus, can partially address these issues.
In the clinical setting, unconjugated antibodies are generally well tolerated, allowing high-dose administration to target receptors on cell layers closer to blood vessels and penetrate deeper into tumor tissue. VHH reduces the size of the conjugate while maintaining affinity and specificity, helping it to enter solid tumors through blood vessels and significantly enhance its therapeutic effect. For example, in a patient-derived organoid (PDO) model, a comparative study of an anti-5T4 VHH-SN38 and a traditional anti-5T4-SN38 found that VHH exhibited superior penetration and greater tumor regression.
Furthermore, unlike mAb that may experience prolonged circulation time due to FcRn interaction, VHH do not interact with FcRn, ensuring their trafficking to lysosomes is unaffected, a property that can lead to more efficient release of the payload after lysosomal degradation.
Delivering biologics to the central nervous system (CNS) presents significant challenges, primarily due to the multiple barriers between the CNS and the peripheral environment, including the blood-brain barrier (BBB), the blood-tumor barrier (BTB), and the binding site barrier (BSB). These barriers are highly selective and regulated, tightly controlling the exchange of substances between the blood and brain parenchyma. This challenge is particularly daunting for antibody-drug conjugates (ADC), as the mAb payloads they carry, modified in structure, size, and interaction with endothelial cells, may prematurely release the drug before reaching the target tumor, increasing the risk of neurotoxicity and leading to serious complications. In contrast, due to ability of VHH to penetrate tumors more rapidly than traditional antibodies and their stability in acidic environments, are ideal delivery vehicles for traversing these barriers and effectively reaching central tumor areas.
Fig 3. Transport of ADC and nADC across the BBB and BSB. The BBB restricts entry of substances from the bloodstream into the brain parenchyma, while the BSB restricts penetration of antibodies (mAb) and VHHs into tumors, resulting in uneven distribution of ADC and nADC. Compared with mAb-based ADC, nADC exhibit enhanced ability to cross the BBB and BSB, facilitating more efficient delivery to brain and tumor tissues. TEM: tumor microenvironment.
Although no VHH-based ADCs have entered clinical trials yet, preclinical research is rapidly expanding.
Common Payloads in nADC Research
● Doxorubicin
● MMAE (Monomethyl auristatin E)
● Cisplatin
● SN38
These studies demonstrate the feasibility of direct nanobody-drug conjugation using diverse linker chemistries, showing encouraging anti-tumor activity.
VHH | Target | Payload | Cancer/Cell Line Models | Linker | Method of Conjugation |
Anti-CD22-VHHs | CD22 | DMI | Lymphoma | Succinimidyltrans-4-maleimidylmethylcyclohexane-1-carboxylate(SMCC) | Maleimide |
n501-SN38 | Oncofetal antigen 5T4 | SN38 | Solid tumor (Pancreas,Breast, Ovarian, Colon) | CIA2 | Maleimide |
B9-S84C | CEACAM5 | Maytansinoid DM4 | Solid tumor (Pancreas) | MC-VC-PAB | Maleimide |
Nb 11-1 | CD147 | Doxorrubicine | CD147-positive tumors | - | Maleimide |
VH1-HLE, VH2-VH1,VH2-VH1-HLE, and J591 | PSMA | DNA-alkylating agent(DGN549) indolinobenzodiazepineDNA-alkylating monoimine | Prostate cancer CWR22Rv1DU145 and DU145-PSMAcell lines | - | Maleimide |
NB7 | PSMA | Doxorrubicine | Prostate cancerPC3-PIP and PC3-flu | pH-sensitive linkerN-(B-maleimidopropionicacid) hydrazide (BMPH) | Maleimide |
VHH7 | aMHC-II | DMI | Lymphoma | - | Sortase-mediatedsite-specific proteinengineering |
HuNbTROP2-HSA | TROP2 | MMAE | Pancreatic cancer | MC-VC-PAB | Maleimide |
VH-Fc3C9 | Mesothelin | MMAE | Solid tumor | VC-PAB | Maleimide |
Tetravalent biparatopicanti-EGFR VHH-drug | EGFR | MMAE | Solid tumor | MC-VC-PAB | Maleimide |
2Rs15d | HER2 | Duocarmycin | HER2 positive tumor | Compound S22 Syntheticduocarmycin linkedto Psyche | VHH fused to Cupid proteinPsyche-duocarmycin |
PEGylated-antiEGFRVHH | BGFR | Pt(IV) (prodrug ofoxaliplatin) | EGFR positive cell lines | Mal-Pi(IV) | Transglutaminase (mTGase)mediated ligation |
11A4 | HER2 | Auristatin F (AF) | - | platinum-based Lx linker | Maleimide |
VHH-conjugated H40-PEG | VEGFR2 | Methotrexate | HEK293 (human embryonic Kidney cells) Breast cancer KDR293 (overexpressed for VEGFR2 receptors) | NHS/EDC | Random lysines |
scPDL1-DM1 | PDL1 | DM1 | PDL1 positive cells | Succinimidy1 trans-4-maleimidy1methy1 cyclohexane-1-carboxylate(SMCC) | Maleimide |
N,7D12-9G8 | EGFR | Cisplatin | A375, A431, Solid tumors | Mal-pt | Maleimide |
Single-chain anti-HER2 | HER2 | Doxorubicine | BT474-M3, NCI-N87 | N-[α-(2-[N-maleimido]propyonylamido)-PEG-omega-oxycarbonyl]-DSPE | Maleimide |
Table 2. Preclinical studies of VHH-drug conjugates
A critical step in ADC efficacy is antibody internalization-the process by which the antibody-antigen complex is taken up into tumor cells and trafficked to lysosomes for drug release.
As a leader in VHH technology, AlpVHHs is dedicated to providing high-quality alpaca-derived single-domain antibodies and cutting-edge research tools. From custom VHH discovery to our Antibody Internalization reagent, we empower biopharmaceutical companies to build the next generation of life-saving nADCs.
AlpVHHs offers a series of VHH (nanobody)-based nano-secondary antibody products specifically designed for evaluating antibody internalization efficiency during the early stage of ADC drug development. This series is particularly suitable for:
● High-throughput screening of antibody internalization efficiency prior to ADC construction
● Rapid functional evaluation and comparison of multiple antibody candidates
● Early-stage ADC drugability assessment in cell-based systems
All products are based on VHH nano-secondary technology, ensuring consistent secondary recognition and reliable assay performance, making them ideal tools for ADC pre-screening workflows.
● Real-time monitoring of antibody internalization
● Quantitative analysis of uptake efficiency
● Compatible with VHH, mAb, and ADC formats
● High sensitivity and reproducibility
● Suitable for high-throughput screening
Before investing in ADC synthesis, it is critical to determine whether candidate antibodies possess sufficient internalization capability. The AlpVHHs Nano-secondary series enables:
● Functional evaluation of antibody internalization
● Rapid comparison of multiple antibody candidates
● High-throughput screening in cell-based systems
● Early-stage ADC drugability assessment
By integrating internalization assays into the workflow, researchers can bridge the gap between binding affinity and functional efficacy, ensuring that candidate antibodies are not only specific-but also therapeutically effective.
Nano-secondary antibody-Drug conjugates
Code | Description | Application |
023-101-101 | Internalization Test | |
023-101-102 | Internalization Test | |
023-101-103 | Internalization Test | |
023-101-113 | Internalization Test |
Code | Description | Application |
001-101-020 | Internalization Test | |
023-101-020 | Internalization Test |
Code | Description | Application |
001-101-014 | Internalization Test | |
023-101-014 | Internalization Test |
