2.2. Strategy for Finding Information

The structured inquiry included pertinent research studies released from 1 January 2014 to 30 June 2024). This specific period was chosen to capture the most recent and relevant developments in the field of ADCs and VHHs. Although the first ADC was approved by the FDA in 2000 (gemtuzumab ozogamicin), the field has significantly evolved since then. Additionally, the first single-domain antibody (caplacizumab) was FDA-approved in 2019. By selecting a period starting from five years prior to the approval of caplacizumab, we aimed to include contemporary studies and advancements that reflect the current state of research and therapeutic applications.

The databases used for the search were PubMed, Cochrane Library, ScienceDirect and LILACS. The search criteria included a mix of medical subject headings (MeSH) and keywords related to ADC and nanoantibodies, such as “nanobody conjugates”, “single-domain antibody conjugates”, “drug-bound VHHs”, “small format antibody-drug conjugate”, “scFv”, “single-chain variable fragment”, “single-domain antibody-drug conjugates”, “fully human single-domain antibody-drug conjugates”, “sdAb”, “NDC”, “small ADC”, “functional heavy (H)-chain antibody”, and “HCAb”. The terms were combined effectively for the search strategy using the Boolean operators “AND” and “OR”. Articles that were published in either English or Spanish and can be fully accessed online were considered. Research covered both clinical and preclinical studies.

2.3. Evaluation and Selection

The first search produced a detailed collection of articles, which were later refined using automated tools to exclude irrelevant records according to the inclusion criteria (Figure 1). After eliminating duplicates, three authors (VMMP, MB, AJS) individually reviewed the titles, abstracts, and full texts of the remaining articles. Additionally, other relevant studies selected by the team outside the specified time range were also incorporated. Full-text reviews were carried out for articles that met the inclusion criteria, and any discrepancies were resolved through consensus-based discussions.

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Figure 1

Strategy for identification, screening and selection of articles for systematic review.

2.4. Determination of Quality

The articles were included in the final analysis after evaluating their quality of evidence. Only peer-reviewed journal articles with rigorous methodologies were reviewed, such as randomized controlled trials and cohort studies, to ensure high-quality evidence synthesis. Case reports, editor letters, and conference studies were excluded, because they typically do not provide the level of rigor and comprehensive data needed for a systematic review. The chosen articles examined different facets of advancements in target VHHs, conjugation methods, payloads, and future prospects in the field of ADCs.

3.1. From Antibody to ADC

3.1.3. Mechanism of Action of Conjugated Antibodies

Pharmacologically, the action of an ADC can be outlined in four steps: systemic circulation, the Enhanced Permeability and Retention (EPR) effect including passive targeting, penetration within the tumor tissue, and action on cells, which encompasses active targeting and controlled release. The canonical model for the mechanism of action of ADCs can be divided into several stages: the binding of the mAb to the target antigen, the internalization of the molecule, and finally, the cleavage of the linker with the release of the cytostatic payload [50,79,80,81] (Figure 3a–c). After antigen binding on the cell surface, the ADC is internalized by the tumor cell and endocytosed to form an early endosome. Here, the mAb binds to the targeted antigens uniquely expressed in cancer cells via one of three main pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, or pinocytosis. The latter is antigen-independent, whereas the first two are antigen-dependent [6,80,81,82,83]. The antigen–ADC complex is then internalized via receptor-mediated endocytosis and trafficked into the lysosome (Figure 3b). Here, the ADC is processed according to the physicochemical properties of the linker, and releases the cytotoxic warhead. Once in the cytoplasm, the released drug ultimately triggers cell death or apoptosis, generally targeting DNA or tubulin. Lipophilic drugs can diffuse from ADC target cells to neighboring cells, killing them independently of their target expression, a mechanism known as the bystander effect (Figure 3d). The antibody can either activate or inhibit target receptors. Designed to specifically recognize and bind to antigens on certain cells, such as tumor cells, ADCs can exert agonistic or antagonistic effects upon binding to their cell surface receptors. This interaction can alter intracellular pathways, potentially inhibiting cell growth or metabolism depending on the design and cellular context (Figure 3e). This effect not only increases the cytotoxicity of ADCs, but also makes it possible to target tumors with heterogeneous antigen expression, increasing the patient population that could benefit [6,22,29,81,82,83,84,85,86]. In addition to the payload-induced killing mechanisms, classical antibody functions, such as the inhibition of the downstream signaling pathways of the target receptor through the Fab region, or Fc-mediated killing mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) (Figure 3f) and complement-dependent cytotoxicity (CDC) (Figure 3g), as well as antibody-dependent phagocytosis (ADCP) (Figure 3h), directly involve innate or complementary immune effectors [6,56,85,87,88].

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Figure 3

ADC mechanism of action. (a) Passive payload diffusion. The drug that has been released from the antibody before binding to the antigen passively diffuses into the surrounding cells. (b) Targeted drug-delivery. The ADC binds to the antigen in the cell and is internalized by endocytosis. Once the conjugated antibody binds to target cells, it can be internalized via endocytosis, carrying along the payload. (c) It is degraded in lysosomes, releasing the drug that carries out its intracellular action. Once bound to the target cells or inside the cell, the conjugate can release its payload (e.g., a cytotoxic drug). This may lead to cell death. (d) Diffusion through the plasma membrane with action on surrounding cells. (e) Antagonism or agonism of the target receptors. The antibody is designed to specifically recognize and bind to the antigens present on the surface of certain types of cells, such as tumor cells. Antibody–drug conjugates can have agonistic or antagonistic effects when binding to their specific receptor on the cell surface, allowing them to modify intracellular pathways and potentially inhibit cell growth or metabolism, depending on their design and specific cellular context. (f) Some antibody–drug conjugates are engineered not only to directly target specific cells by binding to their surface receptors but also to harness the immune system for enhanced cell destruction. This can occur through mechanisms such as (g) antibody-dependent cell-mediated cytotoxicity (ADCC), where immune cells like Natural Killer (NK) are activated to recognize and kill the targeted cells. Additionally, antibody-dependent cellular phagocytosis (ADCP) involves immune cells engulfing and digesting the marked cells, further contributing to their elimination. (h) Complement-dependent cytotoxicity (CDC) is another mechanism employed, where the conjugates activate the complement system to induce cell lysis. These strategies collectively bolster the therapeutic efficacy of antibody–drug conjugates by leveraging immune responses to eliminate target cells more effectively.

However, while these Fc-mediated processes potentially enhance the ADCs’ antitumor effect, they may also adversely affect their safety profile by increasing healthy tissue exposure through nonspecific drug diffusion, Fc-mediated uptake by immune cells, or recycling via neonatal Fc receptors (FcRn) [89,90,91,92,93,94,95,96,97,98,99,100]. Additional factors influencing antibody clearance include the mononuclear phagocyte system and FcRn-mediated recycling. FcRn binds to ADCs within the endocytic vacuole and facilitates their export to the extracellular compartment for recycling [89,90,91,92,93,94,95,96,97,98,99,100].

3.2. ADCs Evolution

4.1. VHH’s Physical, Chemical and Structural Properties

The crystal structure of the VHH domain revealed dimensions of 4 nm × 2.5 nm × 3 nm. VHH domains have been found to be highly soluble and more stable than conventional antibodies. They can be stored at 4 °C or −20 °C for months without significantly losing their antigen-binding capacity. Furthermore, the homology between the VHH and VH domains of the human Ig family VH III was found to be greater than 80%, suggesting that the VHH sequence may induce a mild immunogenic response when used in cancer immunotherapy [111,116].

VHH domains can endure harsh conditions, such as a wide pH range (3–9) and extreme chemical (e.g., 6–8 M urea concentration) and thermal denaturing conditions (e.g., maintaining antigen-binding activity after prolonged incubation at 90 °C). This robustness allows for various administration routes, such as intravenous, oral or intraperitoneal [117,118]. Notably, VHH domains possess a fully hydrophilic surface, enhancing their stability and solubility compared to IgG VH domains, and exhibit significantly less aggregation during production or multimerization (e.g., tandem VHH-based multispecific antibodies). The CDR3 loop in camelid VHH domains is typically longer (3–28 amino acids) than in the conventional VH domains of human IgG (8–15 amino acids). This extended CDR3, which determines recognition specificity, increases the potential interaction surface with a target antigen in the absence of a VL domain (Figure 4b). Interestingly, the longer CDR3 in VHH domains can form a finger-like appendage that fits into a protein cleft, enabling the recognition of epitopes that are inaccessible to larger antibodies such as mAbs. However, the small size of the VHH domain results in rapid renal clearance (half-life ~2 h), which is a significant disadvantage for their application in cancer treatment [111,112,113,114,115,116,119,120]. On the other hand, the VHH fragment of the HCAb in serum shows a unique thermo-reversible stability profile. VHHs can withstand high temperatures due to their ability to refold after heat denaturation [121,122,123,124,125,126]. Furthermore, VHHs remain stable under extreme pH conditions, preserving their bioactivity in the stomach or intestine. This stability allows for the design of treatments using various administration methods, including intravenous injection, inhalation, and oral and intranasal delivery. Overall, the stable biochemical and biophysical properties of VHH support their expanding applications in various therapeutic areas [20,121,127,128,129,130].

4.2. VHH’s Biological Functions

VHHs have exceptional properties, including their small size and ability to penetrate tumors in vivo, which enhance their tumor-targeting capabilities. Unlike conventional antibodies, VHHs retain the same antigen-binding characteristics as a single immunoglobulin variable domain for antigen recognition [120]. Furthermore, VHHs can access epitopes that conventional antibodies cannot, such as clefts on a protein’s surface [131]. Uniquely, the strict monomeric state of VHHs facilitates independent antigen recognition and binding. Another critical difference between the VHH domain of camelid antibodies and the VH domain of conventional antibodies is found in the FR2 fragment. X-ray crystallography analysis of the VHH protein–antigen complex has shown that hydrophobic amino acids in the VHH FR2 are replaced by hydrophilic amino acids in the FR2 of conventional antibodies. Specifically, the amino acids Phe-42, Glu-49, Arg-50, and Gly-52 in the conventional VH-VL cross-linking FR2 are substituted with Val, Gly, Leu, and Trp, respectively [113,132,133,134,135,136,137]. Additionally, the single-variable domains of VHHs can readily form concave surfaces that function as active sites or receptor-binding pockets. While the extension of the HV loop increases flexibility, it can compromise the stability of the VHH domain’s internal structure. To counteract the flexibility introduced by the longer H3 loop, an extra interloop disulfide bond between the H1 and H3 loops reinforces the extended HV loop structure, thereby enhancing antigen binding [138,139]. Additionally, the CDR3 region in VHHs can form an exposed ring structure, acting like a ‘finger’ that inserts into an antigen’s ‘pocket’, unlike conventional antibodies that usually interact with flat surfaces. An extra cysteine residue in CDR3 can also form a disulfide bond with an additional cysteine residue in either CDR1 or the framework region 2 (FR2), which enhances the stability of the VHH structure and lowers the energy required for antigen binding [140,141].

The rate of passive diffusion of a molecule in tissue is inversely proportional to its molecular size. Consequently, monovalent VHHs (12–17 kDa) exhibit faster vascular permeability and better tissue penetration compared to conventional antibodies (~150 kDa) enabling VHHs to achieve a more homogeneous distribution, such as in solid tumors. As detailed in “Section VHHs penetration and transport through barriers”, some VHHs have demonstrated the ability to cross the blood–brain barrier (BBB), offering enhanced potential for the diagnosis and treatment of brain cancer, particularly in cases where the BBB is disrupted [142,143,144,145,146,147,148]. A research study indicates that ¹⁷⁷Lu-labeled anti-CD20 VHHs remain stable in human serum, with over 91% of the complexes intact 144 h post injection [149]. While they are stable, the low molecular weight of VHHs leads to rapid renal clearance. However, VHHs remain bound to the antigen for an extended period [150]. Supporting this, several studies have demonstrated that ¹¹¹In-labeled anti-HER2 VHHs exhibit high specific uptake in HER2-positive brain tumors from 1 h to 3 d after injection. In contrast, ¹¹¹In-labeled mAb Trastuzumab shows high nonspecific uptake in highly vascularized organs, such as the heart, spleen, and liver [147].

CDR3 regions play a crucial role in antigen binding by forming prominent grooves on their surface, while other sections of CDR3 can penetrate deeply into the active site of a lysozyme complex. These distinctive characteristics of the VHH fragment contribute to its higher affinity, solubility, and anti-aggregation properties [141,151,152,153,154].

4.3. Characteristics of VHHs to Develop Novel ADCs

Since their discovery, over 100 VHHs have been isolated, targeting areas relevant to oncology, in vivo imaging, hematology, and infectious diseases, as well as neurological and inflammatory disorders. VHHs are particularly well-suited for these applications because of their small size, target specificity, and long CDR3 loops, which help overcome many of the limitations associated with small-molecule synthetic drugs, such as limited specificity and off-target toxicity. These unique features of VHHs, compared to mAbs, present opportunities for developing VHH drug conjugates (nADCs or “nanoADCs”) with distinct pharmacological benefits (Table 2 and Table S1). nADCs can potentially rival traditional ADCs due to their superior solid tumor penetration, enhanced stability, and their ability to significantly inhibit cancer cell growth [120,131,132,155,156]. A distinctive capability of VHHs is their ability to target epitopes in hard-to-reach locations that larger molecules, such as conventional mAbs, often cannot access. VHHs can bind in a ring-like fashion, allowing them to recognize epitopes typically inaccessible to standard antibodies, such as ion channel domains and intracellular proteins [113,120,133,134,135,136,137].

In summary, VHHs possess several ideal characteristics for drug conjugation, including high thermal and chemical stability, excellent solubility and strict monomeric behavior. Their small size (approximately 2.5 nm in diameter and 4 nm in length, M_W: ~12–17 kDa) facilitates a better tissue penetration, while their relatively low production cost, ease of modification by genetic engineering means, format flexibility, low immunogenicity, and modularity further enhance their suitability for therapeutic applications [132,138,139].

4.4. The Plasticity of the VHH and the Opportunities for Conjugation

VHHs share many characteristics of mAbs that permit their conjugation to payloads, but their simpler structure facilitates genetic modification without losing their affinity. Conjugation can be performed using traditional protocols for lysine, cysteine or site-directed conjugation. Notably, the surface of VHHs is rich in amino acids such as lysine, aspartic acid, and glutamic acid, allowing for higher DARs (drug-to-VHH ratios) through conjugation at these residues [149,151].

The structure of VHHs and their lack of complex post-translational modifications allows for the introduction of non-natural amino acids or cysteines for site-specific conjugation when compared to IgG antibodies. Site-directed conjugation is preferred, as the presence of lysines in the CDR binding sites may compromise their affinity if a drug is conjugated via these sites. Introducing an additional cysteine at a location distant from the paratope, preferably at the C-terminal, can partly solve these issues. Other effective conjugation alternatives include sortase A, transglutaminase, and GTPase enzymes, although they require scaffold modification to be site-specific [141,151,152,153,154].

In clinical settings, unconjugated antibodies are often well tolerated, allowing for high doses that saturate receptors on the cell layers closer to the blood vessel and enable deeper tumor penetration. However, the payload toxicity of ADCs limits the dosage and frequency of administration, which can restrict tumor penetration, allowing for regrowth between doses (typically administered every three weeks in current therapies). Thus, designing treatment strategies that enhance tumor penetration could lead to greater efficacy and improve clinical success rates for ADCs and other protein–drug conjugates [7,11,162,163]. In addition to binding affinity, antibody size influences tumor penetration. Decreasing the size of a conjugate while maintaining affinity and specificity facilitates its entry into solid tumors through blood vessels, significantly enhancing its therapeutic effect. As an example, comparative studies in patient-derived organoid (PDO) models between an anti-5T4 VHH-SN38 and a conventional anti-5T4-SN38 have demonstrated superior penetration and greater tumor regression by the VHH [164].

Moreover, unlike conventional antibodies which may have prolonged circulation due to interaction with FcRn-mediated receptors, VHHs do not interact with FcRn, ensuring that their transport to lysosomes is not compromised [165,166,167]. This characteristic can lead to more effective payload release following lysosomal degradation. Finally, chemical modifications to ADCs can impair stability, making them more sensitive to changes in the intratumoral environment, leading to instability and the potential loss of interaction with the paratope. VHHs, however, are stable under adverse conditions that help maintain their functional integrity and effectiveness in tumor cell elimination [168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185].

4.5. VHHs as Carriers in Antibody–Drug Conjugates (nADCs)

To date, no single class of antibodies perfectly combines several ideal properties for targeting the moieties of ADCs, including low immunogenicity, rapid distribution, the fast clearance of unbound molecules, and high tumor accumulation. Despite substantial interest in ADC therapies, high failure rates have been observed in the clinical setting [186]. Effective tumor accumulation is critical for the antitumor activity of ADCs, especially against solid tumors, and depends on both tumor penetration/retention and antibody pharmacokinetics (Figure 5). Consequently, smaller recombinant antibody fragments such as VHHs and fragment of antigen binding (Fab) of mAbs have been explored as alternatives to intact mAbs for generating ADCs, but their clinical applications have been limited by poor structural stability. Recently, a class of small, soluble antibodies derived from HCAbs such as VHHs have gained significant attention, due to their minimal molecular size and high thermal stability [186,187,188,189,190,191] (Table S1). Along with their high specificity, their solubility, low immunogenicity, and ease of production make VHHs ideal for constructing “nano-ADC” (nADC).

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Figure 5

Graphical comparison of the properties of a conventional mAb versus VHH, including the vascular diffusion, tumor penetration, renal and hepatic clearance rates, and the stability under storage at a wide range of temperature conditions and stability at different pH.

The small size of VHHs facilitates excellent and rapid tumor penetration in vivo. Additionally, this small size is crucial for our design, as it enables VHHs to bring the nADCs construct very close to the membrane after binding to the surface target [113]. This capability makes nADCs a promising approach for next−generation targeted drug conjugates [172,192,193,194,195,196,197,198,199]. Furthermore, their smaller size and aqueous solubility enable quicker tumor infiltration compared to mAbs. VHHs’ superior specificity arises from their ability to bind to epitopes that conventional mAbs cannot reach. Studies indicate that VHHs achieve higher tumor-to-background ratios in molecular imaging in vivo, due to their precise binding and accumulation in tumors, along with the rapid clearance of unbound constructs, resulting in lower background signals and reduced toxicity. Importantly, VHHs exhibit exceptional versatility, making them ideal for integrating various functional modules and enhancing the clinical potential of nADCs. Overall, VHH−drug conjugates or nADCs are emerging as a promising alternative to conventional ADCs [143,164,192,200,201,202,203,204,205,206,207,208,209,210,211,212].

Building on these advantages, VHHs also demonstrate good performance in imaging applications. Their ability to achieve favorable target-to-noise ratios is evident in research settings where VHHs conjugated with radionuclides are used for cancer imaging. For instance, single-positron emission tomography (SPECT) or positron emission tomography (PET) combined with micro-computed tomography (micro-CT) has effectively utilized VHHs for imaging primary tumors and metastatic sites across various cancer models, including melanoma, breast cancer, ovarian cancer, early pancreatic lesions, and advanced pancreatic ductal adenocarcinoma. PET/CT imaging with VHHs provides excellent clarity and signal-to-noise ratios [150,156,173,213,214,215,216].

The advent of new technologies that increase the bioavailability of VHHs will facilitate their transition into clinical use. The main methods include:

• Reduced glomerular filtration rate due to increased glomerular mass or hydrodynamic radius. Strategies such as combining VHHs with nanoparticles and liposomes, or modifying them with polyethylene glycol (PEG), are proving effective in enhancing drug delivery to cancer cells. These approaches improve penetration into solid tumors and reduce systemic toxicity [217,218,219,220,221].

• Binding to plasma proteins with extended half-life. To extend the half-life of VHHs and improve their efficacy, methods are being developed to optimize their affinity and release dynamics. Advances include fusing VHHs with human serum albumin (HSA) or the Fc domain. Other strategies include the “fenobody” platform developed by Kelong Fan and colleagues, where VHHs targeting the H5N1 virus were displayed on a 24-subunit ferritin oligomer. By replacing ferritin’s fifth helix with the VHH, affinity and half-life were significantly improved, offering substantial advantages for large-scale biotechnological applications and promoting the broader adoption of VHH technology [220,222,223,224,225,226].

• Structural and design modifications. Enhancing VHH efficacy against tumor antigens involves techniques such as forming CDR rings, stabilizing secondary structures, or creating bispecific and multispecific VHHs to improve affinity, specificity, stability, and solubility in challenging physiological environments [227,228,229,230,231,232].

4.6. Advances in the Development of nADCs

To date, no VHH–drug conjugates have entered clinical research, and all published studies remain at the preclinical stage. These studies have explored the direct conjugation of the nanoantibody to drugs using multiple forms and linkers, and in some cases, VHHs have been used in conjunction with other formulations to deliver the drug. The main conjugated payloads include doxorubicin, MMAE, cisplatin, and SN38 (Table 3). While nADCs show promise in terms of therapeutic response, a thorough analysis suggests that traditional cytotoxic agents may not be the most suitable choice for advancing their clinical development. Matching the right payload with the appropriate linker and antibody formats is crucial for optimizing nADC efficacy. More potent payloads, such as topoisomerase I inhibitors, could enhance tumor penetrability. Although antimicrotubule inhibitors have shown promising activity in preclinical studies with lymphomas and leukemias, their effectiveness is generally lower in solid tumors compared to antitopoisomerase inhibitors. New payloads, such as pyrrolobenzodiazepines, may be the most suitable for nADCs due to their high potency. Regarding the limitations caused by toxicity [233], combining PBDs with VHHs might reduce toxicity, given VHHs’ shorter plasma retention time. Furthermore, the enhanced penetrability of VHHs in solid tumors compared to conventional antibodies could help maintain a strong antitumor response.

4.7. Disadvantages of VHH for nADCs and Possible Improvements

The authors thank the Molecular Oncology Lab members at IIS Aragón for their critical reading of the manuscript. The contribution of A.J.S. to the review honors the memory of Mª Victoria Arruga.