Solid tumors have long been recognized as an intractable therapeutic barrier for antibody-based therapeutics due to their aberrant stromal density, compromised tissue permeability, and profoundly immunosuppressive tumor microenvironment (TME). Despite the landmark clinical success of conventional monoclonal antibodies (mAbs) in oncology, their large molecular weight (approximately 150 kDa) severely restricts deep tumor penetration, while Fc-domain-mediated non-specific effector functions frequently induce off-target adverse effects and systemic toxicity. As a unique class of single-domain antibodies (sdAbs) derived from camelids and cartilaginous fish, nanobodies (Nbs) exhibit distinct superiorities, including an ultra-small molecular size (12–15 kDa, one-tenth that of conventional mAbs), high antigen affinity, exceptional physicochemical stability, and robust engineering feasibility. These intrinsic properties render Nbs a transformative modality for precision solid tumor therapy.
This review systematically elaborates on the core immunogenicity bottlenecks restricting the clinical translation of Nbs and summarizes state-of-the-art advances in four mainstream Nb-based therapeutic strategies for solid tumor intervention.
1. Mitigation of Zoonotic Immunogenicity: Humanization Strategies for Nanobodies
The zoonotic origin of native Nbs renders them susceptible to immune recognition and rejection in humans. Conjugation with chemical cytotoxins or functional polypeptides further exacerbates immunogenic risks, constituting a major obstacle to long-term in vivo administration and clinical popularization. Building on mature humanization protocols for murine mAbs, current strategies to deimmunize zoonotic Nbs primarily comprise two technical frameworks:
CDR Grafting. This strategy precisely grafts the antigen-specific complementarity-determining regions (CDRs) of xenogeneic Nbs onto the framework regions (FRs) of human immunoglobulin scaffolds. In a representative study, Vaneycken et al. grafted the CDRs of carcinoembryonic antigen (CEA)-targeted NbCEA5 onto a universal human antibody framework. Although surface plasmon resonance (SPR) detection revealed a 30-fold reduction in binding affinity relative to the wild-type Nb, competitive binding assays verified complete retention of the original antigen epitope specificity, and subsequent affinity maturation can effectively restore and optimize its binding potency.
Surface Resurfacing. Based on homologous sequence alignment and structural homology modeling, this approach replaces surface-exposed immunodominant amino acid residues in xenogeneic FRs with corresponding human FR residues. Given that surface residual epitopes serve as the primary targets of host immune surveillance, this modification enables effective immune camouflage of xenogeneic Nbs. Kazemi-Lomedasht et al. performed computer-aided site-directed mutagenesis on nine key surface residues of an anti-VEGF Nb. The humanized Nb43 variant retained equivalent antigen binding affinity validated by enzyme-linked immunosorbent assay (ELISA) and preserved potent inhibitory activity against vascular endothelial cell proliferation.
2. Zero-Immunogenicity Optimization: Development of Fully Human Nanobodies
Fully human Nbs, with entire amino acid sequences derived from human endogenous immunoglobulin repertoires, eliminate xenogeneic immunogenicity fundamentally, representing the optimal safe scaffold for long-term clinical therapy. Considering the absence of natural heavy-chain-only antibodies in the human immune system, the development of fully human Nbs relies on four cutting-edge technical platforms:
Phage Display Technology. As the most mature and widely adopted platform, phage display has facilitated the development of multiple blockbuster antibody drugs (e.g., adalimumab). High-affinity fully human Nb clones can be efficiently panned and screened from high-capacity naïve or synthetic fully human Nb libraries.
Transgenic Mouse Models. Endogenous murine immunoglobulin genes are knocked out and replaced with human immunoglobulin gene clusters, enabling transgenic mice to directly generate fully human antigen-specific Nbs upon targeted immunization.
High-Throughput Single B-Cell Screening. Integrated with microfluidic sorting and microarray chip technologies, this platform enables rapid isolation and identification of antigen-specific Nb clones from single B cells of immunized individuals or convalescent patients.
AI-Guided Computational Design. Emerging deep learning and structural modeling tools (e.g., AlphaFold) have revolutionized antibody engineering. AI-assisted rational design enables precise optimization of Nb structure, driving the intelligent and high-efficiency development of fully human Nbs.
Native human single-domain scaffolds often suffer from insufficient structural stability and poor solubility. To address these defects, multiple optimization strategies have been developed, including the introduction of additional disulfide bonds to enhance conformational rigidity, library panning under extreme denaturing conditions to screen stable variants, and CDR transplantation of high-affinity functional domains onto hyper-stable human scaffolds, thereby balancing therapeutic efficacy and favorable pharmacokinetic (PK) profiles.
3. Multimodal Therapeutic Applications of Nanobodies in Solid Tumors
Solid tumor progression is driven by oncogenic signaling pathway dysregulation and TME remodeling, which collectively construct an immunosuppressive and physically restrictive microenvironment. Effective tumor intervention requires multi-dimensional and synergistic therapeutic strategies. Through genetic engineering and functional modular assembly, antitumor Nbs have evolved into four core therapeutic formats for solid tumor treatment:
3.1 Targeted Cytotoxic Therapy: Nanobody-Drug Conjugates (NDCs)
Conventional antibody-drug conjugates (ADCs) are limited by their large molecular size, which impedes penetration through the dense stroma of solid tumors. NDCs, composed of high-affinity Nb targeting moieties, cleavable linkers, and cytotoxic payloads, have emerged as a superior alternative. NDCs can precisely recognize cryptic tumor-specific epitopes, achieve targeted drug delivery, and minimize off-target toxicities.
Chemotherapeutic and Immunotoxin Conjugates. Platinum-based agents and doxorubicin are the most commonly utilized small-molecule payloads. For instance, anti-EGFR Nb-platinum conjugates can selectively induce apoptotic cell death in EGFR-overexpressing tumor cells. Immunotoxin fusion proteins, constructed by fusing Nb targeting domains with bacterial or fungal cytotoxins, exert potent and specific tumoricidal effects.
Photodynamic Therapy (PDT). Nbs conjugated with photosensitizers (e.g., IRDye700DX) can generate massive reactive oxygen species (ROS) under specific wavelength irradiation, enabling precise ablation of EGFR/HER2-positive solid tumors. Notably, this modality can overcome trastuzumab resistance in HER2-positive malignancies.
Targeted Radionuclide Therapy (TRT). Nbs labeled with α- or β-emitting radionuclides (e.g., Lu, Ac) achieve precise targeted radiotherapy. I-labeled anti-HER2 Nbs significantly prolong the survival of tumor-bearing models, while Ac-labeled Nbs induce melanoma regression and upregulate the expression of antitumor cytokines such as IFN-γ. Additionally, Tc-labeled diagnostic Nb conjugates have entered mid-stage clinical trials for accurate tumor staging and metastatic lesion detection.
Notably, the small molecular size of NDCs leads to rapid renal clearance, which may cause renal reabsorption and potential nephrotoxicity. Rational linker design is critical to balance in vivo structural stability and stimuli-responsive cleavability for optimized safety and efficacy.
Nanoparticle (NP) carriers (diameter < 200 nm), including liposomes, micelles, polymeric NPs, and albumin-based NPs, can significantly optimize the PK behavior and tumor targeting efficiency of therapeutic drugs. Without Fc domains and with ultra-small size, Nbs can be densely modified on NP surfaces, protecting encapsulated cargo from enzymatic degradation, enhancing intratumoral drug accumulation, and reducing systemic immune adverse reactions.
