Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells
is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of ...a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused
a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration
. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors
. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.
NANOBODY® (a registered trademark of Ablynx N.V) molecules (Nbs), also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the
...family. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, nanobodies possess the potential to overcome several of the limitations of conventional monoclonal antibodies. For many years, nanobodies have been of great interest in a wide variety of research fields, particularly in the diagnosis and treatment of diseases. This culminated in the approval of the world's first nanobody based drug (Caplacizumab) in 2018 with others following soon thereafter. This review will provide an overview, with examples, of (i) the structure and advantages of nanobodies compared to conventional monoclonal antibodies, (ii) methods used to generate and produce antigen-specific nanobodies, (iii) applications for diagnostics, and (iv) ongoing clinical trials for nanobody therapeutics as well as promising candidates for clinical development.
To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor ...cells.
Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjμgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc.
One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 μg/mL.
The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.
•One anti-LAG3 nanobody (Nb) sequence was successfully screened.•The specific recombinant anti-LAG3-Nb-Fc was expressed and purified successfully.•The recombinant anti-LAG3-Nb-Fc can promote the killing ability of T cells against tumor cells.
BACKGROUNDInhalable biologics represent a promising approach to improve the efficacy and safety of asthma treatment. Although several monoclonal antibodies (mAbs) targeting IL-4Rα have been approved ...or are undergoing clinical trials, the development of inhalable mAbs targeting IL-4Rα presents significant challenges.OBJECTIVECapitalizing on the distinctive advantages of nanobodies (Nbs) in maintaining efficacy during storage and administration, we sought to develop a novel inhalable IL-4Rα Nb for effectively treating asthma.METHODSThree IL-4Rα immunized Nb libraries were utilized to generate specific and functional IL-4Rα Nbs. LQ036, a bivalent Nb comprising two HuNb103 units, was constructed with a high affinity and specificity for hIL-4Rα. The efficacy, pharmacokinetic and safety of inhaled LQ036 were evaluated in B-hIL4/hIL4Ra humanized mice.RESULTSLQ036 inhibited secreted embryonic alkaline phosphatase (SEAP) reporter activity, TF-1 cell proliferation, and suppressed pSTAT6 in T cells from asthma patients. Crystal structure analysis revealed a binding region similar to Dupilumab but with higher affinity, leading to better efficacy in blocking the signaling pathway. HuNb103 competed with IL-4 and IL-13 for IL-4Rα binding. Additionally, LQ036 significantly inhibited OVA-specific IgE levels in serum, CCL17 levels in BALF, bronchial mucous cell hyperplasia, and airway goblet cell hyperplasia in B-hIL4/hIL4Ra humanized mice. Inhaled LQ036 exhibited favorable pharmacokinetics, safety and tissue distribution, with higher concentrations observed in the lungs and bronchi.CONCLUSIONThese findings from preclinical studies establish the safety and efficacy of inhaled LQ036, underscoring its potential as a pioneering inhalable biologic therapy for asthma.
Nanobodies are single‐domain fragments of antibodies with comparable specificity and affinity to antibodies. They are emerging as versatile tools in biology due to their relatively small size. Here, ...we report the crystal structure of a specific nanobody Nbα‐syn01, bound to a 14 amino acid long peptide of α‐synuclein (αSyn), a 140‐residue protein whose aggregation is associated with Parkinson's disease. The complex structure exhibits a unique binding pattern where the αSyn peptide replaces the N‐terminal region of nanobody. Recognition is mediated principally by extended main chain interaction of the αSyn peptide and specificity of the interaction lies in the central 48–52 region of αSyn peptide. Structure‐guided truncation of Nbα‐syn01 shows tighter binding to αSyn peptide and improved inhibition of α‐synuclein aggregation. The structure of the truncated complex was subsequently determined and was indistinguishable to full length complex as the full‐length form had no visible electron density for the N‐terminal end. These findings reveal the molecular basis for a previously unobserved binding mode for nanobody recognition of α‐synuclein, providing an explanation for the enhanced binding, and potential for an alternate framework for structure‐based protein engineering of nanobodies to develop better diagnostic and therapeutic tools.
Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities ...for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.
Display omitted
•Comprehensive structural characterization of nanobody–antigen complexes.•Nanobodies preferentially recognize structured, rigid, concave and conserved epitopes.•Antigen binding is assisted by residues from nanobody framework regions (“non-CDR”).•Hydrophobic nature of nanobody–antigen interactions•Nanobody–antigen interaction is in some respects more similar to general PPI than to antibody–antigen interaction.
Background/Objectives
Nanobodies are the smallest biologic antigen‐binding fragments derived from camelid‐derived antibodies. Nanobodies effect a peak tumor signal within minutes of injection and ...present a novel opportunity for fluorescence‐guided surgery (FGS). The present study demonstrates the efficacy of an anti‐CEA nanobody conjugated to near‐infrared fluorophore LICOR‐IRDye800CW for rapid intraoperative tumor labeling of colon cancer.
Methods
LS174T human colon cancer cells or fragments of patient‐derived colon cancer were implanted subcutaneously or orthotopically in nude mice. Anti‐CEA nanobodies were conjugated with IRDye800CW and 1–3 nmol were injected intravenously. Mice were serially imaged over time. Peak fluorescence signal and tumor‐to‐background ratio (TBR) were recorded.
Results
Colon cancer tumors were detectable using fluorescent anti‐CEA nanobody within 5 min of injection at all three doses. Maximal fluorescence intensity was observed within 15 min–3 h for all three doses with TBR values ranging from 1.3 to 2.3. In the patient‐derived model of colon cancer, fluorescence was detectable with a TBR of 4.6 at 3 h.
Conclusions
Fluorescent anti‐CEA nanobodies rapidly and specifically labeled colon cancer in cell‐line‐based and patient‐derived orthotopic xenograft (PDOX) models. The kinetics of nanobodies allow for same day administration and imaging. Anti‐CEA‐nb‐800 is a promising and practical molecule for FGS of colon cancer.
Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure ...determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) is a well-characterized biomarker for the clinical diagnosis of various cancers. Nanobodies, considered the smallest antibody ...fragments with intact antigen-binding capacity, have gained significant attention in disease diagnosis and therapy. Due to their peculiar properties, nanobodies have become promising alternative diagnostic reagents in immunoassay. However, nanobodies-based immunoassay is still hindered by small molecular size and low antigen capture efficacy. Therefore, there is a pressing need to develop novel nanobody-based immunoassays with superior performance.
A novel pentameric nanobodies-based immunoassay (PNIA) was developed with enhanced sensitivity and specificity for CEACAM-5 detection. The binding epitopes of three anti-CEACAM-5 nanobodies (Nb1, Nb2 and Nb3) were analyzed. To enhance the capture and detection efficacy of CEACAM-5 in the immunoassay, we engineered bispecific nanobodies (Nb1–Nb2-rFc) as the capture antibody, and developed the FITC-labeled pentameric nanobodies (Nb3-VT1B) as the detection antibody. The binding affinities of Nb1–Nb2-rFc (1.746 × 10−10) and Nb3-VT1B (1.279 × 10−11) were significantly higher than those of unmodified nanobodies (Nb1-rFc, 4.063 × 10−9; Nb2-rFc, 2.136 × 10−8; Nb3, 3.357 × 10−9). The PNIA showed a linear range of 0.625–160 ng mL−1 with a correlation coefficient R2 of 0.9985, and a limit of detection of 0.52 ng mL−1, which was 24-fold lower than the immunoassay using monomeric nanobody. The PNIA was validated with the spiked human serum. The average recoveries ranged from 91.8% to 102% and the coefficients of variation ranged from 0.026% to 0.082%.
The advantages of nanobodies offer a promising alternative to conventional antibodies in disease diagnosis. The novel PNIA demonstrated superior sensitivity and high specificity for the detection of CEACAM-5 antigen. This bispecific or multivalent nanobody design will provide some new insights into the design of immunoassays for clinical diagnosis.
Display omitted
•A novel nanobodies-based immunoassay was developed for CEACAM-5 detection.•Bispecific nanobodies increased the capture efficacy of CEACAM-5.•A simple and efficient purification strategy was developed for pentameric nanobodies.•Pentameric nanobodies increased the sensitivity of immunoassay.•This novel immunoassay was 24-fold more sensitive than conventional immunoassay.
PDF
