Small Antibodies with Big Applications: Nanobody-Based Cancer Diagnostics and Therapeutics

Abstract

Monoclonal antibodies (mAbs) have exhibited substantial potential as targeted therapeutics in cancer treatment due to their precise antigen-binding specificity. Despite their success in tumor-targeted therapies, their effectiveness is hindered by their large size and limited tissue permeability. Camelid-derived single-domain antibodies, also known as nanobodies, represent the smallest naturally occurring antibody fragments. Nanobodies offer distinct advantages over traditional mAbs, including their smaller size, high stability, lower manufacturing costs, and deeper tissue penetration capabilities. They have demonstrated significant roles as both diagnostic and therapeutic tools in cancer research and are also considered as the next generation of antibody drugs. In this review, our objective is to provide readers with insights into the development and various applications of nanobodies in the field of cancer treatment, along with an exploration of the challenges and strategies for their prospective clinical trials.

Keywords: EGFR; HER2; NIR; PD-L1; VHH; immunoPET.

Figure 1

Structures of diverse antibodies. The classical IgG monoclonal antibody is usually generated via animal or human immunization, and consists of two heavy chains and two light chains to form a Y-shape (left). The antigen binding area includes the light-chain variable domain (VL), constant region (CL), heavy-chain variable domain (VH), and constant region 1 (CH1). In contrast, the natural camel antibody only has heavy chains and lacks CH1 (middle). Derived from the IgG antigen binding area, a Fab antibody with a light chain, a VH domain and a CH1 domain, is generated, while a VH domain and a VL domain compose the scFv antibody (right). The nanobody derived from the heavy-chain antibody is the smallest antibody, with a molecular weight of 15 kDa, and has three complementary determining regions.

Figure 2

The construction process of three nanobody libraries. To establish an immune library, camelids are regularly immunized with antigens for two months. Blood is collected from immunized (immune library) and non-immunized (naïve library) camelids, and peripheral lymphocytes are isolated. After extracting mRNAs from lymphocytes, reverse transcription is performed to create libraries containing the desired nanobody gene (cDNA), which is then separated through gel electrophoresis. In the synthetic library (right panel), cloning templates are selected after the computational analysis of the structure and sequence of natural nanobodies. Then, the primers are rationally designed to introduce the sequence diversities for CDR1, CDR2 and CDR3 using PCR technology, while avoiding hydrophobic residues, thus generating highly abundant synthetic libraries.

Figure 3

Schematics of the display technologies for nanobody screening. The phenotypes of the nanobodies were combined with their genotypes by using a number of different methods. (A) The nanobodies were presented on the surface of phages using phage display technology. The phages were screened for binding antigens and re-infected with E. coli using biopanning, and the desired antibodies were identified by combining with ELISA. (B) A library of nanobodies is displayed on the surface of yeast, incubated with fluorescently labeled antigen; then, the nanobody sequences were obtained with flow cytometric sorting. (C,D) Both ribosome display technology and mRNA/cDNA display technology utilize ribosomes to translate nanobody libraries into nanobodies in vitro to form ribosome–nanobody–mRNA complexes (C) and nanobody–puromycin–mRNA complexes (D), relying on tags to screen out nanobody sequences with binding abilities.

Figure 4

The applications of nanobodies in cancer diagnosis and therapy. Imaging probes such as ⁶⁸Ga, ⁸⁹Zr, ⁶⁴Cu, ¹⁸F, and ⁸⁶Y are conjugated with nanobodies to perform immunoPET imaging, while ImmunoSPECT is performed using ⁹⁹mTc, ¹³¹I, and ¹⁷⁷Lu. Microbubbles can transport specific nanobodies to the target site and can be ruptured under ultrasound irradiation, subsequently inducing a tumor-imaging or cancer-killing effect. Near-infrared imaging utilizing different dyes to obtain imaging from a specific tumor or conversion from light to heat to kill target cancer cells (photothermal therapy) is used. The immunotherapy of anti-cancer was usually referred to CAR-T cells, CAR-NK cells and immune checkpoints, such as PD-1/PD-L1. Moreover, nanobodies can be linked with drugs or any other desired materials to bring them to targeted cancer sites.