Protein scaffolds: antibody alternatives for cancer diagnosis and therapy

Abstract

Although antibodies are well developed and widely used in cancer therapy and diagnostic fields, some defects remain, such as poor tissue penetration, long in vivo metabolic retention, potential cytotoxicity, patent limitation, and high production cost. These issues have led scientists to explore and develop novel antibody alternatives. Protein scaffolds are small monomeric proteins with stable tertiary structures and mutable residues, which emerged in the 1990s. By combining robust gene engineering and phage display techniques, libraries with sufficient diversity could be established for target binding scaffold selection. Given the properties of small size, high affinity, and excellent specificity and stability, protein scaffolds have been applied in basic research, and preclinical and clinical fields over the past two decades. To date, more than 20 types of protein scaffolds have been developed, with the most frequently used being affibody, adnectin, ANTICALIN®, DARPins, and knottin. In this review, we focus on the protein scaffold applications in cancer therapy and diagnosis in the last 5 years, and discuss the pros and cons, and strategies of optimization and design.

Fig. 1. Structures of proteins scaffolds. Notes: cartoon-illustrations are generated using PyMol, β-sheets are shown in green, α-helix structures are displayed in yellow and disulfide bonds are colored in red. (A) Affibody (PDB ID:3MZW); (B) ANTICALIN® (PDB ID: 4GH7); (C) adnectin or monobody (PDB ID: 1TTG); (D) Knottin (PDB ID: 2IT7); (E) designed ankyrin repeat proteins (PDB ID: 1MJ0); (F) Nanofitin or Affitin (PDB ID: 4CJ2); (G) centyrins (PDB ID: 5L2H); (H) Kunitz (PDB ID: 1KTH); (I) affimer (PDB ID: 1NB5); (J) avimer (PDB ID:1AJJ); (K) Affilin based on the ubiquitin (PDB ID:1UBI); and (L) affilin based on the γ-B-crystalin (PDB ID: 2JDG).

Fig. 2. HER2-specific-affibody imaging in the clinical trial. Notes: PET imaging of HER2-negative patients (A) and HER2-positive patient (B) with ¹⁸F-FDG (A1, B1) and ⁶⁸Ga-ABY-025 (A2, B2); standard uptake value (SUV) is normalized to 10 for all images; darker colors indicate higher uptake. Specific uptake and high contrast image is shown with ⁶⁸Ga-ABY-025 compared to ¹⁸F-FDG-PET image. Adapted from ref. . Abbreviations: HER2, human epidermal growth factor receptor 2; ABY, affibody; SUV, standard uptake value; PET, positron emission tomography; and FDG, fluorodeoxyglucose.

Fig. 3. In vivo PET study of CD98hc-binding ANTICALIN® tracer. Notes: prostate cancer xenografts mice were injected intravenously with ANTICALIN® probes D11vs-PAS200-Dfo ⁸⁹Zr; PET imaging data are acquired at 24 h, 48 h, and 72 h post-injection. Strong imaging signal is shown at 24 h (arrowhead) and gradually reduced at 48 h and 72 h; * indicated joints. Adapted from ref. . Abbreviation: l, liver; k, kidneys; and bl, bladder.

Fig. 4. PD-L1-targeting adnectin probes for clinical PET imaging. Notes: a patient with advanced NSCLC (PD-L1 expression >50%) was administered with ¹⁸F-FDG PET (left) and ¹⁸F-BMS-986192 adnectin PET tracer (right); compared to the FDG tracer, the PET image of adnectin showed high tumor-to-background contrast and heterogeneous uptake at tumor sites. Adapted from ref. . Abbreviations: NSCLC, non-small-cell lung carcinoma; and PD-L1, programmed death-ligand 1.

Fig. 5. In vivo SPECT imaging of EpCAM-specific DARPins probes. Notes: OVCAR-3 (A) and SKOV-3(B) xenografts models; mice are injected with the DARPin probe [¹²⁵I] I-PIB-Ec1 and imaged at 6 hours post-injection. High contrast tumor imaging and mainly cleared through the kidneys has shown. Adapted from ref. . Abbreviations: SPECT, Single-photon emission computed tomography; EpCAM, epithelial cell adhesion molecules; DARPins, designed ankyrin repeat proteins; K, kidneys; and T, tumors.

Fig. 6. αvβ6-Targeting Knottin tracer of PET/CT imaging in the clinical trial. Notes: a cervical cancer patient was administered with Knottin probe 68Ga-NODAGA-R01-MG; cervical tumor pointed by the cyan arrowhead and SUV_mean reached 4.79 ± 0.37. Adapted from ref. . Abbreviations: Lv, liver; Sp, spleen; Bl, bladder; Kd, kidneys; Si, small intestine; and St, stomach.