Recent progress in the molecular imaging of therapeutic monoclonal antibodies

Abstract

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need. It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented. Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA, and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information, while methods for characterization of antibody's interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development. Recent progress in detecting therapeutic antibodies, in particular, the development of methods suitable for illustrating the molecular dynamics, is described here.

Keywords: Biological function; Molecular imaging; Molecular structure; Therapeutic monoclonal antibodies.

Graphical abstract

Fig. 1

(A) General structure of therapeutic monoclonal antibodies (IgG-type antibodies) [27]. (B) Therapeutic antibodies are glycosylated through the ubiquitous asparagine residue (Asn297) found in the Fc region. The most common oligosaccharides found in current therapeutic antibody products are shown (left). Several engineered oligosaccharides and the resulting impact on functions are shown (right) [28]. The figures were reproduced with permission from Refs. [27,28], respectively.

Fig. 2

Monoclonal antibody-based cancer therapeutic strategies.

Fig. 3

(A) Design of the electrochemical sensor. (B) Binding of mAbs can be measured as a decrease in the peak current. (C) Quantitative detection of six antibodies against contiguous epitopes. The figure was reproduced with permission from Ref. [52].

Fig. 4

(A) Scheme of the SPR-based assay for simultaneous determination of up to six serum samples. Linearity between (B) IFX or (C) ATI concentration and specific SPR signal. The figure was reproduced with permission from Ref. [54].

Fig. 5

(A) EGFR expression in eight non-small cell lung cancer (NSCLC) cell lines (up). Representative ⁶⁴Cu-DOTA-cetuximab PET images in mice xenograft models with NSCLC tumors containing varying EGFR expression levels at 48 h. Arrows indicate the location of tumors (down) [63]. (B) PET-CT of patient with tumor PD-L1 expression < 1%: ¹⁸F-BMS-986192 PET (214.62 MBq, 1 h p.i.) demonstrates low tumor tracer uptake. ⁸⁹Zr-labeled Nivolumab PET (37.27 MBq, 160 h p.i.) demonstrates heterogeneous tracer uptake in the tumor [64]. The figures were reproduced with permission from Refs. [63,64], respectively.

Fig. 6

Maximum intensity projection images at each time point for one of the rhesus monkeys in the ⁸⁹Zr-DFO-squaramide-anti-gD group. The figure was reproduced with permission from Ref. [72].

Fig. 7

(A) Titration curves of nimotuzumab, IRDye800CWnimotuzumab, and control IgG against DLD-1 cells showing percent bound against antibody concentration. (B) Nimotuzumab binding to cell lines expressing various levels of EGFR. (C) IRDye800CW-nimotuzumab stability was assayed in mouse serum at 37 °C over seven days. (D) Biodistribution analysis of IRDye800CW-nimotuzumab in mice bearing DLD-1 and MDA-MB-435 xenografts. Control IgG was injected into mice bearing DLD-1 xenografts. The figure was reproduced with permission from Ref. [76].

Fig. 8

Multi-color imaging targeting CD25, HER1, and HER2, using Alexa700-, Cy5-, and Cy7-labeled respective specific antibodies [79].

Fig. 9

(A) Map of accumulated EGFR trajectories acquired for about 1 min before (up) and after cetuximab treatment (after 3 min; down) in a single COS7 cell. (B-C) Dissociation constant and cooperativity measurement for the interaction of cetuximab with (B) wild-type EGFR and (C) its mutant (L858R) on a COS7 cell membrane. The insets show Scatchard plots to evaluate the interaction cooperativity. (D) Proposed model for the molecular mechanism of the positive cooperativity of cetuximab binding to EGFR L858R. The figure was reproduced with permission from Ref. [88].

Fig. 10

(A) Scheme of experimental procedures. (B) A typical force-distance cycle of the specific interaction between cetuximab and cell membrane EGFR. (C) Histogram of cetuximab-EGFR binding forces. (D) Histogram of EGF-EGFR binding forces (n > 1000). The figure was reproduced with permission from Ref. [89].

Fig. 11

(A) Schematic of the NanoLuc-EGFR/DY605-Cetuximab BRET System. NanoLuc (a 19-kDa luciferase) was fused to the N terminus of the EGFR extracellular domain to generate the BRET donor moiety. A fluorescent dye, DY605, was covalently appended onto cetuximab to generate the BRET acceptor moiety. (B-D) DY605-Cetuximab/NanoLuc-EGFR interaction measurement in vivo. (B) BRET images of mice that received 50 mg/kg (HD)/8.5 mg/kg (MD)/1.0 mg/kg (LD) of DY605-cetuximab or 1.9 mg/kg of control DY605-IgG. (C) Quantified NanoLuc-EGFR/DY605-cetuximab binding according to BRET ratios. (D) Quantified receptor occupancy in living mice. The figure was reproduced with permission from Ref. [90].

Fig. 12

A summary of available activatable mAb-fluorophore conjugates using (A) FRET, (B) acidic pH-sensitivity, (C) AIE, or (D) self-quenching strategies to study the cellular performance of mAbs based on receptor-mediated antibody internalization and degradation.

Fig. 13

Design of the reversible and acidic pH-induced fluorescence activation of BODIPY probe, DiEtNBDP [93].

Fig. 14

(A) Preparation of the mAb-AIEgens conjugate. (B) Schematic representation of the “turn-on” process of mAb-AIEgen conjugates after internalization and catabolism in EGFR overexpressing cells. (C) Live-cell fluorescence images of (a–d) HCC827 cancer cells (EGFR-positive) and (e–h) COS-7 normal cells (EGFR-negative) incubated with cetuximab-CSPP and cetuximab-Cy3 conjugates for 12 h. Images were taken (a, b, e and f) without washing or (c, d, g and h) after washing. The figure was reproduced with permission from Ref. [98].

Fig. 15

(A) Incorporation of TAMRA-C6-AF into scFv in response to a UAG codon in a cell-free translation system. (B) The reaction model of TAMRA-scFv towards antigen. Trp residues, Trp33H, Trp36H, Trp47H, Trp103H, and Trp35L are colored green. (C) Antigen-dependent fluorescence enhancement of TAMRA-labeled anti-BGP scFv upon reaction with BGP-C7. The figure was reproduced with permission from Ref. [106].

Fig. 16

Schematic illustration of N-terminal selective fluorescent labeling of IgG and fluorescence response upon antigen-binding [108].

Fig. 17

(A) Diagram of antigen-responsive antibody-NIR fluorophore conjugates for activatable fluorescence imaging. (B) Real-time fluorescence imaging of membrane binding HER-ATTO680 on SK-BR-3 cells acquired every 1 min (λ_ex = 633 nm, λ_em = 647–754 nm) without washing. (C) In vivo NIR fluorescence images of normal, MDA-MB-231 and Calu-3 tumor-bearing mice upon HER-ATTO680-treatment (λ_ex = 660/20 nm, λ_em = 710/40 nm). The figure was reproduced with permission from Ref. [109].

Fig. 18

(A) The chemical structure of the cleavable crosslinker furnished with a FRET fluorophore pair. (B) Schematic of site-specific modification of trastuzumab via microbial transglutaminase and its “turn-on” response after entering cells [110].

Fig. 19

(A) The rat anti-mouse PD-1 29F.1A12 clone, conjugated to AF647 via NHS ester linkage (left). Diagram depicting intravital imaging setup with labeled aPD-1, MC38 tumor cells, T cells, and TAMs (right). (B) Z-projections of an MC38-H2B-mApple tumor in a DPE-GFP mouse injected intravenously with AF647–aPD-1 after 15 min (left) or 24 h (right). The figure was reproduced with permission from Ref. [111].

Fig. 20

(A) Target glass beads were coated by a lipid bilayer (containing DPPE-biotin, labeled by DOPE-647 in red). Anti-biotin IgG binds to the lipid surface through interaction with the biotin group of DPPE-biotin. Binding between FcγRs on the macrophage surface and the Fc region of anti-biotin IgG is shown. (B) Left: confocal imaging of a RAW 264.7 macrophage-like cell (membrane is labeled with cholera-toxin B 555, cyan) at a contact interface with a target bead in the presence of anti-biotin IgG (labeled with Alexa Fluor 488, green). Right: target beads containing only lipids (up) or pre-incubated with anti-biotin IgG (down) were added to RAW 264.7 cells. (C) Confocal imaging of phagocytosis of target beads bound with biotinylated Fib1L, Fib3L, Fib5L, and Fib7L protein antigens and opsonized with anti-biotin IgG. Cells were labeled with 0.5 μM CellTracker Green (CMFDA) and Hoechst. The figure was reproduced with permission from Ref. [112].