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. 2018 Apr 20;9(1):1580.
doi: 10.1038/s41467-018-03976-5.

Mechanically Interlocked Functionalization of Monoclonal Antibodies

Free PMC article

Mechanically Interlocked Functionalization of Monoclonal Antibodies

Krzysztof P Bzymek et al. Nat Commun. .
Free PMC article


Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope-Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope-Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to "snap" functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.

Conflict of interest statement

J.C.W. and D.A.H. have multiple patents (issued and pending) covering the meditope technology, are co-founders, shareholders, and members of the SAB of Meditope Biosciences, Inc. However, all of the work reported here was funded by charitable contributions to City of Hope and federal grants (i.e., none of the research reported here was supported by Meditope Biosciences, Inc.). The remaining authors declare no competing interests.


Fig. 1
Fig. 1
Increasing the affinity of the meditope site. a Surface representation of an IgG with a bound meditope (yellow). Light blue indicates the light chain and white indicates the heavy chain. b Kinetics and thermodynamics of meditope and antibody modifications (n = 1). Blue points represent data collected at 25 °C. Red points represent data collected at 37 °C (see also Supplementary Table 1). DLE—5-diphenylalanine long meditope ((Ac)CQFDA(Ph)2STRRLRCGGSK) binding to Ile83Glu anti-HER2 memAb; LE—long meditope ((Ac)CQFDLSTRRLRCGGSK) binding to Ile83Glu anti-HER2 memAb; L—long meditope ((Ac)CQFDLSTRRLRCGGSK) binding to anti-HER2 memAb antibody; ori—original meditope (CQFDLSTRRLKC) binding to anti-HER2 memAb. c SPR sensograms of meditope peptide variants binding to immobilized memAb variants at 37 °C (n = 1). Top, left sensogram—CQFDLSTRRLKC meditope (QFD, original unmodified meditope) binding to anti-HER2 memAb (original, meditope-enabled anti-HER2 antibody); top, right sensogram—(Ac)CQFDA(Ph)2STRRLRCGGSK (5-diphenylalanine long meditope) binding to anti-HER2 memAb; bottom, left sensogram—QFD binding to Ile83Glu anti-HER2 memAb; bottom, right sensogram—5-diphenylalanine long meditope binding to Ile83Glu anti-HER2 memAb. Residues highlighted in green correspond to modifications in the meditope (position 5, top panel) or Fab (position 83 LC, left panels)
Fig. 2
Fig. 2
Development of a mechanical bond to interlock the meditope to a Fab. a Back view of Ile83Glu anti-HER2 antibody Fab with (Ac)CQFDA(Ph)2STRRLKC meditope (left) and 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope (right). Light blue surface is the light chain; white surface is the heavy chain. The red arrows indicate the modified residue (arginine 8, left panel) and the reactive azide (right panel). b Chemical structures of the meditope bolt (meditope-containing reactive group) and nut (reactive steric group), the sequence is represented by single letter amino acid code where X represents 5-diphenylalanine and Z represents the modified azido-arginine. R1 and R2 represent potential modification sites. c Proposed scheme for forming a mechanical bond. After binding of the azido-meditope, the reactive azide on the backside of the Fab can react with a reactive cycloalkyne to form a mechanical bond. d Native mass spectrometry analysis of the mechanically interlocked meditope–AF647/Ile83Glu anti-HER2 memAb. The difference in molecular weight (3145 and 6400 Da) corresponds to 1:1 or 2:1 complex of mechanically interlocked meditope:IgG (top panel). The spectrum for unlabeled IgG is shown in the bottom panel. The experimentally determined mass of meditope–DIBO–AF647 is 3293.3 Da (see Supplementary Fig. 4). e Mass spectrometry analysis of “molecular dumbbell”. Top panel: mass spectrum of purified reaction product; calculated mass: 101,634 (2×47730 + 2×2178 + 1818), observed mass: 101,655. Bottom panel: mass spectrum of the Ile83Glu, meditope-enabled Fab used for the reaction above; calculated mass: 47720, observed mass: 47730
Fig. 3
Fig. 3
In vivo imaging. a Analytical flow cytometry analysis of BT474 HER2 overexpressing breast cancer cells incubated with 1, 10, or 100 nM of Ile83Glu anti-HER2 memAb with 5-diphenylalanine meditope–AF647 at a 1:1 molar ratio (blue), Ilu83Glu anti-HER2 memAb directly labeled with AF647 (green) or with mechanically interlocked 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope–DIBO–AF647 Ile83Glu anti-HER2 memAb (red). b Analytical flow cytometry analysis of PBMCs incubated with 1, 10, and 100 nM labeled antibodies. The red trace is the mechanically interlocked, 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope–DIBO–AF647 Ile83Glu anti-CD3 memAb (based on Okt3). The blue traces are cells treated with the anti-CD3 memAb with pre-bound 5-diphenylalanine-meditope-Alexa-647 (e.g., non-covalent). The black trace represents untreated cells. c Xenogen images of mice bearing BT474 xenograft tumors and injected through tail vain with mechanically interlocked meditope–AF647–Ile83Glu anti-HER2 memAb top row, n = 4) or mechanically interlocked meditope–AF647–Ile83Glu anti-CD3 memAb (control, bottom row, n = 4). Images were taken 24, 48, and 72 h after injection. d Tissue distribution of mechanically interlocked AF647–meditope–Ile83Glu anti-HER2 memAb and the mechanically interlocked AF647–meditope–Ile83Glu anti-CD3 memAb 3 days post-injection: tumors (T) and organs (K kidneys, L liver, S spleen, GI gastrointestinal tract)

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