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. Aug/Sep 2017;9(6):1016-1027.
doi: 10.1080/19420862.2017.1342914. Epub 2017 Jun 28.

Chemically-defined Camelid Antibody Bioconjugate for the Magnetic Resonance Imaging of Alzheimer's Disease

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Chemically-defined Camelid Antibody Bioconjugate for the Magnetic Resonance Imaging of Alzheimer's Disease

Matthias Vandesquille et al. MAbs. .
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Abstract

Today, molecular imaging of neurodegenerative diseases is mainly based on small molecule probes. Alternatively, antibodies are versatile tools that may be developed as new imaging agents. Indeed, they can be readily obtained to specifically target any antigen of interest and their scaffold can be functionalized. One of the critical issues involved in translating antibody-based probes to the clinic is the design and synthesis of perfectly-defined conjugates. Camelid single-domain antibody-fragments (VHHs) are very small and stable antibodies that are able to diffuse in tissues and potentially cross the blood brain barrier (BBB). Here, we selected a VHH (R3VQ) specifically targeting one of the main lesions of Alzheimer's disease (AD), namely the amyloid-beta (Aß) deposits. It was used as a scaffold for the design of imaging probes for magnetic resonance imaging (MRI) and labeled with the contrastophore gadolinium using either a random or site-specific approach. In contrast to the random strategy, the site-specific conjugation to a single reduced cysteine in the C-terminal part of the R3VQ generates a well-defined bioconjugate in a high yield process. This new imaging probe is able to cross the BBB and label Aß deposits after intravenous injection. Also, it displays improved r1 and r2 relaxivities, up to 30 times higher than a widely used clinical contrast agent, and it allows MRI detection of amyloid deposits in post mortem brain tissue of a mouse model of AD. The ability to produce chemically-defined VHH conjugates that cross the BBB opens the way for future development of tailored imaging probes targeting intracerebral antigens.

Keywords: Alzheimer's disease diagnosis; amyloid deposits; chemically-defined imaging probe; magnetic resonance imaging; single-domain antibody; site-specific conjugation.

Figures

Figure 1.
Figure 1.
MS analyses of compounds involved in random (A, C) and site-specific approaches (B, D). Analyses (deconvoluted spectra) of starting VHHs 1 (expected Mr = 15,752.3949) (A) and 3 (expected Mr = 15,724.2820) (B), and their respective DOTA/Gd conjugates 2a (expected Mr = 16,293.0421 (DOTA/Gd)1, 16,833.6735 (DOTA/Gd)2) (C) and 5 (expected Mr = 18,113.0720) (D) showed the polydisperse mixture obtained with 2a as opposed to the well-defined conjugate 5. MS analyses of R3VQ-SH 3 showing the presence of a single reduced cysteine and of a stable disulfide bond (E). Analyses (deconvoluted spectra) were realized on 3 without treatment (expected Mr = 15,724.2820), after reduction/alkylation (expected Mr = 15,781.3339 with 1 alkylated cysteine), and after denaturation/reduction/alkylation experiments (expected Mr = 15,895.4378 with 3 alkylated cysteines). The magnified overlay (top) showed the shifts due to alkylation of the thiol functions depending on conditions.
Scheme 1.
Scheme 1.
Overview of random (A) and site-specific (B) chemical conjugationa. aReagents and conditions: (a) Diafiltration, PBS/NaCl, pH 7.3; (b) DOTA-NHS, 3 h, 4 °C; (c). Diafiltration, NaAc, pH 5; (d) GdCl3, 3 h, 4 °C; (e) Compound 4, PBS/NaCl, 3 h, 4 °C, pH 6.8. The site-specific conjugation (B) was performed with (method 1) or without (method 2) initial buffer exchange. Synthesis of compound 4 is described in Scheme 2. Table C summarizes the number of steps and the overall yield of each approach (include all the synthetic process from the starting protein in the affinity column elution buffer). n = average amount of DOTA/Gd per VHH (randomly distributed on different sites). m = exact amount of DOTA/Gd per VHH (located on a single site).
Scheme 2.
Scheme 2.
Synthesis of maleimide-(DOTA/Gd)3 4a. aReagents and conditions: (a) piperidine/DMF 20%; (b) Fmoc-Lys(DOTA(OtBu)3)-OH, HATU, DIEA, DMF or Fmoc-Gly-OH, DIC, DMF; (c) 6-Maleimidohexanoic acid, HATU, DIEA, DMF; (d) TFA/H2O/TIS, 95/2.5/2.5, 4 h; (e) Gd(OAc)3, pH 5, 25 min, 95 °C.
Figure 2.
Figure 2.
Immunostaining of amyloid deposits by R3VQ-SH 3 (left) and R3VQ-S-(DOTA/Gd)3 5 (right) on brain tissue from mouse model of amyloidosis, showing the preserved properties of the site-specific conjugate (see Figure S7 for IHC controls).
Figure 3.
Figure 3.
Immunostaining of amyloid plaques after intravenous injection of R3VQ-S-(DOTA/Gd)3 5, highlighting its ability to cross the BBB in vivo. PS2APP mice (15 month-old) were injected in the tail vein with compound 5 at 50 mg/kg (left panel, n = 3) or with PBS (right panel, n = 2), and sacrificed after 4 hours. IHC were realized with an anti-His-tag antibody. Whereas only unspecific background was observed in mice brains injected with PBS, a specific labeling of amyloid deposits was detected in those injected with 5 thus confirming the ability of the conjugate to cross the BBB after in vivo injection.
Figure 4.
Figure 4.
MR relaxometric parameters at three different magnetic fields. Measures of r1 and r2 (normalized per mM of Gd) showed the high relaxivities of the contrast agent R3VQ-S-(DOTA/Gd)3 5 that reach values until 10 times higher than those of the reference contrast agent DOTAREM. Triplicate measures of r1 and r2 (in mM−1.s−1) are expressed as mean +/− SEM (detailed values in Fig. S9).
Figure 5.
Figure 5.
In vitro MRI revelation of amyloid deposits by the contrast agent R3VQ-S-(DOTA/Gd)3 5. (A) PS2APP or wild-type (amyloid free) brains were incubated with PBS or 5 before MRI acquisitions (left frames). IHC were realized with 4G8 antibody as a reference anti-beta amyloid antibody (middle frames), or an anti-His-tag antibody to reveal the amyloid deposits labeled by the contrast agent 5 (right frames). MRI acquisitions were performed with a 25 µm isotropic resolution (n = 2/group). Red squares show the magnified areas used for the registration. Scale bar = 500 µm. See Fig. 6 for hypointense spots quantification. (B) Registration was done between MRI, 4G8 and anti-His-tag IHC on PS2APP tissues incubated with 5. Hypointense spots on MR images correspond to amyloid deposits labeled by 5 on the anti-His-tag IHC and by 4G8 (red arrows). White dotted lines represent landmarks that delimited the corpus callosum and the hippocampus. Scale bar = 250 µm.
Figure 6.
Figure 6.
Quantification of hypointense spots detected on MR images. Measures from MR images obtained on post mortem brain tissues (Fig. 5A) confirmed the major increase in amyloid plaques detection in PS2APP brains incubated with the R3VQ-S-(DOTA/Gd)3 5 compared to the controls, i.e., PS2APP brains incubated with PBS or wild-type amyloid-free brains incubated with R3VQ-S-(DOTA/Gd)3 5.

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