MAbs. 2013 Sep-Oct;5(5):665-77.

Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of Alzheimer disease

Lydia Giménez-Llort, Geovanny Rivera-Hernández, Marta Marin-Argany, José L. Sánchez-Quesada and Sandra Villegas.

 

Abstract

The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid β (Aβ) peptide in neuroblastoma cell cultures by withdrawing Aβ oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia-like behaviors. Cognitive deficits included short- and long-term learning and memory deficits, and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aβ oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.

KEYWORDS: Alzheimer disease; amyloid β oligomers; apoE; apoJ; behavior; clusterin; immunotherapy; scFv

PMID: 23884018

 

Supplement

The World Health Organization and Alzheimer’s Disease International have estimated that 44 million people were living with dementia in 2013, with Alzheimer’s disease (AD) being the most common form [1]. Future projections are shattering, with 135 million people estimated to be living with dementia by 2050.  The magnitude of this 21st century pandemic, together with the suffering caused to patients and caregivers make urgent solutions required.

Current drugs, consisting of several cholinesterase inhibitors (donepezil, galantamine, and rivastigmine) and an antagonist of NMDA receptors (memantine), do not actually halt the progression of the disease, have benefit only during some stages, and are very individual-dependent.

The most remarkable histological features of an Alzheimer’s diseased brain are a dramatic reduction in the number of cortical cells as well as the occurrence of distinctive extracellular plaques and intracellular neurofibrillary tangles. It is widely accepted that the main component of the amyloid plaques, the Aβ-peptide, triggers the pathology and that it exerts the highest neurotoxicity in the form of the soluble aggregates known as Aβ-oligomers. Thus, clearance of Aβ-oligomers in early stages of the disease should preclude cell death and the triggering of the amyloid cascade leading to disease.

The most direct way of targeting Aβ-oligomers is by using administered specific antibodies, a therapeutic intervention known as pasive immunotherapy. In the 2000 Bard et al. first showed that the systemic injection of an IgG2b monoclonal antibody, specific for the first five N-terminal residues of the Aβ-peptide, to the PDAPP mice model for Alzheimer disease resulted on the transfer of the antibody to the brain, the binding of the antibody to amyloid plaques, and the subsequent induction of microglial phagocytosis (mediated by the antibody Fc fraction). This antibody, 3D6-mAb, is the precursor of AAB-001 or bapineuzumab, which has been tested in several clinical trials. However, Phase 3 studies were contradictory and the test was discontinued in August 2012. It has been claimed that these trials failed because they were performed at late stages of the disease and that more attention must be paid to the side effects of high doses. Because these side effects included meningoencephalitis, microhemorrhage and vasogenic edema, some laboratories claim that the use of a fragment of the antibody loosing the Fc fraction, but still recognizing its target by its variable domains, should be safer.

Our laboratory engineered a single chain antibody fragment derived from bapineuzumab, scFv-h3D6, which is composed of the variable domain of the heavy chain linked to the variable domain of the light chain (Figure 1) [2-3].

 

fig1Figure 1: Molecular model for the three-dimensional structure of scFv-h3D6 [3].

fig22

Figure 2: Viability assay in neuroblamtoma cell-cultures. C bar, cells in the absence of the Aβ-peptide (100% viability). 0-10N bars, cells in the presence of the Aβ-peptide (10 μM) and the indicated concentracion of scFv-h3D6.

 

The complementary determining regions, CDR, are in magenta for the VL domain and in cyan for the VH domain. These regions recognize the first five N-terminal residues of the Aβ-peptide.

Once the design of the molecule was done, we synthesized the corresponding gene and inserted it in an Escherichia coli plasmid. After recombinant expression, the scFv was purified by several chromatographic techniques and tested for its ability to preclude the toxicity induced by the Aβ-peptide in human neuroblastoma cell-cultures (Figure 2). No significant differences were obtained between untreated cell-cultures in the absence of the Aβ-peptide and those in the presence of an equimolar ratio Aβ-peptide:scFv-h3D6 [2]. Therefore, scFv-h3D6 completely precluded the cytotoxicity induced by the Aβ-peptide.

To gain insight into the conformational changes underlying the prevention of Aβ toxicity by this antibody fragment, the conformational landscape of scFvh3D6 upon temperature perturbation was studied by biophysical methods as CD, FTIR and TEM [2]. We described for the first time, that the scFv-h3D6 binds Aβ-oligomers and the resulting complex escapes from the amyloid, -and toxic-, pathway to enter a different aggregation pathway featured by curved fibrils, known as worm-like fibrils (Figure 3), that, as shown in Figure 2, turned out to be non-toxic.

 

fig33Figure 3: Transmission Electron Microscopy (TEM) micrographs of amyloid and worm-like fibrils. The bar is 0.5 μm.

fig4Figure 4: Three-dimensional structure of Aβ-oligomers extracted from diseased human brain [6].

 

Next, the efficacy of a single dose of intraperitoneally administered scFv-h3D6 was tested on the 3xTg-AD mouse model for Alzheimer disease [4-5]. This triple-transgenic mouse combines three human genes, two of them found in familiar forms of Alzheimer’s disease and responsible for the accumulation of the Aβ-peptide in the form of amyloid plaques. Untreated five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia-like (BPSD-like) behaviors. Cognitive deficits included short- and long-term learning and memory deficits, and high swimming navigation speed. After treatment the swimming speed reverted to normal levels and the learning and memory deficits were ameliorated. Analysis by westernblot of brain extracts of these animals revealed the occurrence of trimeric Aβ-oligomers, i.e trimers, hexamers, nonamers and dodecamers, particularly in the cortex and olfactory bulb; and more excitingly, their global decrease upon scFv-h3D6 treatment. The trimeric nature of the Aβ-oligomers found in the 3xTg-AD mouse model is in agreement with the recently solved three-dimensional structure of Aβ-oligomers extracted from diseased human brain [6]. In this structure, each monomer adopts a hairpin conformation and packs against other two monomers in a head-tail fashion (Figure 4), with the C-terminal end of each peptide in the core and the 5 first N-terminal residues exposed to the solvent. Each trimer packs against another trimer in parallel, but with a small shift perpendicular to the fibril’s axis of 0.5 nm (not shown, see ref [6]). This shift generates fibrils where the 5 first N-terminal residues of all of the monomers are completely exposed to the solvent, and this explains how the interaction between the scFv-h3D6, specific for these residues, and the trimeric oligomers occurs.

Each monomer in the trimer adopts a hairpin conformation and packs against other two monomers in a head-tail fashion. The five N-terminal residues are rendered as atoms, whereas the rest of the peptide is shown as ribbon diagram. The C-terminal ends of the monomers are located in the core of the trimer.

Our next question was how the Aβ-peptide:scFv-h3D6 complex was being cleared from the brain. When we studied in vitro the physicochemical nature of the Aβ-scFv complex, we determined that it shows a high content of hydrophobic residues exposed to the solvent [2]. Therefore, our next hypothesis was that it could be bound and cleared by apolipoproteins in the CNS.  Then we determined that the concentrations of both apoE and apoJ (also known as clusterin) in the 3xTg-AD mouse were rather increased in the cortex, as well as in the hippocampus for apoE. ScFv-h3D6 treatment significantly recovered the non-pathological levels of these apolipoproteins, plausibly because the complex has already been cleared and the lack of Aβ-oligomers down-regulates apoE and apoJ concentrations.

On the other hand, we did not observe any molecular difference in the cerebellum of the mouse model, but its size was quite smaller than the corresponding to the control mice. A preliminary histological study revealed a dramatic death in young 3xTg-AD Deep Cerebellar Nuclei (DCN) neurons and, more exciting, that scFv-h3D6 had the capability to prevent it [5].

As a general conclusion, this antibody fragment is effective at the behavioral, cellular and molecular levels, and more research is needed in order to eventually enter the first trials in humans.

fig5Sandra Villegas, BSc, MSc, PhD

Group leader of the Protein Folding and Stability Group

Biosciences Unit of the Biochemistry and Molecular Biology Department, Universitat Autònoma de Barcelona, Spain.

E-mail: sandra.villegas@uab.cat

Web page: http://bioquimica.uab.es/paginas_cat/recerca_grup_detall.php?idgrup=29&idarea=8

 

References

[1] Alzheimer’s Disease International. World Alzheimer Report 2013: Journey of Caring. 2013. Available at: http://www.alz.co.uk/research/world-report-2013. Accessed October 12, 2014

[2] Marín-Argany M, Rivera-Hernández G, Martí J, Villegas S. An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formation and cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway. Biochem J. 2011;437(1):25–34. doi:10.1042/ BJ20101712

[3] Rivera-Hernández G, Marín-Argany M, Blasco-Moreno B, Bonet J, Oliva B, Villegas S. Elongation of the C-terminal domain of an anti-amyloid beta single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency. MAbs. 2013; 5(5): 678-689. doi:10.4161/mabs.25382

[4] Giménez-Llort L, Rivera-Hernández G, Marin-Argany M, Sánchez-Quesada JL, Villegas S. Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of Alzheimer disease. MAbs. 2013;5(5):665–77. doi:10.4161/mabs.25424

[5] Esquerda-Canals G, Martí J, Rivera-Hernández G, Giménez-Llort L, Villegas S. Loss of deep cerebellar nuclei neurons in the 3xTg-AD mouse and protection by an anti-Abeta antibody fragment. MAbs. 2013; 5(5):660. doi:10.4161/mabs.25428

[6] Lu JX, Qiang W, Yau WM, Schwieters CD, Meredith SC, Tycko R. Molecular Structure of β-Amyloid Fibrils in Alzheimer’s Disease Brain Tissue. Cell, 2013; 154, 1257–1268. doi:10.1016/j.cell.2013.08.035

 

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