Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer's Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety

Both Amyloid-β Peptide and Tau Protein Are Affected by an Anti-Amyloid-β Antibody Fragment in Elderly 3xTg-AD Mice

Altres ajuts: ARR received an FPU fellowship and GSM a PIF-UAB fellowshipAlzheimer's disease (AD) is the most common dementia worldwide. According to the amyloid hypothesis,the early accumulation of the Aβ-peptide triggers tau phosphorylation,synaptic dysfunction, and eventually neuronal death leading to cognitive impairment, as well as behavioral and psychological symptoms of dementia. ScFv-h3D6 is a single-chain variable fragment that has already shown its ability to diminish the amyloid burden in 5-month-old 3xTg-AD mice. However, tau pathology is not evident at this early stage of the disease in this mouse model. In this study, the effects of scFv-h3D6 on Aβ and tau pathologies have been assessed in 22-month-old 3xTg-AD mice. Briefly, 3xTg-AD female mice were treated for 2 weeks with scFv-h3D6 and compared with 3xTg-AD and non-transgenic (NTg) mice treated with PBS. The treatment with scFv-h3D6 was unequivocally effective in reducing the area of Aβ staining. Furthermore, a tendency for a reduction in tau levels was also observed after treatment that points to the interplay between Aβ and tau pathologies. The pro-inflammatory state observed in the 3xTg-AD mice did not progress after scFv-h3D6 treatment. In addition, the treatment did not alter the levels of apolipoprotein E or apolipoprotein J. Thus, a 2-week treatment with scFv-h3D6 was able to reduce AD-like pathology in elderly 3xTg-AD female mice

Blood phospho-tau in Alzheimer disease: analysis, interpretation, and clinical utility

Well-authenticated biomarkers can provide critical insights into the biological basis of Alzheimer disease (AD) to enable timely and accurate diagnosis, estimate future burden and support therapeutic trials. Current cerebrospinal fluid and molecular neuroimaging biomarkers fulfil these criteria but lack the scalability and simplicity necessary for widespread application. Blood biomarkers of adequate effectiveness have the potential to act as first-line diagnostic and prognostic tools, and offer the possibility of extensive population screening and use that is not limited to specialized centres. Accelerated progress in our understanding of the biochemistry of brain-derived tau protein and advances in ultrasensitive technologies have enabled the development of AD-specific phosphorylated tau (p-tau) biomarkers in blood. In this Review we discuss how new information on the molecular processing of brain p-tau and secretion of specific fragments into biofluids is informing blood biomarker development, enabling the evaluation of preanalytical factors that affect quantification, and informing harmonized protocols for blood handling. We also review the performance of blood p-tau biomarkers in the context of AD and discuss their potential contexts of use for clinical and research purposes. Finally, we highlight outstanding ethical, clinical and analytical challenges, and outline the steps that need to be taken to standardize inter-laboratory and inter-assay measurements

Quantification of SNAP-25 with mass spectrometry and Simoa: a method comparison in Alzheimer's disease

BACKGROUND: Synaptic dysfunction and degeneration are central to Alzheimer's disease (AD) and have been found to correlate strongly with cognitive decline. Thus, studying cerebrospinal fluid (CSF) biomarkers reflecting synaptic degeneration, such as the presynaptic protein synaptosomal-associated protein 25 (SNAP-25), is of importance to better understand the AD pathophysiology. METHODS: We compared a newly developed Single molecule array (Simoa) immunoassay for SNAP-25 with an in-house immunoprecipitation mass spectrometry (IP-MS) method in a well-characterized clinical cohort (n = 70) consisting of cognitively unimpaired (CU) and cognitively impaired (CI) individuals with and without Aβ pathology (Aβ+ and Aβ-). RESULTS: A strong correlation (Spearman's rank correlation coefficient (rs) > 0.88; p < 0.0001) was found between the Simoa and IP-MS methods, and no statistically significant difference was found for their clinical performance to identify AD pathophysiology in the form of Aβ pathology. Increased CSF SNAP-25 levels in CI Aβ+ compared with CU Aβ- (Simoa, p ≤ 0.01; IP-MS, p ≤ 0.05) and CI Aβ- (Simoa, p ≤ 0.01; IP-MS, p ≤ 0.05) were observed. In independent blood samples (n = 32), the Simoa SNAP-25 assay was found to lack analytical sensitivity for quantification of SNAP-25 in plasma. CONCLUSIONS: These results indicate that the Simoa SNAP-25 method can be used interchangeably with the IP-MS method for the quantification of SNAP-25 in CSF. Additionally, these results confirm that CSF SNAP-25 is increased in relation to amyloid pathology in the AD continuum

Longitudinal blood biomarker trajectories in preclinical Alzheimer's disease

INTRODUCTION: Plasma biomarkers are altered years prior to Alzheimer's disease (AD) clinical onset. METHODS: We measured longitudinal changes in plasma amyloid-beta (Aβ)42/40 ratio, pTau181, pTau231, neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) in a cohort of older adults at risk of AD (n = 373 total, n = 229 with Aβ and tau positron emission tomography [PET] scans) considering genetic and demographic factors as possible modifiers of these markers' progression. RESULTS: Aβ42/40 ratio concentrations decreased, while NfL and GFAP values increased over the 4-year follow-up. Apolipoprotein E (APOE) ε4 carriers showed faster increase in plasma pTau181 than non-carriers. Older individuals showed a faster increase in plasma NfL, and females showed a faster increase in plasma GFAP values. In the PET subsample, individuals both Aβ-PET and tau-PET positive showed faster plasma pTau181 and GFAP increase compared to PET-negative individuals. DISCUSSION: Plasma markers can track biological change over time, with plasma pTau181 and GFAP markers showing longitudinal change in individuals with preclinical AD. HIGHLIGHTS: Longitudinal increase of plasma pTau181 and glial fibrillary acidic protein (GFAP) can be measured in the preclinical phase of AD. Apolipoprotein E ε4 carriers experience faster increase in plasma pTau181 over time than non-carriers. Female sex showed accelerated increase in plasma GFAP over time compared to males. Aβ42/40 and pTau231 values are already abnormal at baseline in individuals with both amyloid and tau PET burden

Plasma p-tau231 and p-tau217 as state markers of amyloid-β pathology in preclinical Alzheimer’s disease

Blood biomarkers indicating elevated amyloid-β (Aβ) pathology in preclinical Alzheimer's disease are needed to facilitate the initial screening process of participants in disease-modifying trials. Previous biofluid data suggest that phosphorylated tau231 (p-tau231) could indicate incipient Aβ pathology, but a comprehensive comparison with other putative blood biomarkers is lacking. In the ALFA+ cohort, all tested plasma biomarkers (p-tau181, p-tau217, p-tau231, GFAP, NfL and Aβ42/40) were significantly changed in preclinical Alzheimer's disease. However, plasma p-tau231 reached abnormal levels with the lowest Aβ burden. Plasma p-tau231 and p-tau217 had the strongest association with Aβ positron emission tomography (PET) retention in early accumulating regions and associated with longitudinal increases in Aβ PET uptake in individuals without overt Aβ pathology at baseline. In summary, plasma p-tau231 and p-tau217 better capture the earliest cerebral Aβ changes, before overt Aβ plaque pathology is present, and are promising blood biomarkers to enrich a preclinical population for Alzheimer's disease clinical trials

P-tau235: a novel biomarker for staging preclinical Alzheimer's disease

Alzheimer's disease (AD) is characterised by a long preclinical phase. Although phosphorylated tau (p-tau) species such as p-tau217 and p-tau231 provide accurate detection of early pathological changes, other biomarkers capable of staging disease progression during preclinical AD are still needed. Combining exploratory and targeted mass spectrometry methods in neuropathologically confirmed brain tissue, we observed that p-tau235 is a prominent feature of AD pathology. In addition, p-tau235 seemed to be preceded by p-tau231, in what appeared to be a sequential phosphorylation event. To exploit its biomarker potential in cerebrospinal fluid (CSF), we developed and validated a new p-tau235 Simoa assay. Using three clinical cohorts, we demonstrated that (i) CSF p-235 increases early in AD continuum, and (ii) changes in CSF p-tau235 and p-tau231 levels during preclinical AD are consistent with the sequential phosphorylation evidence in AD brain. In conclusion, CSF p-tau235 appears to be not only a highly specific biomarker of AD but also a promising staging biomarker for the preclinical phase. Thus, it could prove useful tracking disease progression and help enriching clinical trial recruitment

Immunotherapy for alzheimer’s disease: from antibody engineering to combined therapy with apolipoproteins.

ScFv-h3D6 és un fragment variable de cadena lleugera, derivat de l’anticòs bapineuzumab, capaç d’evitar la citotoxicitat induïda pel pèptid Aβ, retirant els oligòmers de la via amiloide cap a una via d’agregació worm-like (WL). A més, s'ha demostrat que és eficaç a nivell cognitiu, cel·lular i molecular en el tractament del model murí d’Alzheimer 3xTg-AD. Tot i el seu gran potencial, la producció del scFv-h3D6 estava limitada per alguns colls d'ampolla com la presència de conformacions scrambled i la seva contaminació amb endotoxines, que reduïen el rendiment de producció i n’augmentaven el cost. En el present treball, diverses formes per millorar la producció i l'estabilitat termodinàmica han estat explorades. En primer lloc, els ponts disulfur s'han eliminat per tal d'evitar conformacions scrambled i, d'aquesta manera, millorar el rendiment de purificació de la proteïna, assegurant una conformació homogènia. L’eliminació del pont disulfur del domini més estable, va resoldre el problema de l’scrambled, duplicant el rendiment de producció. També es va canviar la ruta d'agregació des de la morfologia de protecció WL a una amiloide. Després, es va estabilitzar el scFv-h3D6 mitjançant la introducció de la mutació VH-K64R i combinant-la amb l'extensió anteriorment descrita (C3). Les estabilitats de les diferents construccions del scFv-h3D6 van resultar en l'ordre C3> K64R / C3> VH-K64R ≥ scFv-h3D6, demostrant que la combinació d'ambdues mutacions no era additiva sinó que es cancel·laven parcialment entre si. En els assajos de citotoxicitat, tots els mutants evitaven la citotoxicitat del pèptid Aβ d'una manera dependent de la dosi i amb eficiències que es correlacionaven amb l'estabilitat. Finalment, es va realitzar un canvi en el sistema d'expressió del scFv-h3D6: d’Escherichia coli a Pichia Pastoris. A més de mantenir les mateixes propietats termodinàmiques, es van millorar d’altres: homogeneïtat de la mostra, l'absència de conformacions scrambled, mostra lliure d'endotoxines, un millor rendiment i fàcil extrapolació a una escala més gran. L'eficàcia terapèutica es va demostrar in vitro i in vivo. D'altra banda, la immunoteràpia contra el pèptid Aβ ha estat molt estudiada, però no com altres molècules implicades en la malaltia d’Alzheimer poden afectar al rendiment de l'anticòs. Per entendre completament les possibles interaccions i efectes entre Aβ, scFv-h3D6, apoE i apoJ; es va realitzar un estudi complet des de diferents perspectives: biofísica, cel·lular i in vivo. Enlloc d'apolipoproteïnes senceres, es va combinar el scFv-h3D6 amb els pèptids mimètics (MP) de les apolipoproteïnes (apo) E i J, compostos d'estructures essencials per a la unió a altres molècules. Els estudis biofísics van mostrar que el apoE-MP impedia la formació de les fibres WL pel complex scFv-h3D6/Aβ i parcialment interferia en la reducció induïda per scFv-h3D6 en la captació d’Aβ pels astròcits; mentre que el apoJ-MP permetia la formació de fibres WL i no interferia en la captació d’ Aβ. En mirar la inflamació associada amb l'Alzheimer, el apoE-MP va desenvolupar un paper antiinflamatori en cultiu cel·lular i en el model de ratolí 3xTg-AD; mentre apoJ-MP va mostrar un perfil antiinflamatòri només quan es combinava amb scFv-h3D6. ScFv-h3D6 sol no va induir l'activació microglial, prèviament observada amb anticossos complets, sinó més aviat la va reduir. Els nivells d’apoE i J es van reduir amb el scFv-h3D6, però van augmentar amb apoE-MP i apoJ-MP. Com a conclusió general, el tractament amb scFv-h3D6 disminueix la càrrega d’Aβ, disminueix la captació d’ Aβ en astròcits, restaura els nivells de apoE i apoJ endògens i redueix la inflamació. Encara que la teràpia combinada amb MPs s'ha d'analitzar més, aquest treball demostra que tots els efectes del scFv-h3D6 estan dirigits a prevenir la patologia de l’Alzheimer, sense provocar els efectes perjudicials que es mostren amb el bapineuzumab en assajos clínics.ScFv-h3D6 is a single chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aβ-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway towards the worm-like one. In addition, it was shown to be effective at the behavioral, cellular, and molecular levels in the treatment of the mouse model of Alzheimer’s disease (AD) 3xTg-AD. Although its great potential, scFv-h3D6 production was limited by some bottlenecks like the presence of scrambled conformations generated in the refolding process and its contamination with endotoxins, which dramatically reduce the production yield and increase the cost. In the current work, several ways to improve protein production and thermodynamic stability have been explored. Firstly, disulphide bridges were eliminated to avoid scrambled conformations and, in this way, improve the protein purification yield and assure an homogenous protein conformation. Elimination of the disulphide bridge of the most stable domain, VH, solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm-like morphology to an amyloid one. Then, we have stabilized the scFv-h3D6 fold by introducing the mutation VH-K64R and combining it with the previously described elongation of the VL domain (C3). The stabilities of the different scFv-h3D6 constructs resulted in the order C3 > K64R/C3 > VH-K64R ≥ scFv-h3D6, showing that combination of both mutations was not additive but rather they partially canceled each other. Cytotoxicity assays showed that all the mutants recovered cell viability of Aβ-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody. Finally, a change in the expression system of the scFv-h3D6 was performed. The novel protein obtained in the eukaryotic system Pichia Pastoris maintained the same thermodynamic properties as the protein obtained from Escherichia coli. However, other properties were improved: homogeneity of the sample, absence of scrambled conformations, endotoxin free sample, better protein yield, easy purification and extrapolation to a larger scale. Therapeutic effectiveness was also demonstrated in vitro and in vivo. On the other hand, Aβ-Immunotherapy has long been studied in the treatment AD, but not how other molecules involved in the disease can affect antibody performance. To completely understand the possible interactions and effects among Aβ, scFv-h3D6, apoE and apoJ, we performed a complete study from all different perspectives: biophysical, cellular and in vivo. Instead of full-length apolipoproteins, we combined scFv-h3D6 with apolipoproteins (apo) E and J mimetic peptides (MP) composed of essential structures within these apolipoproteins for binding to other molecules. Biophysical studies showed that apoE-MP precluded the formation of protective WL fibrils by the scFv-h3D6/Aβ complex, and partially interfered with the scFv-h3D6-induced reduction in Aβ-uptake by astrocytes; whereas apoJ-MP allowed the formation of WL fibrils and did not interfere with the reduction in Aβ uptake. When looking at the inflammation associated with AD, apoE-MP developed an anti-inflammatory role in cell culture and in the 3xTg-AD mouse model; while apoJ-MP showed an anti-inflammatory profile only when combined with scFv-h3D6. ScFv-h3D6 alone did not induce microglial activation, contrary as previously shown with complete antibodies, but rather reduced it. Apolipoprotein E and J levels were reduced by scFv-h3D6, but increased by apoE-MP and apoJ-MP, affecting each other. As a general conclusion, scFv-h3D6 treatment decreases Aβ burden, decreases astrocytic Aβ uptake, restores endogenous apoE and apoJ levels and ameliorates inflammation. Although, combined therapy with MPs should be further analyzed, this work demonstrates that all the effects of scFv-h3D6 are addressed to preclude AD pathology, without eliciting the detrimental effects shown by bapineuzumab in clinical trials

Effects of an Aβ-antibody fragment on Aβ aggregation and astrocytic uptake are modulated by apolipoprotein E and J mimetic peptides

Aβ-Immunotherapy has long been studied in the treatment of Alzheimer’s disease (AD), but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aβ-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE) and J (apoJ) concentrations to non-pathological values, and Aβ internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aβ aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs). Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aβ were studied by CD. The conformational change induced by the interaction of Aβ with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aβ complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aβ uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aβ uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides