update 2013-08-31: this post is deprecated.  For a more recent treatment of this subject please see the following posts:

Anti-PrP vaccines November 18, 2012

Passive immunization against PrP April 8, 2013

MRC Prion Unit discusses possible clinical trial of PRN100 antibody July 31, 2013


IMMUNOTHERAPY

Abstracts/selections from papers addressing immunotherapy treatment possibilities for prion diseases in general.

 

“Monoclonal antibodies inhibit prion replication and delay the development of prion diseases,” White et al., Nature, 2003

http://www.ncbi.nlm.nih.gov/pubmed/12621436

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are fatal, neuro-degenerative disorders with no known therapy. A proportion of the UK population has been exposed to a bovine spongiform encephalopathy-like prion strain and are at risk of developing variant CJD. A hallmark of prion disease is the transformation of normal cellular prion protein (PrP(C)) into an infectious disease-associated isoform, PrP(Sc). Recent in vitro studies indicate that anti-PrP monoclonal antibodies with little or no affinity for PrP(Sc) can prevent the incorporation of PrP(C) into propagating prions. We therefore investigated in a murine scrapie model whether anti-PrP monoclonal antibodies show similar inhibitory effects on prion replication in vivo. We found that peripheral PrP(Sc) levels and prion infectivity were markedly reduced, even when the antibodies were first administered at the point of near maximal accumulation of PrP(Sc) in the spleen. Furthermore, animals in which the treatment was continued remained healthy for over 300 days after equivalent untreated animals had succumbed to the disease. These findings indicate that immunotherapeutic strategies for human prion diseases are worth pursuing.

 

MRC Prion Unit, London

http://www.prion.ucl.ac.uk/clinic-services/research/drug-treatments/

There are several other potential treatment options in the pipeline including agents that target the healthy form of the prion protein.

Immunotherapy has become increasingly relevant to neurodegenerative disorders over the past decade as successful strategies have been translated from murine models to human trials in the treatment of Alzheimer’s disease (AD).  Many thousands of AD patients are involved in advanced clinical trials of several different antibody therapies. Immunotherapy also represents one of the most promising possibilities for prion diseases as treatment of infected mice prior to the onset of clinical signs has a dramatically beneficial effect.  Passive immunotherapy refers to giving antibodies against the prion protein directly to patients.  A large number of anti-prion antibodies have been generated, two of these (ICSM18 and ICSM35) are currently being manufactured for clinical trials in the UK by the MRC Prion Unit.

 

“PAW35 Anti-prion protein monoclonal antibodies at low doses effectively treat prion disease in mice without side effects,”  Carswell et. al. inc. Collinge, J Neurol Neurosurg Psychiatry (2010)

https://iris.ucl.ac.uk/research/browse/show-publication?pub_id=269653&source_id=2

There has been intense interest in treating neurodegenerative conditions by passive immunotherapy. Here we describe the treatment of mice infected intraperitoneally (ip) with 104.6 infectious units of Rocky Mountain Laboratory (RML) prions, with bi-weekly injections of ip anti-prion monoclonal antibodies (mAbs). Mab ICSM18 (prion protein (PrP) epitope 143-153) raised to recombinant PrP, and ICSM35 (prion protein epitope 93-105) raised to an alternatively folded form of PrP, were both used at doses of 1 mg/week, or 0.25 mg/week. Treatment was given when prion infection was well established, 30 days after inoculation. Treatment resulted in a reduction in disease-related PrP (PrPSc) in the spleen and a significant extension in survival with both mAbs. ICSM18 showed a dose response and was more potent than ICSM35 with a 40% survival at 300 days postinfection using a dose of 1 mg/week. mAb treatment did not disrupt the normal function of the immune system. We have successfully demonstrated that the course of RML prion infection in mice can be safely attenuated by the early treatment with anti-PrP mAbs, at lower doses than previously reported. Humanised versions of these two mAbs will undergo safety-testing prior to manufacture for first-in-man study.

 

“CJD drugs could help Alzheimer’s patients,” The Telegraph, 2011

http://www.telegraph.co.uk/health/healthnews/8561836/CJD-drugs-could-help-Alzheimers-patients.html

Researchers already knew that in CJD, a “toxic substance” known as amyloid beta attaches itself to cells in the brain causes memory loss.

They also knew that two antibodies, ICSM-18 and ICSM-35, play a “crucial” role in stopping the amyloid proteins taking hold, and clinical trials to see if drugs based on them can help CJD patients are due to start in 2012.

Now, in a paper published in the journal Nature Communications, researchers say their experiments suggest that the two antibodies may also help block the onset of Alzheimer’s, the most common form of dementia.

Prof Dominic Walsh at University College Dublin said: “The use of these specific antibodies is particularly exciting since they have already undergone extensive pre-clinical testing for use in treating CJD. Thus a lot of basic work has already been done and could fast-track these antibodies for use in humans.

 

“Anti-PrP antibodies block PrP(Sc) replication in prion-infected cell cultures by accelerating PrP(c) degradation,” Perrier et. al., Journal of Neurochemistry (2004)

http://hal.archives-ouvertes.fr/docs/00/14/32/26/XHTML/index.xhtml

Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein and SAF61 directed against PrP amino acid residues (144–152), not only inhibited PrPSc formation in prion-infected neuroblastoma cells but also decreased the PrPC levels in non infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased the PrPC and the PrPSc levels in the cells, synergistically. In presence of both antibodies, our results showed that the mode of action which leads to the disappearance of the PrPSc in cells is directly coupled to PrPC degradation by reducing the half-life of the PrPC protein.

Co-treatment experiments using antibodies possessing epitopes quite far from one another in the PrP sequence maximizes the inhibitory effect. Indeed, treatment with both SAF34 and SAF61 antibodies decreased the PrPC and the PrPSc levels synergistically suggesting that combining several antibodies in treatments would be a more efficient in vivo therapeutic strategy

 

 

“A camelid anti-Prp antibody abrogates Prp(sc) replication in prion-permissive neuroblastoma cell lines,” Jones et. al., Plos One, (2010) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009804

The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrPSc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the “proof-of-principle” for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrPC. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrPSc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.

In this report, we show that PrioV3 anti-prion antibody was effective in crossing BBB, reduce peripheral prion replication in vivo and cured chronically scrapie-infected N2a cells and was also able to abolish prion replication. Finally, we also demonstrate here that PrioV3 antibody failed to trigger neurotoxic effects as previously shown with conventional anti-prion antibodies raised in mouse ([26], M. Tayebi and M. David, submitted).

The mechanism(s) of prion-mediated neuronal degeneration are not fully understood but it is believed that this is caused by increased PrPSc neurotoxicity, so-called “gain of function hypothesis” as prn-p−/− mice displayed no obvious phenotype [47], or by loss of PrPC function. It is likely that the apoptotic phenomenon observed after cross-linking of PrPC with ‘toxic’ antibodies mimics the prion-mediated degenerative process and leads to antibody-mediated loss of function. The favourable efficiency and toxicity property observed for PrioV3 together with its ability to cross the BBB and to enter the plasma membrane of affected neurons makes it an attractive weapon in the fight against prion and other related diseases.

 

Scripps Research Institute

http://www.scripps.edu/philanthropy/priondiseases.html  (2001)

The antibody Burton, Williamson and colleagues designed seems to halt the infection all together. The antibody, called Fab D18, binds to the normal form of prion protein and prevents the infectious form from binding in cell culture. Significantly, the normal cellular machinery degraded whatever infectious prions remained, suggesting that the antibody has the potential to cure established infection. The finding is also significant because it provides a potential therapeutic target – a highly effective human drug might be designed to bind to the same place as Fab D18.

The research article, “Antibodies inhibit prion propagation and clear cell cultures of prion infectivity,” appears in the August 16, 2001 issue of the British journal Nature and is authored by David Peretz, R. Anthony Williamson, Klotoshi Kaneko, Julie Vergara, Estelle Leclerc, Gerold Schmitt-Ulms, Ingrid R. Mehlhorn, Gluseppe Legname, Mark R. Wormald, Pauline M. Rudd, Raymond A. Dwek, Dennis R. Burton, and Stanley B. Prusiner.

 

“Antibodies inhibit prion propagation and clear cell cultures of prion infectivity,” Peretz et al., Nature (2001)

http://www.nature.com/nature/journal/v412/n6848/abs/412739a0.html\

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions1, 2. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

 

“Immunization delays the onset of prion disease in mice,” Sigurdsson et. al., American Journal of Pathology (2002)
http://www.ncbi.nlm.nih.gov/pubmed/12107084

The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has been shown to be effective in mouse models of another neurodegenerative condition, namely Alzheimer’s disease. Here we report that vaccination with recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized before exposure. The increase in the incubation period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a similar approach may work in humans or other mammalian species at risk for prion disease.

 

“Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo.”  Sadowski et. al., Neurobiological Disorders (2009)
http://www.ncbi.nlm.nih.gov/pubmed/19385058

In this study, we show that 6D11 anti-PrP monoclonal antibody (Mab) prevents infection on a FDC-P1 myeloid precursor cell line stably infected with 22L mouse adapted scrapie strain. Passive immunization of extracerebrally infected CD-1 mice with Mab 6D11 resulted in effective suppression of PrP(Sc) replication in the LRS. Although, a rebound of PrP(Sc) presence occurred when the Mab 6D11 treatment was stopped, passively immunized mice showed a prolongation of the incubation period by 36.9% (pb0.0001) and a significant decrease in CNS pathology compared to control groups receiving vehicle or murine IgG. Our results indicate that antibody-based therapeutic strategies can be used, even on a short-term basis, to delay or prevent disease in subjects accidentally exposed to prions.

 

“A prion protein epitope selective for the pathologically misfolded conformation,” Paramithiotis et. al. Nature Medicine (2003)

http://www.ufrsdv.u-bordeaux2.fr/siteIML/Maste1biosante/Master1biosantecours/Immunochimie-2oct-article1.pdf

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains.  We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine.  Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry.  Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein.  Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

 

“Antibody-mediated neural apoptosis: therapeutic implications for prion diseases,” Tayebi, Immunology Letters, 2006
http://www.sciencedirect.com/science/article/pii/S0165247806000897

Recent in vivo studies indicate that prion replication can be inhibited by anti-PrP monoclonal antibodies that led to the indefinite delay in the development of prion disease.

The recent report by Solforosi and colleagues has increased the need to understand pathway(s) leading to prion-associated apoptosis and neuronal death thought to be the cause of death in transmissible spongiform encephalopathy (TSE) individuals.

Furthermore, these reports increased momentum about the use of antibody-based therapy in prion diseases, although great caution should be exerted when using anti-prion antibodies directly into the central nervous system (CNS) with special emphasis on refined strategies such as specific targeting of regions of the prion protein thought not to be involved in signalling pathways.

 

“A role for B lymphocytes in anti-infective prion therapies?”  Tayebi, Expert Review of Anti-Infective Therapy, 2007
http://www.ingentaconnect.com/content/ftd/eri/2007/00000005/00000004/art00013

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of a number of neurodegenerative conditions affecting the nervous system. These disorders include prion and Alzheimer’s diseases and are of enormous importance for public health. It has become apparent over the last 20 years that specificity and application in prion diseases’ diagnostic and therapeutic situations are the most important considerations in designing strategies for the generation of antiprion antibodies. Specific antiprion therapeutics have been suggested and the establishment of the `proof-of-principle’ that the use of epitope-specific antiprion antibodies leads to indefinite delay of disease onset, has increased momentum for its use, although caution should be exerted prior to the application of new therapeutic strategies in a clinical set up. Furthermore, in vivo stimulation of immune-competent cells to specifically recognize and neutralize the abnormally folded isoform should also be pursued.

 

“Detection of prion epitopes on PrP(c) and PrP(Sc) of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP,” Yuan et al., Immunology and Cell Biology, 2005

http://www.nature.com/icb/journal/v83/n6/full/icb200588a.html

Amino acid residues 90–120 of the prion protein (PrP) are likely to be critical for the conversion of PrPc to PrPsc in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90–120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrPc and PrPsc. Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90–120 could detect PrPsc in ‘native’ and denatured forms and PrPc in normal cells, as well as recognize epitopes within PrP93–112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90–120 peptide, could detect PrPc and recognize epitopes within PrP93–107 residues. Four of these reagents could also detect denatured PrPsc on western blots but not PrPsc plaques in brain tissue. The results indicate that residues PrP93–102 are exposed in PrPc but are buried upon conversion to the PrPsc isoform. Furthermore, PrP103–107 residues are partially buried in PrPsc while only the PrP107–112 epitope remains exposed, suggesting that the region PrP93–112 undergoes conformational changes during its conversion to PrPsc.

“Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment,” Skrlj et al., Plos One (2010)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3024399/

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.

The present data describe the humanization of the antibody single-chain fragment (scFv) V5B2 and its characterization. To our knowledge, this is the first report of an anti-PrP mAb being humanized. We rationally designed four variants of humanized scFvs V5B2 by resurfacing of variable regions guided by computer modelling. By site-directed mutagenesis, human amino acid residues were stepwise introduced into murine variable regions. After being produced in E. coli using pMD204 expression vector [32], humanized antibody fragments were purified from the periplasm and their antigen-binding properties were analysed. The optimized construct was a scFv with 13 mutations introduced at key positions in the structure, which retained stability, binding specificity and affinity of the parent antibody. We believe that the recombinant humanized scFv with preserved functional properties of V5B2 could be used for designing new compounds with potentially diagnostic and therapeutic anti-prion properties.

Even though several antibodies have been resurfaced in the last decade, their immunogenicity remains undetermined, since no clinical data on resurfaced antibodies has been published yet [52]. Since the amino acid sequence of the humanized scFv was carefully designed and was found to be highly similar to human sequences, we believe that we efficiently removed all major immunogenic epitopes on the murine antibody. However, the actual immunogenicity could only be determined in clinical trials.

Immunotherapy based on anti-PrP antibodies is a promising strategy for the treatment of prion diseases. It has already been shown that some anti-PrP mAbs can antagonize prion propagation in vitro and in vivo, but only outside the brain, most likely due to very limited entry of large molecules into the central nervous system.

Single-chain fragments are much smaller than whole antibodies, but usually they retain specific monovalent antigen-binding affinity of the parent antibody, with improved pharmacokinetics for tissue penetration. Antibody fragments have already been reported to be successfully delivered to the central nervous system by intranasal administration [53], by virus-mediated gene transfer system [54] or by re-engineering as fusion proteins with BBB molecular Trojan horses [55]. Besides, antibody fragments appear to be more appropriate for TSE treatment than full antibodies, since bivalent anti-PrP antibodies have been shown to cross-link PrPC molecules and trigger neuronal apoptosis in certain neuronal populations [56]. It was also demonstrated that constant domains are unnecessary for antiprion effect, since Fab D18 [22], scFv 6H4 [57] and scFv D18 [58] all exhibited antiprion activity. A construct that targets PrPSc specifically could even be more efficient.

 

“Development of antibody fragments for immunotherapy of prion diseases,” Campana et al. inc. Prusiner, Biochemistry Journal (2009)

http://www.ncbi.nlm.nih.gov/pubmed/19000036

Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrP(C) and inducing its conversion into the disease-causing isoform denominated PrP(Sc). Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrP(Sc) content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv-PrP(C) complex. Interestingly, according to the docking results, Arg(PrP)(151) (Arg(151) from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrP(C) to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

 

Donofrio G, Heppner FL, Polymenidou M, Musahl C, Aguzzi A. Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies. J Virol. 2005;79:8330–8338.

http://www.ncbi.nlm.nih.gov/pubmed/19000036

Prion diseases are characterized by the deposition of PrPSc, an abnormal form of the cellular prion protein PrPC. A growing body of evidence suggests that antibodies to PrPC can antagonize deposition of PrPSc. However, host tolerance hampers the induction of immune responses to PrPC, and cross-linking of PrPC by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrPSc, even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.

Conversely, administration of antibodies generated in Prnp-ablated animals (“passive immunization”), while feasible and effective (43), suffers from the intrinsic problem of poor diffusion from vessels into tissues, particularly into the central nervous tissue. This may explain why administration of monoclonal antibodies has been shown to prevent prion pathogenesis only when applied simultaneously or shortly after peripheral prion infection (43). In addition, production of the large amounts of monoclonal antibodies necessary for the treatment of human patients is technically challenging and expensive (18, 25, 28).

Worryingly, intracerebral injection of anti-PrP immunoglobulin G (IgG) antibodies was found to provoke neurotoxicity by cross-linking PrPC (40).

We find that secretion of anti-PrP scFvs by mammalian cells cures chronically prion-infected N2a neuroblastoma cells (ScN2a). These results suggest that open reading frames (ORFs) encoding anti-PrP scFvs may be used in gene therapy against prion diseases.

Intracerebellar or intrahippocampal injection of monoclonal anti-PrP holoantibodies induces neurotoxicity, whereas injection of monovalent F(ab)1 fragments prepared from the same monoclonal antibodies was innocuous, suggesting that antibody-induced cross-linking of PrPC may be toxic (40). However, if toxicity is inherent to the bivalent character of monoclonal IgG holoantibodies, as the results mentioned above appear to suggest, one would expect that anti-PrP scFvs, which are monovalent and therefore cannot cross-link PrPC, should be safer than bivalent full-fledged antibodies.

The comparatively small size of the cDNA that encodes for the scFv described here (957 base pairs) makes it easy to efficiently package it into a variety of viral vectors.

 

“Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein,” Skrlj et. al, Applied Biochem Biotechnology (2009)
http://www.ncbi.nlm.nih.gov/pubmed/19597999

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

 

“Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases,” Padiolleau-Lefevre et al., Molecular Immunology (2007)
http://www.ncbi.nlm.nih.gov/pubmed/17140664

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.

 

“Applications of single chain variable fragment antibodies in therapeutics and diagnostics,” Weissner et al., Biotech Advances (2009)
http://www.ncbi.nlm.nih.gov/pubmed/19374944

Antibodies (Abs) are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical applications. This review will show the tremendous versatility and importance of scFv fragments as they provide the basic antigen binding unit for a multitude of engineered Abs for use as human therapeutics and diagnostics.

 

Other:
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Féraudet C, Morel N, Simon S et al. Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J. Biol. Chem. 2005; 280: 11 247–58.

Piccardo P, Langeveld JP, Hill AF et al. An antibody raised against a conserved sequence of the prion protein recognizes pathological isoforms in human and animal prion diseases, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Am. J. Pathol. 1998; 152: 1415–20.

Demart S, Fournier JG, Creminon C et al. New insight into abnormal prion protein using monoclonal antibodies. Biochem. Biophys. Res. Commun. 1999; 265: 652–7.