FC conjugated oligothiophenes

Abstract

A first aspect of the invention relates to a conjugate comprising an immunoglobulin Fc polypeptide, and an oligothiophene moiety. The invention further relates to the use of a conjugate according to the invention as set forth above in treatment and / or prevention of a disease, particularly in treatment and / or prevention of a neurodegenerative disease.

Description

Field The present invention relates to Fc conjugated oligothiophenes and their use as a pharmaceutical agent, particularly for treatment of a protein misfolding disease, e.g. a neurodegenerative disease associated with formation of plaques, such as prion diseases and Alzheimer’s disease. Background The nervous system is a complex network of cells and proteins that regulates bodily processes and cognitive functions. The delicate balance of this system can be disrupted by the accumulation of misfolded proteins, leading to a cascade of pathological events. Recent decades have witnessed significant advances in understanding the molecular mechanisms underlying neurodegenerative diseases, among which the aggregation of aberrantly folded proteins plays a central role. Diseases such as Alzheimer's and prion diseases or peripheral amyloidosis are characterized by the accumulation of toxic protein aggregates, leading to neuronal death and the progressive loss of neurological function. These aggregates, typically composed of specific misfolded proteins, exhibit a propensity to form beta-sheet rich fibrils that are resistant to proteolytic degradation, thereby persisting within neural tissues. The pathogenic process is further compounded by the cellular stress responses to these protein aggregates, including inflammation and oxidative stress, exacerbating neuronal damage and disease progression. Despite considerable research efforts, the precise mechanisms by which protein misfolding initiates and propagates within the brain remain only partially understood. Moreover, current therapeutic strategies have proven largely ineffective in reversing or halting disease progression, underscoring the urgent need for novel approaches. Oligo- or polythiophenes represent a class of conjugated polymers that consist of repeating units of thiophene, which imparts the material with semiconducting properties. The versatility of polythiophenes has led to their exploration in a variety of applications, ranging from organic solar cells and light-emitting diodes (LEDs) to field-effect transistors and sensors. In the context of cellular biology, the application of polythiophenes has extended into the realm of biosensor and imaging agent development. The conductive and fluorescent properties of certain polythiophene derivatives make them suitable for detecting biological molecules, including proteins and nucleic acids, thereby facilitating the study of cellular processes and the diagnosis of diseases. Moreover, the biocompatibility of some polythiophene derivatives has prompted investigations into their use as scaffolds for tissue engineering and as vehicles for targeted drug delivery. Notably, the interaction of polythiophenes with biological molecules, such as the aggregates of misfolded proteins involved in neurodegenerative diseases, has been explored to develop novel diagnostic tools. Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to treat or prevent protein misfolding diseases. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification. Summary of the Invention A first aspect of the invention relates to a conjugate comprising an immunoglobulin Fc polypeptide, and an oligothiophene moiety. The invention further relates to the use of a conjugate according to the invention as set forth above in treatment and / or prevention of a disease, particularly in treatment and / or prevention of a neurodegenerative disease. Terms and definitions General For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control. The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.” Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise. "And / or'' where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods. The term sortase in the context of the present specification relates to a class of enzymes that are responsible for attaching specific proteins to the peptidoglycan layer of the bacterial cell wall. The mechanism by which sortases operate involves recognizing and cleaving a specific peptide sequence within a target protein, and then catalyzing the covalent attachment of this protein to the cell wall. Several types of sortase enzymes are known, classified based on their structure and the specific functions they perform. The most well-studied type is Sortase A, which primarily attaches proteins involved in virulence and colonization to the cell wall. Other types, such as Sortase B and Sortase C, have roles in processes like pilus assembly and iron acquisition. The term terminal moiety in the context of the present specification relates to a chemical moiety capping the remaining chemical structure. The term prion fibril in the context of the present specification relates to a type of p-sheet-rich protein which binds to another in the same conformation, thereby stabilizing it and forming fibrils, leading to abnormal protein aggregates called amyloids. The term PBS in the context of the present specification relates to Phosphate Buffered Saline: NaCI: 8.0 g / L; KCI: 0.2 g / L; Na2HPO4: 1.44 g / L KH2PO4: 0.24 g / L, pH 7.4. Any patent document cited herein shall be deemed incorporated by reference herein in its entirety. Sequences Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ). One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et aL, J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively. Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence). Particular embodiments make use of the sequences as disclosed herein (i.e. 100% identical). General Biochemistry: Peptides, Amino Acid Sequences The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. The term peptide or oligopeptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15 amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds. Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd ed. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). The term variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in its primary amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. In the context of the present specification, the term dimer refers to a unit consisting of two subunits. In the context of the present specification, the term homodimer refers to a dimer comprised of two subunits that are either identical or are highly similar members of the same class of subunits. In the context of the present specification, the term amino acid linker or peptide linker refers to a polypeptide of variable length that is used to connect two polypeptides to generate a single chain polypeptide. Exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 1,2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. There is no constraint on the amino acid composition of the linker. In certain embodiments, the linker consists of amino acids selected from the group of G, S, A and D. An important characteristic of the conjugate peptide linkers as specified above are low immunogenicity, and a peptide length that allows the domains which are joined by the linker, to interact to form a functional entity as disclosed herein. In particular desirable embodiments of the domain peptide linkers specified above, the sequences are primarily made up of stretches of amino acids such as glycine (G) and serine (S). In certain embodiments peptide linker is >15 amino acids in length, particularly 15 to 30 amino acids in length wherein the amino acids are selected from G, S, A and D. A non-limiting example of an amino acid linker is a monomer or di-, tri- or tetramer of a peptide motif composed of three or four glycine and one serine. Any embodiments relating peptide linkers as disclosed herein, encompass structures in which amino acids with similar characteristics are exchanged, for example, the amino acids V, L, I, P, S, C, or M may replace G, S, or A, and D may be replaced by E. General Molecular Biology: Nucleic Acid Sequences, Expression The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art. The term transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another. In the present context, the term may also refer to transfer of the natural or physiologically intact variant of a genetic sequence into tissue of a patient where it is missing. It may further refer to transfer of a natural encoded sequence the expression of which is driven by a promoter absent or silenced in the targeted tissue. The term recombinant in the context of the present specification relates to a nucleic acid, which is the product of one or several steps of cloning, restriction and / or ligation and which is different from the naturally occurring nucleic acid. A recombinant virus particle comprises a recombinant nucleic acid. The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and / or protein product. The term nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or -in the case of an RNA construct being transfected- to translate the corresponding protein of interest from a transfected mRNA. For vectors operating on the level of transcription and subsequent translation, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell. The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of < 10'8mol / L (particularly < 10'9mol / L) when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure. In the context of the present specification, the term dissociation constant (Kd) is used in its meaning known in the art of chemistry and physics; it refers to an equilibrium constant that measures the propensity of a complex composed of [in most cases, two] different components to dissociate reversibly into its constituent components. The complex can be e.g. an antibodyantigen complex AbAg composed of antibody Ab and antigen Ag. Kd is expressed in molar concentration [mol / l] and corresponds to the concentration of [Ab] at which half of the binding sites of [Ag] are occupied, in other words, the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated according to the following formula: _ [Ab] * [Ag] D " [AbAg] [Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]: concentration of antibody-antigen complex In the context of the present specification, the terms off-rate (KOff;[1 / sec]) and on-rate (Kon; [L / (sec*mol)]) are used in their meaning known in the art of chemistry and physics; they refer to a rate constant that measures the dissociation (Koff) or association (Kon) of an antibody with its target antigen. KOff and Kon can be experimentally determined using methods well established in the art. A method for determining the KOff and Kon of an antibody employs surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore® or the ProteOn® system. They can also be used to determine the dissociation constant Kd by using the following formula: *D = KJ The natural upper limit for the on-rate Kon is 109 L / (sec*mol). In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen-binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (Vh) and a heavy chain constant region (Ch). The heavy chain constant region of IgG is comprised of three domains, Ch1, Ch2 and Ch3. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain. In the context of the present specification, the term fragment crystallizable (Fc) region refers to a fraction of an antibody comprising, if applied to IgG, two identical heavy chain fragments consisting of a Ch2 and a Ch3 domain, covalently linked by disulfide bonds. The heavy chain fragments, while being identical, in some embodiments may be linked or conjugated to distinct functional moieties to provide for multifunctional constructs. Organic Chemistry The formulae of the present specification follow the convention of organic chemistry to not show hydrogen atoms on carbon scaffolds. Carbon is tetravalent and bonds not shown are assumed to be hydrogen unless shown otherwise. Hydrogen can be exchanged for deuterium without changing the bulk chemical properties of the molecule; however, in the case of dye or drug molecules, the exchange of hydrogen for deuterium may lead to changes in the spectral properties or receptor interactions of the molecule. Unless explicitly stated otherwise herein, the disclosure of a formula showing, explicitly or implicitly by the convention restated in the first sentence of this paragraph, encompasses molecules in which one or several of the hydrogen atoms are exchanged for deuterium. The term alkyl in the context of the present specification relates to a saturated linear, branched or (partially or completely) cyclic hydrocarbon, wherein in certain embodiments one carboncarbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). The term unsubstituted Cn alkyl when used herein in the narrowest sense relates to the moiety -CnH2n-if used as a bridge between moieties of the molecule, or -CnH2n+i if used in the context of a terminal moiety. It may still contain fewer H atoms if used in the context of a cyclical structure. The term C1-C4 alkyl in the context of the present specification relates to a saturated linear or branched hydrocarbon having 1, 2, 3 or 4 carbon atoms. Non-limiting examples for a C1-C4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, cyclo-butyl, cyclo-propyl, methyl-cyclo-propyl. In certain embodiments, a C1-C4 alkyl is a methyl, ethyl, propyl or butyl moiety. The term alkene in the context of the present specification relates to a hydrocarbon comprising a double bond. Unsubstituted alkene is of formula -CH=CH- when being located intramolecularly, and of formula -CH-CH2 when being a terminal moiety. An unsubstituted alkene consists of C and H only. A substituted alkene may comprise substituents as defined herein for substituted alkyl. The term alkyne in the context of the present specification relates to a hydrocarbon comprising a triple bond. Unsubstituted alkyne is of formula -CHC- when being located intramolecularly, and of formula -CHCH (-C2H) when being a terminal moiety. An unsubstituted alkyne consists of C and H only. A substituted alkyne may comprise substituents as defined herein for substituted alkyl. The term carboxyl-substituted [group] refers to a moiety or group that is modified by one or several carboxyl groups COOH (also written as CCVor COONa). The term carboxyl substituted alkyl refers to an alkyl according to the above definition that is modified by one or several carboxyl groups COOH, or derivatives thereof, particularly carboxylamides CONH2, CONHR and CONR2, or carboxylic esters COOR. Particular embodiments of alkylcarboxyl (C1-C4 carboxyl) encompass formate (-COO') and acetate (-CH2 COO') in its acid or salt form. As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration. As used herein, the term pharmaceutically acceptable earner includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). The invention also encompasses nanoparticles, liposomes, or cellular carriers within the meaning of pharmaceutically acceptable carrier. Detailed Description of the Invention A first aspect of the invention relates to a conjugate comprising an immunoglobulin Fc polypeptide, and an oligothiophene moiety. The term conjugate in this context relates to a covalent linkage of the two components. As described herein, the oligothiophene moiety can bind to certain pathological protein aggregates, and the immunoglobulin Fc polypeptide facilitates clearance of these structures in a way that ameliorates or prevents disease associated with these pathological protein aggregates. In certain embodiments, the oligothiophene moiety is conjugated to the immunoglobulin Fc polypeptide through a linker moiety. The linker moiety may be present to allow some steric flexibility between the oligothiophene part and the Fc, and may likewise serve as an adaptor to link multiple oligothiophenes to one Fc moiety. In certain embodiments, the oligothiophene moiety is conjugated to a C-terminus of the immunoglobulin Fc polypeptide, optionally through a linker. In certain embodiments, the immunoglobulin Fc polypeptide and the oligothiophene moiety are linked covalently through a linker that is not cleavable under physiological conditions. The term physiological conditions in the context of the present specification relates to conditions inside the human body. In certain embodiments, the term physiological conditions relates to a pH-neutral (~pH 7) condition. The term not cleavable in the context of the present specification relates to stable covalent bonds of the linker moiety which remain stable under physiological conditions. Typical cleavable linkers can be classified into two subclasses, that is chemically labile linkers and enzyme-cleavable linkers. Chemically labile linkers can be further classified as acid-cleavable linkers and reducible or disulfide linkers. The linker of the present invention is neither chemically labile nor cleavable by an enzyme under physiological conditions. This means that the linker does not comprise a recognition site for a protease or a glucuronide bond, and does not comprise a disulfide bond or an acid-cleavable site. Suitable non-cleavable linkers are exemplary provided herein, as described also in Example 6. In certain embodiments, the (non-cleavable) linker is a poly-(AK)-linker. In certain embodiments, the (non-cleavable) linker comprises a polyethylene moiety. In certain embodiments, the (non-cleavable) linker is a poly-(AK)-linker and comprises a polyethylene moiety linking the Lys side chain to the oligothiophene moiety. In certain embodiments, the (non-cleavable) linker is a linker as depicted in Figure 22. In certain embodiments, the immunoglobulin Fc polypeptide is or comprises an immunoglobulin G fragment crystallizable region (IgG-Fc). Immunoglobulin gamma Fc are well explored as stabilizing moieties for therapeutic proteins, and ample knowledge exists with regard to their engineering. In certain embodiments, the IgG-Fc is a human IgG-Fc. Human sequences are preferred in applications relating to treatment of human patients for reasons of function and avoidance of anti-drug antibody formation. In certain embodiments, the IgG-Fc is an lgG1-Fc. In certain embodiments, the IgG-Fc is an lgG4-Fc. The inventors performed experiments shown in the Example section with murine lgG1-Fc, which bears functional similarities to human lgG4-Fc. In certain embodiments, the immunoglobulin Fc polypeptide consist of an amino acid sequence >85% identical, particularly >90% identical, more particularly >95% identical, even more particularly >97% identical, yet even more particularly >98% identical, most particularly >99% identical to (SEQ ID NO: 2) (Fc sequences without sortase recognition site). In certain embodiments, the immunoglobulin Fc polypeptide further comprises a C-terminal sortase A binding motif portion. Sortase enzymes recognize and cleave specific amino acid sequences in their substrate proteins, generally located near the C-terminus. The recognition sequence varies slightly among different types of sortase enzymes, but they typically include a conserved motif. For Sortase A, the most studied type, the recognition sequence is often described as LPXTG (X can be any amino acid). After recognizing this sequence, Sortase A cleaves between the T and G residues, and then catalyzes the formation of a covalent bond between the carboxyl group of Threonine and an amino group within the peptidoglycan layer of the cell wall, effectively anchoring the protein to the bacterial surface. Other sortase enzymes have variations on this motif. In certain embodiments, the sortase A binding motif portion is LPXT, wherein X is E or A, particularly X is E. In certain embodiments, the sortase A binding motif portion is linked to the immunoglobulin Fc polypeptide through an amino acid linker, particularly a 5 to 20 amino acid linker, more particularly a 5 to 10 amino acid linker, even more particularly GGGGS (SEQ ID NO: 3). The inventors employed a pentamer of glycine linked to an ethine moiety through an ethylene glycol moiety (see Fig. 3). The inventors determined experimentally that the optimal linker size is < 20 residues. In certain embodiments, the immunoglobulin Fc polypeptide is a homodimeric Ig-Fc. In the context of the present specification, the term immunoglobulin Fc refers to a portion of an antibody comprising a single heavy chain fragment consisting of a Ch2 and a Ch3 domain. When applied to IgG, immunoglobulin Fc comprises one of the two identical heavy chain fragments of a fragment crystallizable (Fc) region. In the context of the present specification, the term homodimeric Ig-Fc refers to two identical immunoglobulin Fc. These two immunoglobulin Fc may be covalently linked by disulfide bonds. In certain embodiments the conjugate comprises a homodimeric Ig-Fc. In certain embodiments, a homodimeric Ig-Fc comprises two identical immunoglobuline Fc. In certain embodiments, a homodimeric Ig-Fc comprises two identical immunoglobuline Fc, wherein the two identical immunoglobuline Fc are covalently linked by disulfide bonds. In certain embodiments, each monomer of the homodimeric Ig-Fc is conjugated to at least one oligothiophene moiety. In certain embodiments each monomer of the homodimeric Ig-Fc is conjugated to an oligothiophene moiety. In certain embodiments an oligothiophene moiety is conjugated to each monomer of the homodimeric Ig-Fc. In certain embodiments, a plurality of oligothiophene moieties are conjugated to each monomer of the homodimeric Ig-Fc. In certain embodiments, the oligothiophene moiety comprises a sub-moiety (compound) of a general formula (I): n wherein n is an integer denoting a number of thiophene monomer subunits selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, wherein -  each R1 is independently selected from: H, C1-C4 carboxyl; -  each R2 is independently selected from: H, C1-C4 carboxyl; - Rl designates the bond to the immunoglobulin Fc polypeptide, optionally through a linker, particularly a manifold linker; - Re is a terminal moiety. In particular embodiments thereof, R1 and R2 are H. In other particular embodiments thereof, one of R1 and R2 is H and the other one is acetic acid / acetate (-CH2COOH I-CH2COO). In some embodiments of an oligothiophene moiety used herein, in some of the thiophene residues of the oligothiophene, both R1 and R2 are H and in other thiophene residues of the same oligothiophene, one of R1 and R2 is H and the other one is acetic acid / acetate (-CH2COOH I-CH2COO ). In certain embodiments, the terminal moiety RE is selected from H, Ci to C4 alkyl or Ci to C4 carboxyalkyl or a salt thereof or wherein 0 Rw, Rx, RY and Rz are independently selected from CH, N, NRS, CRT; 0 Ru is selected from CH2, S, NH, NRS, CRT, wherein ■ Rs and RT are independently selected from C1-C4 alkyl or C2-C6 alkene; and wherein 0 only one of CH, N, NRS or CRT is covalently bound to a thiophene directly 5                      preceding the terminal moiety RE. In certain embodiments, RE is C-i-Ce carboxyl, particularly COOH. In certain embodiments, RE is an azaindole, particularly 6-azaindole. In certain embodiments, RE is benzothiazole. The heterocycle Re may influence the selectivity of the ligand. Azaindole (HS-276) is selective 10 for A-beta, while benzothiazole (b-TVBT2) is selective for A-tau, see Klingstedt et al. ChemBioChem 2021,22, 2568 - 2581. In certain embodiments, RE is the oligothiophene moiety is selected from a group consisting of: In certain embodiments, RE is the oligothiophene moiety is In certain embodiments, RL comprises an oligo(ethylene glycol) conjugating the oligothiophene moiety to the immunoglobulin Fc polypeptide. This connection may optionally be made through a linker. In certain embodiments, more than one oligothiophene moiety is present in the conjugate per immunoglobulin Fc polypeptide. The principle is illustrated by a IgG Fc, containing two Fc chains as a homodimer, to which five oligothiophene moieties are attached each, resulting in a total often oligothiophene moieties. In certain embodiments, 1 to 25 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. In certain embodiments, 2 to 20, particularly 5 to 15 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. In certain embodiments, wherein 5 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. In certain embodiments, the conjugate further comprises a linker, covalently connecting the immunoglobulin Fc polypeptide and the oligothiophene moiety. In certain embodiments, the linker is a manifold linker that allows attachment of more than one oligothiophene moiety to one immunoglobulin Fc polypeptide. Polyvalent (manifold) linkers are known to the skilled artisan and include, but are not limited to, star-shaped oligo(ethylene glycol) and side-chain functionalized poly-lysine moieties. In certain embodiments, the manifold linker comprises an oligopeptide, and an N-terminus of the manifold linker binds the C-terminus of the immunoglobulin Fc polypeptide. Le. the manifold linker connects the oligothiophene moiety to the C-terminus of the immunoglobulin Fc polypeptide. The manifold linker may be connected to the C-terminus of the immunoglobulin Fc polypeptide through a short (2 to 15) amino acid linker sequence as defined in the terms section above; the inventors used a pentamer of glycine moieties to connect the manifold linker to the sortase motif as shown in the Examples. In certain embodiments, the manifold linker comprises an oligopeptide moiety constituted of amino acids bearing an oligothiophene moiety on their side chain. In certain embodiments, the of amino acid bearing an oligothiophene moiety on their side chain is a lysine bearing an oligothiophene moiety connected to the e-amino group of the lysine through a bridging moiety consisting of 2 to 50 atoms selected from C, O, N and S. In certain embodiments, an immunoglobulin Fc polypeptide to manifold linker ratio Fc / L is 1 / 1. In certain embodiments, a homodimeric Ig-Fc to manifold linker ratio Ig-Fc / L is 1 / 2. The manifold linker is distinct from the sortase sequence that may enable the regiospecific covalent attachment of the manifold linker to the Fc immunoglobulin. Triazole is part of the linker in the Examples shown herein, but the skilled artisan will realize that other linker chemistries are possible and may indeed be preferable both from an economic and physiochemical point of view, as they should be less likely to pose regulatory issues. Other possible linkers or components from which linkers may be constituted include, but are not limited, to alkyl moieties, alkylether, peptide / peptoid (e.g. poly-L-lysine (PLL), poly glycine (PG)), esters such as pentafluorophenyl (PFP); combinations of the afore mentioned once, also with polyethylene glycol (PEG). Linker chemistry: The oligothiophene used in the examples is functionalized with an azide moiety, so that the linker can be added by copper-catalyzed click chemistry (alkyne) or DBCO-NHS-ester chemistry. Coupling to the Fc may also be affected in different ways, including but not limited to amide linker chemistry, SMCC (NHS ester and maleimide), EDC. To avoid interference with the antibody effector functionalities imparted by the Fc sequence, the inventors chose to link the thiophene bearing moiety through the sortase reaction, as it is controlled and site specific. In certain embodiments, the conjugate comprises a first affinity to a Fc receptor, particularly a Fey receptor, more particularly to a Fcyl receptor, most particularly a human Fcyl receptor or a murine Fcyl receptor. In certain embodiments, the conjugate comprises a first affinity to a Fc receptor, wherein the affinity is characterized by the following values: Probe Analyte ko„[M-1s1] Ms’1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 1*E+04 to 1+E06 3*E-03 to 3*E-05 3*E-07 to 3*E-11 50 to 60 0.1 to 0.5 In certain particular embodiments, the first affinity to a Fc receptor is characterized by the following values: Probe Analyte MM’1 s’1] Ms’1] Ko[M] RU (max) Chi2 Fcyl receptor conjuga te 9.072*E04 2.65*E-04 2.92*E-09 56.4 0.21 In certain embodiments, the conjugate comprises a second affinity to a prion fibril. In certain embodiments, the conjugate is characterized by a phagocytic index (PIconjugate), characterized in that Plconjugate > PllgG1-Fc and / or Plconjugate > Plp-FTAA. In certain embodiments, PlCOnjugate > (PhgGi-Fc + PIp-ftaa). Another aspect of the invention relates to the use of a conjugate according to the invention as set forth above, in a method of treatment and / or prevention of a disease. Yet another aspect of the invention relates to the use of a conjugate according to the invention as set forth above, in a method of treatment and / or prevention of a neurodegenerative disease or an amyloidosis. In certain embodiments, the neurodegenerative disease is selected from a group consisting of: Alzheimer’s disease, a prion disease and a systemic amyloidosis. In certain particular embodiments, the prion disease is selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS). Likewise, the invention provides the use of the conjugate according to the invention for manufacture of a medicament for treatment and / or prevention of a neurodegenerative disease. Likewise, the invention provides the use of the conjugate according to the invention for manufacture of a medicament for treatment and / or prevention of amyloidosis. Also provided is a composition comprising a conjugate according to any one the embodiments disclosed herein, and a pharmaceutically acceptable excipient. Pharmaceutical Compositions, Administration / Dosage Forms and Salts According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient. The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc. In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product. Similarly, the invention provides a dosage form for the prevention or treatment of an indication selected from Alzheimer’s disease, a prion disease and an amyloidosis. Particular embodiments relate to a dosage form for treatment of a prion disease selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS), comprising a conjugate according to any of the above aspects or embodiments of the invention. Certain embodiments of the invention relate to a dosage form for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present. The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892). Method of Manufacture and Method of Treatment according to the invention The invention further encompasses, as an additional aspect, the use of a conjugate agent as identified herein, for use in a method of manufacture of a medicament for the treatment or prevention of a condition selected from the group consisting of Alzheimer’s disease, a prion disease and an amyloidosis. Particular embodiments relate to a method of manufacture of a medicament for the treatment of a prion disease selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS). Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with Alzheimer’s disease or a prion disease. This method entails administering to the patient an effective amount of a conjugate agent as identified herein. Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. The specification further encompasses the following items: Items: 1. A conjugate comprising a. an immunoglobulin Fc polypeptide, and b. an oligothiophene moiety. 2. The conjugate according to item 1, wherein the immunoglobulin Fc polypeptide and the oligothiophene moiety are linked covalently through a linker that is not cleavable under physiological conditions. 3. The conjugate according to item 1 or 2, wherein the oligothiophene moiety is conjugated to a C-terminus of the immunoglobulin Fc polypeptide. 4. The conjugate according to any one of the preceding items, wherein the immunoglobulin Fc polypeptide comprises an immunoglobulin G fragment crystallizable region (IgG-Fc). 5. The conjugate according to item 4, wherein the IgG-Fc is a human IgG-Fc. 6. The conjugate according to any one of the preceding items 4 or 5, wherein the IgG-Fc is an lgG1-Fc. 7. The conjugate according to any one of the preceding items 4 or 5, wherein the IgG-Fc is an lgG4-Fc. 8. The conjugate according to any one of the preceding items, wherein the immunoglobulin Fc polypeptide consists of an amino acid sequence 285% identical, particularly >90% identical, more particularly >95% identical, even more particularly 297% identical, yet even more particularly 298% identical, most particularly 299% identical to (SEQ ID NO: 2). 9. The conjugate according to any one of the preceding items, wherein the immunoglobulin Fc polypeptide further comprises a C-terminal sortase A binding motif portion. 10. The conjugate according to any one of the preceding items, wherein the sortase A binding motif portion is LPXT, wherein X is E or A, particularly X is E. 11. The conjugate according to any one of the preceding items, wherein the sortase A binding motif portion is linked to the immunoglobulin Fc polypeptide through an amino acid linker, particularly a 5 to 20 amino acid linker, more particularly a 5 to 10 amino acid linker, even more particularly GGGGS (SEQ ID NO: 3). 12. The conjugate according to any one of the preceding items, wherein the immunoglobulin Fc polypeptide is a homodimeric Ig-Fc. 13. The conjugate according to any one of the preceding items, wherein each monomer of the homodimeric Ig-Fc is conjugated to an oligothiophene moiety. 14. The conjugate according to any one of the preceding items, wherein the oligothiophene moiety comprises a sub-moiety of a general formula (I): n wherein n is an integer denoting a number of thiophene monomer subunits selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, wherein - R1 is independently selected from: H, C1-C4 carboxyl; - R2 is independently selected from: H, C1-C4 carboxyl; - RL designates the bond to the immunoglobulin Fc polypeptide, optionally through a manifold linker; - Re is a terminal moiety. 15. The conjugate according to item 14, wherein the terminal moiety RE is selected from H, II Ci to C4 alkyl or Ci to C4 carboxyalkyl or a salt thereof or             K , wherein 0 Rw, Rx, Ry and Rz are independently selected from CH, N, NRS, CRT; 0 Ru is selected from CH2, S, NH, NRS, CRT, wherein ■ Rs and RT are independently selected from C1-C4 alkyl or C2-C6 alkene; and wherein 0 only one of CH, N, NRS or CRT is covalently bound to a thiophene directly preceding the terminal moiety RE. 16. The conjugate according to any one of the preceding items 14 to 15, wherein RE is selected from: - Ci-Ce carboxyl, particularly COOH or COO'; - an azaindole, particularly 6-azaindole; - benzothiazole. 17. The conjugate according to any one of the preceding items 14 to 16, wherein the oligothiophene moiety is selected from a group consisting of: 10 19. The conjugate according to any one of the preceding items 14 to 18, wherein RL 15          comprises an oligo(ethyleneglycol) conjugating the oligothiophene moiety to the immunoglobulin Fc polypeptide. 20. The conjugate according to any one of the preceding items, wherein more than one oligothiophene moiety is present in the conjugate per immunoglobulin Fc polypeptide. 21. The conjugate according to item 20, wherein 1 to 25 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. 22. The conjugate according to item 21, wherein 2 to 20, particularly 5 to 15 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. 23. The conjugate according to item 20, wherein 5 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide. 24. The conjugate according to any one of the preceding items, wherein the conjugate further comprises a manifold linker, connecting the immunoglobulin Fc polypeptide and the oligothiophene moiety. 25. The conjugate according to item 24, wherein the manifold linker allows attachment of more than one oligothiophene moiety to one immunoglobulin Fc polypeptide. 26. The conjugate according to item 24 or 25, wherein the manifold linker comprises an oligopeptide, and an N-terminus of the linker binds the C-terminus of the immunoglobulin Fc polypeptide. 27. The conjugate according to any one of the preceding items 24 to 26, wherein the manifold linker comprises an oligopeptide moiety constituted of amino acids bearing an oligothiophene moiety on their side chain. 28. The conjugate according to item 27, wherein the of amino acid bearing an oligothiophene moiety on their side chain is a lysine bearing an oligothiophene moiety connected to the s-amino group of the lysine through a bridging moiety consisting of 2 to 50 atoms selected from C, O, N and S. 29. The conjugate according to any one of the preceding items 24 to 28, wherein an immunoglobulin Fc polypeptide to linker ratio Fc / L is 1 / 1. 30. The conjugate according to any one of the preceding items 24 to 29, wherein a homodimeric Ig-Fc to linker ratio Ig-Fc / L is 1 / 2. 31. The conjugate according to any one of the preceding items, wherein the conjugate comprises a first affinity to an Fc receptor, wherein the affinity is characterized by the values in the following table: Probe Analyte kon[M-1s1] Aoff[s-1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 1*E+04 to 1+E06 3*E-03 to 3*E-05 3*E-07 to 3*E-11 50 to 60 0.1 to 0.5 32. The conjugate according to item 30, wherein the first affinity to a Fc receptor is characterized by the values in the following table: Probe Analyte MM'1 s'1] Aoff[s’1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 9.072*E04 2.65*E-04 2.92*E-09 56.4 0.21 33. The conjugate according to any one of the preceding items, wherein the conjugate comprises a second affinity to a prion fibril. 34. The conjugate according to any one of the preceding items, wherein the conjugate is characterized by a phagocytic index (Plconjugate), characterized in that Plconjugate > PhgGi-Fc and / or PI conjugate > Plp-FTAA. 35. The conjugate according to any one of the preceding items, wherein Pleonjugate > (PhgGi-Fc + Plp-FTAA). 36. The conjugate according to any one of the preceding items, for use in treatment and / or prevention of a disease. 37. The conjugate for use according to item 36, wherein the disease is a neurodegenerative disease or amyloidosis. 38. The conjugate for according to item 36 and 37, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer’s disease and a prion disease. 39. The conjugate for use according to any one of items 36 to 38, wherein the prion disease is selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS). 40. Use of the conjugate according any one of items 1-35 in a method of manufacture of a medicament for treatment and / or prevention of a neurodegenerative disease or amyloidosis. 41. The use according to item 40, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer’s disease and a prion disease. 42. The use according to item 40 and 41, wherein the prion disease is selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS). 43. A composition comprising a conjugate according to any one of items 1-35 and a pharmaceutically acceptable excipient. The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. Description of the Figures Fig. 1 shows production of Sortase. A) SDS-PAGE of bacteria culture supernatant during sortase expression. Sample were collected 3h, 5h and overnight before and after IPTG induction; B) SDS-PAGE of selected fractions of sortase purification using Ni-NTA affinity chromatography; C) SDS-PAGE of purified sortase with different amount. Fig. 2 shows production of modified Fc. A) Modified mouse Fc plasmid including the IL2 signal sequence, the sortase recognition site and His-tag for affinity purification. Original plasmid included an EF-1a core promoter and resistance to Zeocin. B) SDS-PAGE of fractions from Fc affinity purification. Fig. 3 shows A) Chemical structure of p-FTAA-azide. B) Sortase-mediated transpeptidation to tag the linker to the C-terminal of the Fc; the sortase recognizes the LPETG (SEQ ID NO: 5) sequence in the modified Fc, cleaves the peptide after ‘G’ and attaches itself onto the terminal as the intermediate; then a polyglycine linker attacks the bond after LPET (SEQ ID NO: 8) and forms the final product Fc-linker.; GGHHHHHH (SEQ ID NO 19), HHHHHHGGTEPLSGGGG (SEQ ID NO 24), GGGGSLPETGGHHHHHH (SEQ ID NO 4), GGGGSLPET (SEQ ID NO 21), TEPLSGGGG (SEQ ID NO 22), GGGGG (SEQ ID NO 20). C) Simplified scheme of the alkyne-azide cycloaddtion click chemistry reaction; under the catalysis of Cu(l), the azide on the p-FTAA-azide and the alkyne on the Fc-linker forms a 1,2,3- triazole. This scheme only represents one alkyne on the Fc-linker but in real reaction all the 10 alkynes can be coupled. Fig. 4 shows production of an exemplary luminescent conjugated polythiophene (LCP) Fc-LCP conjugate. A) Sortase reaction with only Fc resulted in a Fc- intermediate. B) Sortase reaction with the Fc and different concentrations of linkers. The highest concentration (400 fold) of linker gave the highest yield of Fc-linker. C) Azide-alkyne cycloaddition click chemistry reaction between the Fc-linkerand the p-FTAA-azide. The Fc-LCP has a higher molecular weight. D) Full reaction steps to generate the Fc-LCP conjugate from Fc, the Fc-intermediate, the Fc-linker and the Fc-LCP. Fig. 5 shows measurement of the concentration of the Fc-LCP by spectrometer. A) The wavelength of absorbance is set from 230-500nm. Fc has a peak at 280nm, and LCP (p-FTAA) has a peak at 420nm. B) Linear model to calculate the concentration of LCP (p-FTAA) based on the absorbance at 420nm. Fig. 6 shows Fc-LCP has high affinity to the Fcyl receptor. SPR results show that the affinity between the Fc-LCP to the Fcyl receptor and the affinity between unconjugated Fc to the Fcyl receptor. Fig. 7 shows that the Fc-LCP can recognize and bind to prion and A0 fibrils. A) Immunoprecipitation of the Fc-LCP with RML6 and NBH showed that Fc-LCP binds to prion fibrils but not prion monomers. Non-PK digested brain homogenates were used as references. B) The sandwich ELISA results demonstrated that the Fc-LCP binds to A0 fibrils but not A0 monomer. Fig. 8 shows Fc-LCP can bind to A0 plaques in APP / PS1 mouse brain slides. A) Staining on APP / PS1 mouse brain slides with the Fc-LCP. Fc is counterstained with Alexa647 conjugated goat anti-mouse IgG; upper lane and lower lane with different magnificence; scale bar: 20pm. B) Upper lane: staining of LCP on APP / PS1 mouse brain slides; lower lane: staining of Fc on APP / PS1 mouse brain slides; scale bar: 20pm C) Upper lane: staining of LCP on B57C6J mouse brain slides; lower lane: staining of Fc-LCP on B57C6J mouse brain slides; scale bar: 20pm. DAPI channel: 405nm, LCP channel: 488nm, anti-Fc channel: 647nm. Fig. 9 shows that Fc-LCP stopped the primary nucleation of prion and a-syn fibril formation. A,B) The Fc-LCP stopped rPrP fibril formation in a dose-dependent manner and had a better performance compared to LCP only; C,D) The Fc-LCP stopped a-syn fibril formation in a dose-dependent manner and had a better performance compared with LCP only. Black: ThT, Thioflavin T; yellow: blank; green: LCP dilutions; pink: Fc-LCP dilutions (n = 3 replicates per group). Fig. 10 shows imaging flow cytometry-based phagocytosis assay and controls. A) Panels in imaging flow cytometry (channel 1: bright field; channel 2: 488nm for LCP). B) Imaging flow cytometry gated for BV2 phagocytosis with rPrP fibrils + LCP, only LCP, and rPrP monomers + LCP. C) Phagocytosis with BV2 could be completely blocked by cytochalasin D. D) Pilot BV2 phagocytosis assay using insulin fibrils at different concentrations and different time points to set up the 5                experimental parameters showed that 2.4pM and 1h are optimal for the phagocytosis assay. Fig. 11 shows Fc-LCP enhanced microglial phagocytosis for rPrP and A0-42 fibrils. A-B). C) Fc-LCP enhanced phagpcytosis for 1.5 pM rPrP fibrils in BV2. D) Fc-LCP enhanced primary microglia phagocytosis for 1.5 pM rPrP fibrils. E) Fc-LCP 10                enhance BV2 phagocytosis for 5pM A0-42 fibrils. F) Dose-dependent curve of BV2 phagocytosis for 5pM A0-42 fibrils with Fc-LCP, normalized with 1pM A0-42 fibrils labeled with LCP. Fig. 12 shows schematic of COCS experiment for therapeutic efficacy of Fc-LCP in prion disease. 15 Fig. 13 shows The Fc-LCP treatment reduced the prion load and protected neurons from prion toxicity in RML6-infected COCS. A) Representative PKWB image showed that the Fc-LCP treatment reduced the amount of prions in RML6 infected COCS compared to the controls including PBS, Fc and LCP, respectively. B) Representative WB image showed that the Fc-LCP treatment 20                 also reduced the level of total PrP in RML6-infected COCS. C) Representative WB image and statistical analysis showed that the Fc-LCP treatment did not affect the level of neurons in NBH treated COCS, but protected neurons in RML6 infected COCS compared with the other three controls. Actin was used as loading standard, and POM1 and NeuN as detection antibody. For statistical 25                 analysis, the NeuN was normalized to the actin. Fig. 14 shows Fc-LCP protected neurons from prion toxicity in RML6 infected COCS. A) Immunostaining using the neuronal marker NeuN and DAPI on NBH-treated COCS treated with PBS, Fc, LCP, and the Fc-LCP, respectively. There is no significant difference of the NeuN level between the Fc-LCP and other three 30                control groups (PBS, Fc and LCP). scale bar: 200pm. B) Immunostaining using the neuronal marker NeuN and DAPI on RML6-infected COCS treated with PBS, Fc, LCP, and the Fc-LCP. The NeuN area is significantly higher for the Fc-LCP treated and RML6 infected COCS than for treatment with the other three groups (PBS, Fc and LCP). scale bar: 200pm. C) Immunostaining using 35               the neuronal marker NeuN and DAPI on NBH treated COCS and RML6-infected COCS treated with the Fc-LCP. The effective concentration of the Fc-LCP is below 0.25|jM. scale bar: 500|jm. D). Immunostaining using the neuronal marker NeuN and DAPI on NBH treated COCS and RML6-infected COCS treated with the Fc-LCP. The Fc-LCP protected neurons at early (14dpi) and late stage (30dpi) from prion toxicity, scale bar: 500pm. Fig. 15 shows the effect of microglia depletion on the Fc-LCP treatment in RML6-infected COCS. A) Treatments (DMSO, DMSO + 1pM Fc-LCP, 1pM PLX5622, 1 pM PLX5622 + 1 pM Fc-LCP) were started 14 dpi to RML-6 infected COCS. B) PLX5622 treatment resulted in higher prion levels compared to DMSO treatment, indicating microglia’s protective role in prion pathology. C) After PK digestion, no prions were detected in all Fc-LCP treated groups, despite the PLX5622 treatment. D) PLX5622 + Fc-LCP treatment resulted in more total prion protein compared to DMSO + Fc-LCP treatment. However, when compared to the PLX5622 treated groups, the Fc-LCP cleared more prions despite the presence of PLX5622. E) WB for the neuronal marker NeuN, microglial marker Iba1, astrocyte marker GFAP and actin. For NeuN, Fc-LCP treatment rescued more neurons whereas there was no difference for the treatment with DMSO and PLX5622, DMSO + Fc-LCP and PLX5622 + Fc-LCP. For Iba1, PLX5622 depleted most microglia which could partially by the treatment with the Fc-LCP. For GFAP, both PLX5622 and Fc-LCP treatment reduced the level of GFAP. NeuN, Iba 1 and GFAP were normalized to the actin level used as loading control. Fig. 16 shows Fc-LCP protected neurons from prion toxicity despite of microglia depletion A) DMSO and 1 pM PLX5622 treatment to RML6-infected COCS started on the same day of prion infection (day 0); DMSO + 1pM Fc-LCP and 1 pM PLX5622 + 1 pM Fc-LCP treatment to RML6-infected COCS started 14 dpi. B) Immunostaining using the neuronal marker NeuN and DAPI on RML6-infected COCS treated with DMSO, 1pM PLX5622, DMSO + 1pM Fc-LCP and 1pM PLX5622 + 1pM Fc-LCP. PLX5622 treated RML6-infected COCS showed more disrupted morphological changes compared with DMSO treated COCS; neurons were protected from prion toxicity in the Fc-LCP treated RML6-infected COCS and the effect was partially cancelled by PLX5622 induced microglia depletion. Fig. 17 shows that early PLX5622 treatment completely depleted microglia and partially cancelled the efficacy of the Fc-LCP. Immunostaining using the microglial marker Iba1, the neuronal marker NeuN and DAPI on RML6-infected COCS treated with DMSO, 2pM PLX5622, DMSO + 1pM Fc-LCP and 2pM PLX5622 + 1pM Fc-LCP. A) Images taken at the middle level of COCS showed that PLX5622 induce complete microglia depletion despite the Fc-LCP treatment but only partially cancelled the prion removal efficacy of the Fc-LCP. B) Images taken at the surface level of COCS showed that PLX5622 did not completely deplete microglia when the Fc-LCP is present Fig. 18 shows original Linker-LCP design. Peptide backbone is GGGGGKKKKK (SEQ ID NO 23) (the 5x glycine is the recognition site of Sortase). Each lysine is supposed to be coupled to one p-FTAA molecule via click chemistry. Fig. 19 shows new Linker-LCP. Peptide backbone contains GGGGG (SEQ ID NO 20). Different numbers of lysine are added at C terminal. Each lysine is supposed to be coupled to one p-FTAA molecule via DBCO-mediated click chemistry. This linker-LCP allows to add different numbers of LCPs and the product can be homogeneous. Fig. 20 shows cysteine-based conjugation. The thiol group on the engineered cysteine at C-terminal of Fc will react with linker-LCP which starts with a maleimide head. The rest of the linker remains the same as previous linker-LCP. Fig. 21 shows the Q tag method. Fig. 22 shows PEG-comprising linkers. Fig. 23 shows BV2 phagocytosis assay on Abeta40 / 42 fibrils. Both lgG1 Fc-LCP and lgG2a Fc-LCP enhanced the phagocytosis of Abeta40 / 42 fibrils. Fig. 24 shows BV2 phagocytosis assay on tau fibrils. Both IgG 1 Fc-LCP and lgG2a Fc-LCP enhanced the phagocytosis of tau fibrils. Fig. 25 shows staining of Fc-LCP on human tissue samples from A4 amyloidosis brain, ATTR amyloidosis heart and AA amyloidosis testicle (The Zurich Ethics Committee approved this part of the project, BASEC2019-01479). Examples Example 1: Generation of Fc-LCP conjugate Production of the sortase enzyme A modified Ca2+-dependent Staphylococcus aureus (S. aureus) sortase A (Chen et al., Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404) was firstly produced to transfer the linker onto the Fc. Sortase was expressed by transforming the sortase plasmid into competent BL21(DE3) E. coll. 3h, 5h and overnight bacteria samples including before and after IPTG induction were collected to check the IPTG-induction efficiency (Figure 1A). A C-terminal 6x histidine tag enabled sortase purification with nickel affinity chromatography (Figure 1B and C). After purification, sortase aliquots were snap frozen and stored at - 80°C. Production of modified Fc To make the mouse lgG1-Fc suitable for sortase reaction, I modified the pFUSE-mlgG1-Fc plasmid, which was designed to construct Fc-fusion protein. Firstly, the plasmid does not allow the protein to be secreted extracellularly. Therefore, an IL2 signal sequence was inserted into the Fc gene. Sortase transpeptidation is based on the recognition of a special amino acid sequence called LPETG (SEQ ID NO: 5) (Guimaraes et al., Nat Protoc. 2013 Sep;8(9):1787-99). I therefore designed another insertion GGGGSLPETGGHHHHHH (SEQ ID NO: 4), which included the recognition sequence and a His-tag to the plasmid. GGGGS (SEQ ID NO: 3) serves as the amino acid linker, LPETGG (SEQ ID NO: 6) as the recognition site for the sortase, and HHHHHH (SEQ ID NO: 7) enables the purification of the Fc by nickel affinity chromatography (Figure 2A). The modified Fc plasmid was formulated with 40kD linear polyethyleneimine (PEI) and transfected into Expi293 cells. Valproic acid and sodium propionate were added to enhance the expression. After 7 days of expression, the supernatant was harvested and purified with Nickel-NTA affinity chromatography. The yield of 1L culture was around 40mg protein in total (Figure 2B). Preparation of the Fc-LCP conjugate The conjugation of the LCP (p-FTAA-azide) (Figure 3A) to the mouse IgG-Fc requires 2 chemical reactions. Firstly, to generate the Fc-linker, a peptide-based linker GGGGG-(K-PEG2-alkyne)s (SEQ ID NO: 10) was added to the C terminus of the Fc by the sortase reaction (Figure 3B). Secondly, the p-FTAA-azide was coupled to the Fc-linker using copper catalyzed alkyne-azide (CuAAC) click chemistry reaction (Figure 3C). In the sortase transpeptidation reaction (Figure 3B), the sortase recognizes the amino acid sequence LPETG (SEQ ID NO: 5) in the Fc and cleaves the bond between ‘T’ and ‘G’, and then couples a nucleophile molecule which is the linker GGGGG-(K-PEG2-alkyne)s (SEQ ID NO: 10) to the LPXT end (Guimaraes et al., Nat Protoc. 2013, ibid). When the linker is not in the reaction, the sortase cleavage to Fc results in an intermediate product: Fc without His-tag (Figure 4A, fourth lane). When the linker is in the reaction, the 5x Glycine part in the linker will attack the Fc intermediate at the C-terminus sortase recognition site to form the Fc-linker. 1 unit of Fc would be labeled with 2 units of linker. Because this is a competitive enzymatic reaction, we added different concentrations of linker (from 5 to 400 fold of the Fc concentration) into the reaction to identify the best concentration for the coupling. I found that the highest concentration of the linker gave the best coupling yield of the Fc-linker (Figure 4B). Since the Fc-linker does not have a His-tag on its C-terminus after the coupling anymore, the product was purified by Nickel-NTA beads to separate the His-tagged sortase and unreacted Fc from the Fc-linker, which was collected in the flow-through. For the conjugation of LCP (p-FTAA-azide) to the Fc-linker, alkyne-azide cycloaddition was performed (Figure 3C). 1 unit of the linker GGGGG-(K-PEG2-alkyne)s (SEQ ID NO: 10) has 5 units of free alkyne, which is the reaction site to couple the ‘p-FTAA-azide’ to the Fc-linker by CuAAC. An excessed p-FTAA-azide was added to the reaction to saturate the alkynes (Figure 4C). After the reaction, the mixture was purified, because azide is toxic and might disturb the use of the conjugate for the therapeutic treatment afterwards. SEC was performed to remove the unreacted p-FTAA-azide from the Fc-LCP. The products from all reaction steps of the Fc-LCP coupling were confirmed by the molecular weight shifts on an SDS-PAGE (Figure 4D). After the purification, the Fc-LCP was successfully prepared for the following characterization. Concentration of Fc-LCP When measured as individual molecules, Fc has an absorbance peak at 280nm, and LCP (p-FTAA) has an absorbance peak at 420nm. We found that when measured as a conjugate, Fc-LCP retained the spectral properties of both Fc and LCP (Figure 5A). The concentration of LCP (p-FTAA) as an individual to the absorbance value at 420nm was regressed as a linear model (Figure 5B), which was used to calculate the concentration of LCP (p-FTAA) as in the Fc-LCP conjugate. On average, one unit of Fc was coupled with 5 units of LCP. Note that in this specification, for convenience, the concentration of the Fc-LCP is documented as the concentration of the Fc part in the Fc-LCP. Therefore, the concentration of the LCP part is 5 times of the concentration of the Fc-LCP. Example 2: Characterization of Fc-LCP After conjugation, I wondered if the Fc-LCP retains the properties of both Fc and LCP. By performing various experiment, I confirmed that the Fc-LCP i), has high affinity to the Fcyl receptor and ii), recognizes and binds to multiple protein aggregates. Affinity between Fc and Fcyl receptor Fc is the effector domain in Fc-LCP, which could be recognized by FcyRI, that initiates the phagocytosis by phagocytes. The affinity between Fc-LCP and FcyRI determines the final therapeutic effect and potential side effects. Therefore, we measured the affinity of Fc-LCP and the unlabeled Fc to FcyRI. Sensorgrams shown in (Figure 5) demonstrate a high affinity of both analytes with very slow dissociation that was calculated to be below the experimental limit of 10'5 s’1. For this reason, only an upper limit of the equilibrium constant, KD, can be specified. Kinetic data are collected in Tab. 1. There are two significant differences in the kinetics of binding. Fc binds to FcyRI by first-order kinetics (1 + 1 model) whereas for Fc-LCP a model of higher order kinetics (heterogeneous ligand) was needed to fit the data. This leads to a second kinetic component in the low nanomolar range with a weigth of about 10% that is characterized by a faster association and dissociation of Fc-LCP (Figure 6). As a conclusion, the modification with LCP has a strong effect on the binding kinetics of Fc. Although the modification significantly reduces the affinity of FcyRI, Fc-LCP can be considered a high affinity binder. Table 1: Kinetic characterization of Fc-LCP Probe Analyte kon[M-1s1] Aoff[s-1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 9.072*E04 2.65*E-04 2.92*E-09 56.4 0.21 Binding of the Fc-LCP to various protein aggregates It has been shown that p-FTAA can bind to multiple protein aggregates including prion, Ap, a-syn and tau (Brelstaff et al., Neural Regen Res. 2015 Nov;10(11 ):1746-7). To further confirm that the LCP (p-FTAA) is a pan-amyloid fluorescent dye, I performed different experiments to evaluate the aggregate binding properties of the LCP in the Fc-LCP conjugate. To confirm the detection of prion aggregates by the Fc-LCP, immunoprecipitation was performed. The Fc-LCP was incubated with RML6 infected mouse brain homogenate and NBH, overnight respectively. The incubation was digested with PK and analyzed by western blotting to detect PK resistant PrPSc bands, as a surrogate marker for the presence of prions. I found that the Fc-LCP bound to the prion aggregates in RML6 but not to the native prion protein, PrP0, in NBH (Figure 7A). To evaluate the detection of Ap aggregates by the Fc-LCP, a sandwich ELISA was performed. A 384-well plate was coated with either recombinant Ap monomers or Ap fibrils. Fc-LCP, Fc and LCP were added at serial diluted concentrations to bind to the fibrils. HRP-conjugated goat anti mouse IgG antibody was then added as secondary antibody to detect the Fc domain in the Fc-LCP. The ELISA results showed that the Fc-LCP recognizes and binds to Ap fibrils but not to the monomer (Figure 7B). To further confirm the binding between the Fc-LCP and Ap aggregates, I also immunohistochemically stained brain slides from APP / PS1 mouse containing amyloid plaques with the Fc-LCP. The Fc-LCP was able to stain the Ap plaques mediated through the LCP. The presence of the Fc domain was confirmed with Alexa647 conjugated goat anti-mouse IgG antibody (Figure 8A). As control, I also stained the APP / PS1 mouse brain slides with the LCP alone and an antibody directed against the Fc, respectively (Figure 8B). No positive results were found when wild-type mouse brain slides were stained with the LCP and Fc-LCP (Figure 8C). Collectively, these results showed that the Fc-LCP can recognize and bind to multiple protein aggregates from multiple platforms. The Fc-LCP stops primary fibril formation Previous research by Margalith et al (Margalith etal., J Biol Chern. 2012 Jun 1 ;287(23): 1887287) has shown that LCPs can inhibit prion propagation after binding to prion aggregates. I therefore investigated if the Fc-LCP has the same ability in stabilizing prion aggregates, and the potential in stabilizing other protein aggregates. By repeating the same conversion assay performed by Margalith et al, I incubated a serial concentration of diluted Fc-LCP with monomeric mPrP under slightly denaturing conditions. The aggregate formation was monitored at an absorbance of 350nm for 48h under shaking. I observed that in the blank and Thioflavin T controls, the mPrP aggregated in an exponential manner followed by a plateau. In the presence of 0.08 pM Fc-LCP, aggregation was completely blocked in the primary nucleation phase (Figure 9B). Similar to the results by Margalith et al, the LCP inhibited prion aggregate formation in a dose dependent manner and completely blocked fibril formation at 50 pM (Figure 9A). I then performed the conversion assay for a-syn. The effect of the Fc-LCP did not repeat itself in blocking a-syn aggregate formation. The addition of the Fc-LCP reduced the growth rate of a-syn aggregation at a concentration of 0.4 pM. From 2 pM, the Fc-LCP can completely block the fibril formation of a-syn (Figure 9D). Although it is consistent with the findings from Margalith et al that the LCP’s ability of inhibiting fibril formation increased with an increasing dose, the LCP alone was less pronounced compared to the Fc-LCP (Figure 9C). Example 3: Bioactivity of the Fc-LCP After characterizing the molecular properties of the Fc-LCP in binding multiple protein aggregates and recognizing the Fcyl receptor, I explored the bioactivity of the Fc-LCP in a cellbased assay. As proposed in the hypothesis, the Fc-LCP is a pan-amyloid bi-functional molecule and can enhance the phagocytosis of multiple protein aggregates by immune cells, including microglia. To prove this hypothesis, I tested the phagocytic ability of the Fc-LCP using an in vitro microglial phagocytosis assay. Imaging flow cytometry The phagocytosis assay was performed using imaging flow cytometry. By combining the features of flow cytometry and microscopy, imaging flow cytometry shows the details of each cell in an individual event. All staining information could be visualized in different channels and merged with the bright field channel. As mentioned previously, the LCP is a fluorescent dye, that binds to multiple protein aggregates and emits a fluorescent signal at 488nm with a sharp green signal. Therefore, in this phagocytosis assay, the LCP served as both binder and dye, making it unnecessary to label the aggregates with a different dye or antibody. In channel 2, LCP-bound aggregates could be seen (Figure 10A). Phagocytosis assay controls Before testing the function of the Fc-LCP, several control experiments need to be performed. It is known that the LCPs bind to aggregates, but not monomers. However, when using the LCPs as the indicator for aggregates in the phagocytosis assay, I need to ensure that the LCPs do not stain anything other than aggregates. I therefore incubated the LCP with mPrP fibrils and monomers separately and let BV2 cells phagocyte for 1h. In addition, I also set a group where only LCPs are added to the microglia. I found that while microglia which phagocyted LCP-stained mPrP aggregates can be positively shown in the imaging flow cytometry gate, there are no positive cells for the LCPs when incubated with mPrP monomers (Figure 10B right). In addition, the LCPs did not induce unspecific staining from any cell components in microglia (Figure 10B middle). Then I introduced another control to test the phagocytosis process using cytochalasin D. Cytochalasin D is the inhibitor of actin polymerization and disrupts actin filament, resulting in immobility of the cell membrane, which can be used to block the microglial phagocytosis. When microglia were treated with cytochalasin D 15min before the phagocytosis assay, phagocytosis was completely blocked (Figure 10C). This finding confirmed that the LCP signal in channel 2 is intracellular and results from proper phagocytosis by microglia. Phagocytosis assay setup To find the optimal concentration of the fibrils and time for phagocytosis, a pilot phagocytosis assay was conducted. I incubated different amounts of insulin fibrils (2.4pM, 4.8pM and 12p.M) with corresponding concentrations (2.4|iM, 4.8p.M and 12|iM) of the LCPs and fed to microglia. After 1h of phagocytosis, cells were harvested for imaging flow cytometry and the phagocytic ability of the microglia was measured by the total number of microglia that have LCP-stained insulin fibrils internalized. I also found that a higher amount of fibrils led to an increased phagocytic ability. Therefore, I chose 2.4|iM for future phagocytosis assays, because it allows space for the Fc-LCP to show its enhancement under this concentration (Figure 10D). For the following experiment, I incubated 2.4p.M insulin fibrils with 5|iM LCP and added the mixture to the microglia for phagocytosis. I harvested the cells at different time points, which were 1 h, 4h and 8h, respectively. The phagocytic ability increased with time but reached a plateau after 4h. To give enough capacity for the Fc-LCP to show the enhancement, the phagocytosis time was set at 1h (Figure 10D). Fc-LCP enhanced microglial phagocytosis After completing the control experiments and setting up the experimental parameters, I investigated the bioactivity of the Fc-LCP in microglial phagocytosis enhancement. I utilized a function termed phagocytic index (Park et al., Front. Immunol., 18 February 2020, Sec. Comparative Immunology Volume 11 - 2020) to calculate the amount of protein aggregates that were taken up by microglia. Phagocytic index (PI) = phagocytic ability * phagocytic capacity The phagocytic ability is the amount of microglia that are engaged in the phagocytosis process, which can be calculated by the ratio of the amount of microglia that have finished phagocytosis to the total number of microglia that are fed with protein aggregates. The phagocytic capacity is the amount of aggregates that are taken up by the phagocytic microglia, which can be shown by the mean amount of aggregates per phagocytic microglia. As mentioned above, the LCPs can be used as a dye for the detection of various protein aggregates. Therefore, the intensity of the LCP in channel 2 (Figure 10A), could be correlated with the amount of aggregates that were labeled with it. The intensity value thus will indicate how many aggregates are phagocytosed by one microglia cell. I first tested the phagocytosis capacity of the Fc-LCP in BV2 microglia which is a immortalized murine microglial cell line (Henn et al., 2009). 1.5pM mPrP fibrils were incubated with three groups of reagents: 5uM LCP, 1|j.M Fc + 5p.M LCP and 1p.M Fc-LCP, respectively, for 15min at 37°C and then add to the BV2 cells. In the first group, mPrP fibrils were incubated only with the LCP, to set the phagocytic baseline for the phagocytic index of the BV2 cells. The baseline indicates the amount of aggregates microglia are expected to take up under natural conditions. In the second group, the LCP and Fc were added as non-conjugated, single molecule to the mPrP fibrils, to serve as controls for the Fc-LCP. The third group was the experimental group in which the Fc-LCP was tested for its phagocytic capabilities. I found that the Fc-LCP significantly increased the amount of mPrP aggregates that were taken up by the BV2 cells (Figure 11 A). The experiment with the same settings was repeated with primary mouse microglia and the same enhanced phagocytosis induced by the Fc-LCP was observed (Figure 11B). The results from mPrP fibrils demonstrated the bi-functional property of the Fc-LCP in that it can bind to mPrP aggregates and enhance the phagocytosis by microglia. To confirm the panamyloid properties of the Fc-LCP, I validated the same phagocytosis assay with A0 aggregates. Ap is the most common extracellular protein that forms aggregates in neurodegenerative diseases. AP42 aggregates were produced by shaking, and then mixed with the LCPs and the Fc-LCP, respectively. In the presence of the Fc-LCP, BV2 microglia engulfed more AP42 aggregates (Figure 11C). Then, BV2 microglia were fed with different amounts of AP42 fibrils stained with corresponding amounts of LCPs and Fc-LCP, respectively. The phagocytic index of BV2 treated with the Fc-LCP-stained aggregates from each concentration was normalized to the phagocytic index of BV2 with those stained with the LCPs. I plot the normalized phagocytic ratio and conclude that the phagocytic enhancement by the Fc-LCP treated aggregates increased in a dose-dependent manner (Figure 11D). Example 4: Ex vivo therapeutic effect of Fc-LCP in prion disease Cultured organotypic cerebellar slices (COCS) COCS have been well established and used for research in PrD because they represent the 3-dimensional physiological structure of the brain and recapitulate all necessary features of PrD, including the prion propagation and neurotoxicity. As demonstrated in Figure 12, cerebellar slices were generated from Tga20 mouse pups, and infected with 30ug RML6 infected brain homogenates on day 0 to produce the ex vivo prion model. NBH were added as negative control for prion infection. Treatment of the Fc-LCP started from 14 days post infection (dpi), which allowed for enough time for the prions to propagate. Previous research has shown that prion induced neuronal pathology did not start until 28 dpi. Therefore, a later treatment starting from 30 dpi was performed to evaluate the therapeutic efficacy of the Fc-LCP at the late stage of prion progression. The main purpose of this experiment was to test: a) If the Fc-LCP could reduce the infectious prion load by enhancing microglial phagocytosis. b) If the Fc-LCP could protect neurons from prion toxicity. The experiment included three control groups: treated with PBS, Fc and LCP after RML6 infection. The PBS treated group represented the pathophysiological process in prion infection, which served as a baseline for the therapeutic treatment. The Fc and LCP alone were used as non-conjugated, single controls for the Fc-LCP treatment in the COCS. The starting concentration of the Fc-LCP is 1p.M (equivalent of the Fc concentration). Because on average there are 5 LCP per Fc molecule, the concentration of the control LCP is 5p.M to match the Fc-LCP and the concentration of control Fc is 1p.M. The Fc-LCP reduced prion load and protected neurons from prion toxicity in RML6 infected COCS For purpose a), I performed a PK-WB using POM1 antibody (Polymenidou et aL, 2008, PLOS ONE 3(12): e3872) to detect the infectious prion load in the COCS homogenates. After PK digestion, no prions were detectable in the NBH treated COCS homogenates. However, since infectious prions are partially resistant to PK digestion, I could clearly see that the Fc-LCP treatment efficiently reduced the amount of prions in RML6 infected COCS (Figure 13A). A WB was also performed to detect the total prion protein level in all treatment groups. While the Fc- LCP did not affect the total prion protein level in NHB treated COCS, it reduced the total prion protein level in RML6 infected COCS, which is consistent with the PK WB results (Figure 13B). For purpose b), the viability of neurons was checked after the different treatments in COCS. The NeuN protein is a neuronal marker and can be detected immunohistochemically with the anti-NeuN antibody. WB on the COCS homogenates detected that in all NBH treated groups, there was no difference in the NeuN level, indicating that no neuronal toxicity was induced by the treatment with the Fc-LCP, whereas the NeuN level was significantly higher in the Fc-LCP treated RML6-infected COCS than any of the three control groups, demonstrating a neuronal protective efficacy of the Fc-LCP. Besides WB, I also performed immunostaining on all COCS to show any morphological changes due to prion-infection and treatments. The results of the immunostaining are consistent with the findings from the WB. The Fc-LCP did not induce any toxicity in NBH treated COCS (Figure 14A). At the terminal stage of the RML6-infected COCS, the Fc-LCP treatment starting from 14dpi protected neurons from prion toxicity, whereas all neurons in non-, Fc-, or LCP-treated RML6-infected COCS were dead. The level of neurons in the Fc-LCP treated RML6 infected COCS was similar to those of NBH treated COCS, indicating that the Fc-LCP treatment from 14dpi has a preventative effect in prion infection (Figure 14B). In order to find the minimum effective concentration of the Fc-LCP, I then added a serial concentration from 0.25p.M, 0.5|a.M to 1p.M of Fc-LCP to RML6-infected COCS on 14 dpi. However, I did not see any difference in the numbers of survived neurons between the three concentrations (Figure 14C), indicating that the effective concentration of the Fc-LCP is below 250nM. As mentioned above, the prion induced neuronal toxicity appears after 28dpi. Therefore, the efficacy from the treatment on 14dpi is protective rather than therapeutic. However, I found that the neuron level in the Fc-LCP treatment starting from 30dpi is comparable to the treatment from 14dpi, demonstrating that the Fc-LCP also has therapeutic efficacy at late stage of prion infection (Figure 14D). Example 5: Mechanism of the Fc-LCP treatment The role of microglia in Fc-LCP efficacy The hypothesis of the Fc-LCP that the Fc-LCP binds to protein aggregates and enhances the clearance of the aggregates by microglia relies on the Fey receptors on microglia. To verify this hypothesis, I tested the efficacy of the Fc-LCP in the absence of microglia. PLX5622 is a highly selective cluster stimulating factor-1 receptor (CSF-1R) inhibitor, which can deplete 95% of microglia at 1 p.M in slices. Therefore, I added PLX5622 to RML6-infected COCS to suppress microglia’s function. Since PLX5622 was dissolved in DMSO, the following treatments were used as controls: 1pM PLX5622, DMSO, 1pM PLX5622 + 1pM Fc-LCP and DMSO + 1pM Fc-LCP. All reagents were added from 14 dpi (Figure 15A). The loss of microglia resulted in a higher prion load in RML6 infected COCS, when compared to DMSO treated RML6 infected COCS (Figure 15B). This finding added to the evidence that microglia are protective in prion diseases. The data showed that after PK digestion there was no difference in the prion load in Fc-LCP treated RML6-infected COCS despite the PLX5622 (Figure 15C). Although there is slightly more total prion protein in Fc-LCP + PLX5622 treated COCS than in only Fc-LCP treated COCS, it can be noted that the amount of prions in PLX5622 treated COCS is much higher than in PLX5622 + Fc-LCP treated COCS (Figure 15D). The PLX5622 induced microglia depletion was confirmed by WB using an anti-lba1 antibody which binds to Iba1, a microglia specific protein (Figure 15E). However, when combined with the Fc-LCP treatment, less microglia were depleted compared to PLX5622 treatment alone (Figure 15E). In addition, the Fc-LCP treatment protected neurons from prion toxicity, which is consistent to my results described above. However, the Fc-LCP related neuronal protection also presented in PLX5622 treated RML6-infected COCS. Combined with the results of the prion load, I assume that the Fc-LCP treatment can reverse the effect of PLX5622 to a certain level. Lastly, I did not find any significant difference in the expression of glial fibrillary acidic protein (GFAP) among all groups, which validated the high selectivity of PLX5622 (Figure 15E). Collectively, these results showed that either the Fc-LCP could preserve microglia from PLX5622 depletion, or the Fc-LCP does not fully rely on microglia to clear prions. The effect of Fc-LCP is independent of microglia Since the microglia depletion by PLX5622 did not completely block the efficacy of the Fc-LCP, I assume that the possible reason could be that PLX5622 was added at the same time with the Fc-LCP, which did not allow enough time for microglia depletion. I adjusted the scheme by adding PLX5622 on the same day of prion infection, which is two weeks ahead of the Fc-LCP treatment. Besides, I also increased the concentration of PLX5622 to 2 pM (Figure 16A). The continuous treatment of PLX5622 throughout the prion infection course leaded to complete death of the infected COCS, accompanied with morphological changes including the disrupted brain structure (Figure 16B). Although there are more neurons in the Fc-LCP treatment than Fc-LCP + PLX5622 group, Fc-LCP + PLX5622 could still significantly protect neurons from prion toxicity compared with only PLX5622 treatment, indicating that PLX5622 could partially blocked the efficacy of the Fc-LCP (Figure 16B). I then stained the COCS with the microglial marker I ba 1 antibody to see if the treatment of the Fc-LCP and / or PLX5622 resulted in any morphological changes to microglia. In DMSO treated COCS which represents the natural pathophysiological process of prion infection, there was evident microglial activation (Figure 17A). Indeed, the Fc-LCP treatment did not cause enhanced microglial activation compared with DMSO treated COCS, whereas no microglia can be detected in both PLX5622 and PLX5622 + Fc-LCP treated COCS (Figure 17A). There is a large amount of microglia accumulated on the surface of COCS due to scar formation, so I looked for if there is any difference in the amount of microglia in the surface level. While dense microglia were in the scar level in both DMSO and Fc-LCP treated COCS, I did not find any microglia in PLX5622 treated COCS, which confirms the successful depletion of microglia by PLX5622 (Figure 17B). Interestingly, compared with only PLX5622 treatment, there were some microglia remained in the COCS when the Fc-LCP was present in the PLX5622 treatment, but only in the scar area (Figure 17B). I speculate that the Fc-LCP kept the microglia active in the scar level which partially contributed to the protection of neurons from prion toxicity. Collectively, these results showed that PLX5622 can completely deplete microglia. However, the Fc-LCP may not be microglia dependent to exert its function in removing prions and protecting neurons from prion toxicity. Example 6: Linkers Based on sortase-mediated transpeptidation conjugation, we produced various linker-LCP with different numbers of LCP. (Fig 18 and 19) Besides Sortase based transpeptidation conjugation, we also included another conjugation method. We inserted a cysteine at the C terminal and react with maleimide leading Linker-LCP (Fig. 20). This method is more time and cost efficient compared to sortase based conjugation. Another method is Q-tag conjugation. This method uses microbial transglutaminase (mTG) to label protein with a Glutamine (Q-tag). The linker payload is in Fig. 21. To summarize, whichever conjugation method is used, the linker between Fc and LCP should be non-cleavable. Cleavable linkers are designed including chemical (hydrazone bond and disulfide bond) and enzyme (glucuronide bond and peptide bond) cleavable linkers. In contrast, non-cleavable linkers (e.g., thioether or maleimidocaproyl group) remain stable in common chemical and enzymatic environment. In our design, the linker between Fc and LCP should be non-cleavable. It is based on the mechanism of action of Fc-LCP: only when Fc-LCP remains intact in the circulation, it can bridge between the immune cells and aggregates. The insertion of PEG (polyethylene glycol) into the linker as spacer allows more flexibility for the binding between LCP and protein aggregates. To answer the question that if conjugated LCP has the same / better effect as Fc-LCP, flow cytometry-based phagocytosis assay was performed. In short, BV2 cells were equally seeded in 6-well plates as phagocytes. Except for the blank, all groups were treated with the same amount of Abeta40 / 42 and tau fibrils. Nothing was added in the blank group, which serves as the control for LCP signals. LCP can bind to Abeta40 / 42 fibrils and is a fluorescent indicator. In each group, Abeta40 / 42 and tau fibrils were incubated with conjugated LCP, mouse IgG 1 Fc-LCP or mouse lgG2a Fc-LCP, respectively. The concentrations of LCP in all groups were controlled the same. The drug to antibody ratio of lgG1 FcLCP and lgG2a FcLCP is similar, both at around 7, so the equivalent concentration of Fc domain is comparative among two groups. (Fig. 23 and 24) The results showed that Fc-LCP (both IgG 1 Fc-LCP and lgG2a Fc-LCP) can enhance the phagocytosis of both Abeta40 / 42 and tau fibrils, compared with conjugated LCP. Specifically, mlgG2a Fc-LCP has stronger effect in enhancement than mlgG1 Fc-LCP. In mouse, lgG2a is the most abundant isotype of IgG, and is the functional equivalent of human IgG 1 antibody. Human lgG1 isotype antibody has been mostly used in monoclonal antibody therapy. We stained human tissues from different amyloidosis patients with Fc-LCP. The results showed that Fc-LCP bind to the A4 aggregates in human brain sample (Fig. 25, first row), transthyretin aggregates in human heart sample, amyloid A aggregates in human testicle sample (Fig. 25). Example 7: Material and Methods Materials - Animal strains C57BL / 6J mice were used for primary microglia cultures and Tga20 mice for cerebellar organotypic cultured slices. - Cell lines Expi293F Mammalian cell line, derived from HEK293T cell line. Optimized for Gibco™         high-yield protein production. BV2              Murine microglial cell line. Bacteria strains 5 E.coli DH5a               fhuA2 lac(del)U169 phoA glnV44 080' lacZ(del)M15 ...   _ ।   .    । .        gvrA96 recA1 relA1 endA1 thi-1 hsdR17 New England BioLabs E.coli BL21DE3pLys       F- dem ompT hsdS(rB- mB-)gal A(DE3) In-house - Plasmid Plasmid                              Source Sortase A pentamutant (eSrtA) in pET29 Addgene pFUSE-mlgG1-Fc                       Invivogen - Protein production and purification materials Materials / chemicals Source Ni-NTA resin Qiagen Chromatography column for self-package CrystalCruz HILoad 16 / 600 Superdex 75pg column Cytiva Lysozyme Sigma Imidazole Sigma Isopropyl-R-D-thiogalactopyranoside Sigma LB Broth Base Invitrogen LB agar Invitrogen Kanamycin Sigma Zeocin Invitrogen Cell culture and other experimental materials Consumables / medium / supplement / chemicals Supplier / source Corning Erlenmeyer flask 125ml / 500ml / 1L Sigma / Aldrich TPP 6 / 24-well plate UZH-AUL Webshop TPP-flask, 25 / 75cm2, with filter cap UZH-AUL Webshop Serological pipette, 2ml / 5ml / 10ml / 25ml / 50ml UZH-AUL Webshop Filtering sterile mesh Merck Expi293TM Expression Medium Gibco Opti-MEM™ 1 Reduced Serum Medium, no phenol red Gibco Basal Medium Eagle (BME) Gibco MEM, glutamine, HEPES, powder Gibco Dulbecco's Modified Eagle Medium (DMEM) Gibco Fetal Bovine Serum, Heat Inactivated Cytiva Penicillin-Streptomycin (10,000 U / mL) Gibco GlutaMAX™ Supplement Gibco 1 M HEPES buffer solution Gibco Poly-D-Lysine Gibco HBSS, calcium, magnesium Gibco Trypsin-EDTA (0.05%), phenol red Gibco D-(+)-Glucose Solution 45% In H2O Sigma / Aldrich Deoxyribonuclease I Sigma / Aldrich Trypsin inhibitor Sigma / Aldrich TMB substrate Sigma / Aldrich Immobilon Crescendo Western HRP-Substrate Sigma / Aldrich - Antibodies Antibodies for immunoblotting Primary Antibody        Species Dilution Supplier Anti-actin-HRP          Mouse 1:10000 Sigma Anti-NeuN              Rabbit 1:1000 Abeam Anti-prion (POM 1)       Mouse 1:5000 Home-made Secondary Antibody     Species Dilution Supplier HRP anti-rabbit IgG      Goat 1:10000 Invitrogen HRP anti-mouse IgG    Goat 1:10000 Thermos Fisher Antibodies for immunofluorescence Antibody Species Dilution Supplier Alexa Fluor 647 conjugated anti-NeuN Rabbit 1:1000 Abeam Alexa Fluor 488 conjugated anti-lba1 Rabbit 1:1000 Abeam - Kits Kits supplier Q5 site-directed mutagenesis kit New-England labs Phusion DNA polymerase New-England labs HiFi DNA assembly New-England labs QIAprep Spin Miniprep Kit QIAGEN EndoFree® Plasmid Maxi Kit QIAGEN - Equipment Name Manufacturer AKTA go protein purification system Cytiva Centrifuge Invitrogen GE AKTA Prime Liquid Chromatography System Cytiva Image Stream X (ISX) Mark II imaging flow cytometer Luminex Fluoview confocal microscope Olympus Stellaris 5 upright microscopy Leica Sonicator Bandelin electronic GmbH Western blot imaging Fuji VT1000 S vibrating blade microtome Leica - Software Snap gene Nanodrop Image J - win64 GraphPad Prism 9.5.1 IDEAS Microsoft office 2016 Affinity Designer 2 Endnote 20 Fuji LAS-3000 Imaging system Methods - Animals Mice were bred and maintained in the Laboratory Animal Service Center (LASC) Schlieren under specific-pathogen-free conditions and 12h light I 12h dark cycle. 3-5 animals were placed in one cage, provided with sterilized food and water ad libitum. All experiments were approved by the Veterinary Office of the Canton of Zurich (Permits ZH236 / 2019, ZH084 / 2023). - Isolation of sortase plasmid Sortase A pentamutant (eSrtA) with 6xHis-tag in vector pET29 (Addgene #75144) was shipped transformed in E. coli DH5alpha strain as agar stab. A dip of bacteria was cultured into 5ml LB Broth Base (Invitrogen #12780053) medium with 100ng / ml kanamycin overnight at 37°C with shaking at 180rpm. The next day, the culture was harvested and centrifuged at 16,000g. QIAprep Spin Miniprep kit (Qiagen #27104) was used to extract and purify the plasmid. Briefly, the bacterial pellet was resuspended in 250pl of P1 suspension buffer supplemented with RNase A. 250(11 of P2 lysis buffer was added and mixed by inverting the tube for 4-6 times, and incubating it for 5min. 350(11 of N3 neutralizing buffer was added to the suspension and after inverting for 4-6 times, the suspension was centrifuged at 16,000g for 10min. The supernatant was loaded to QIAprep 2.0 spin column by pipetting, followed by centrifuging for 30s. The column was washed with 500|il of buffer PB, turned and washed with 750|il of Buffer PE in turn and centrifuged for 60s to completely remove any residual liquid. 50|il of milliQ water was added to the column to elute the plasmid by centrifuging for 1min. Nanodrop at 260nm was used to measure the concentration of the eluted plasmid. - Transformation and Sortase expression The protocol was adapted from (Guimaraes, 2013, ibid). The plasmid harboring the sortase gene was transformed into E. coli BL21DE3pLys strain by heat shock. Briefly, 50pil of E. coli was thawed on ice. 100ng of the purified plasmid was added to the bacteria and gently mixed. After 30min of incubation on ice, the mix was incubated in a thermomixer set at 42°C for 45 seconds, followed by 2min on ice. 950|il of super optimal broth with Catabolite repression (SOC) medium was added to the bacteria mix and then placed in the thermomixer set at 37°C and shaking at 500rpm for 1h. The transformed E. coli culture was then plated on a LB agar plate with lOOpg / ml kanamycin and let grown overnight at 37°C to get single colonies. The next day, a single colony was picked and inoculated into 100ml LB broth medium with 100|ig / ml Kanamycin and let grown overnight and shaking at 180rpm. On the day of expression, 10ml of the pre-culture was inoculated into 1L LB medium with 100pg / ml Kanamycin and let grown at 37 °C under shaking at 120rpm. When the value of optical density (OD) at 600nm of the culture reached to 0.8, 1ml of 1M isopropylthio-p-galactoside (IPTG) was added to the culture to induce protein expression. The temperature of the incubator was lowered to 25°C after the induction. 16 hours after induction, the culture was harvested and centrifuged at 15000g at 4°C for 30min to collect the bacterial pellet. - Sortase enzyme purification The pellet was resuspended in 100ml lysis buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM MgCI2, 10 mM imidazole, 10% glycerol, 1 mg / ml DNase and 1 mg / ml lysozyme) and sonicated for 30min on ice. 20ml of Qiagen Ni-NTA agarose was loaded into a Qiagen column, followed by washing with 50ml of milHQ water and 50ml of washing buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM MgCI2, 10 mM imidazole and 10% glycerol). The cell lysate was centrifuged at 20,000g for 30min at 4°C. The supernatant was collected and loaded into the Ni-NTA column by gravity. After loading, the column was washed with 100ml washing buffer. The sortase was eluted with elution buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 500 mM imidazole and 10% glycerol). Fractions were analyzed on a Coomassie blue-stained SDS-PAGE. The fractions with pure sortase were collected and dialyzed against Nickel-binding buffer (50 mM Tris-HCI, pH 7.5, and 150 mM NaCI) twice (3h and overnight, respectively). The purified Sortase was concentrated with 10k cutoff concentrator (Amicon), aliquoted, snap frozen and stored in -80°C. Fc plasmid modification pFUSE-mlgG1-Fc plasmid (InvivoGen) to express wild type mouse IgG-Fc was purchased as lyophilized DNA. In order to make Fc suitable for sortase reaction, several mutations were introduced into the plasmid: 1. A GGGGSLPETGGHHHHHH (SEQ ID NO: 4) sequence for the recognition by sortase and His-tag purification. 2. Signal sequence for the production in mammalian cell lines. For the first mutation, polymerase chain reaction (PCR) by Phusion DNA polymerase (M0530L) followed by HIFI DNA assembly (E5520S) were performed to build the new construct. The primers for the GGGGSLPETGGHHHHHH (SEQ ID NO: 4) sequence insertion were synthesized by Microsynth as following: The capital letters are sequence existing in the original plasmid and the small letters are newly introduced. Internal forward: AACCCCGGGAGGAGCAGTTCgctAGCACTTTCCGCTCAGTCAG (SEQ ID NO: 11) Internal reverse: gtccaccagtttcaggaagagaacctccacctccACCAGGAGAGTGGGAGAGG (SEQ ID NO: 12) vector forward: ctcttcctgaaactggtggacatcaccatcaccatcactaaTGATCCCAGTGTCGCTAG (SEQ ID NO: 13) vector reverse: GAACTGCTCCTCCCGGGGTTG (SEQ ID NO: 14) 5 The reaction setup for the PCR is described below: Table 2: PCR reaction setup Components Volume 5x HF buffer 10pl 10mM dNTPs 1 pl 1 ng / ul template plasmid 1pl DNA polymerase 0.5pl 10 pM Forward Primer 1pl 10 pM Reverse Primer 1 pl ddH2O 35.5pl In total 50 pl The thermo-cycling condition for the reaction were as following: Table 3: PCR thermocycling Step Temperature Time Initial denaturation 98°C 30s Cycles 26 rounds 98°C 10s 60°C 30s 72°C 2min for vector, 25s for insert Final extension 72°C 5min hold 4°C - 10 The PCR products were fused using the following protocol: Table 4: PCR product protocol Components Volume Vector 2.5pl Insert 0.6pl Mix master 10)11 ddH2O 6.9pl In total 20|il The mix was incubated at 50°C for 15min. The assembly product was transformed into E.Coli DH5alpha bacterial strain cells and incubated on a LB agar plate with 25pg / ml Zeocin. A single colony was picked and inoculated into 5ml LB medium with 25pg / ml Zeocin and shaked at 5 180rpm overnight. The next day, the plasmid was extracted with QIAprep Spin Miniprep kit (#27104) (for detailed protocol see section sortase production). Then we inserted the signal sequence using Q5 Site-Directed Mutagenesis Kit (E0554S). The primers were synthesized by Microsynth: Signal sequence insertion forward: 10 GCACTAAGTCTTGCACTTGTCACGAATTCGGGTTGTAAGCCTTGCATATG (SEQ ID NO: 15) Signal sequence insertion reverse: AATGCAAGACAGGAGTTGCATCCTGTACATAGATCTAACCATGGTGCTCG (SEQ ID NO: 16) 15 The reaction setup was as below: Table 5: Mutagenesis reaction setup Components Volume 5X HF buffer 10pl 10mM dNTPs 1 pl 1 ng / ul template plasmid 1pl DNA polymerase phusion 0.5pl 10 pM Forward Primer 1pl 10 pM Reverse Primer 1pl ddH2O 35.5pl In total 50pl The thermocycling time for the reaction was as follows: Table 6: Thermocycling protocol Step Temperature Time Initial denaturation 98°C 30s Circles 26 rounds 98°C 10s 66°C 30s 72°C 2min Final extension 72°C 5min hold 4°C - The PCR product was transformed, extracted, and sent for sequencing (protocol seen above). The sequencing primers are: EF1a Fwd sequencing: CGCAACGGGTTTGCCGCCAG (SEQ ID NO: 17) Fc Rev sequencing: CCATACCACATTTGTAGAGG (SEQ ID NO: 18) - Isolation of the Fc plasmid The modified Fc plasmid was transformed into the E.coli DH5a strain cells by heat shock methods as described above. A single colony was picked and expanded in 250ml LB broth medium complemented with 25pg / ml zeocin. The overnight culture was harvested and centrifuged to collect the pellet at 4°C. Qiagen Endo free Plasmid Maxi Kit (#12362) was used for bulk extraction of the modified Fc plasmid. 10ml P1 suspension buffer was added to suspend the pellet. 10ml P2 lysis buffer complemented with LyseBlue reagent was then added to the suspension. After inverting 5 times, the solution became blue and was incubated at room temperature for 5min. After adding chilled P3 neutralizing buffer, the mixed solution was inverted for 5 times until became colorless. The lysate was poured into the barrel of the QIAfilter Cartridge and incubated at room temperature for 10min. The supernatant was filtered from the lysate by inserting a plunger into the cartridge. 2.5ml ER buffer was added to the filtered solution and incubated on icefor30min. After washing the QIAGEN-tip with 10ml buffer QBT, the lysate was loaded into a column by gravity, followed by washing with buffer QC twice. Plasmid DNA was eluted with 15ml buffer QN and precipitated with 10.5ml isopropanol at room temperature (RT). The pellet was collected by centrifuging at 3,500 rpm at 4°C for 60min followed by washing with 70%endo-free ethanol at RT and centrifuging at 3,500 rpmfor30min. The pellet was let air-dried overnight and resuspended in 200p.l of milliQ water. Nanodrop was used to measure the concentration of the plasmid DNA. - Fc production and purification The Expi293 cell line was used for transient Fc expression and production. 107 Expi293 cells stored in liquid nitrogen were thawed in 37°C water bath, added into 4ml Expi293 expression medium and suspended. Cells were centrifuged at 1,000 rpm at 4°C for 5min. The cell pellet was resuspended in 30ml pre-warmed medium and shaking at 120rpm and 37°C. When a density of 3 million viable cells per ml were reached, cells were passaged. Cells were ready for transfection after 3 passages. 1 day before transfection, cells were seeded at a density of 2 million viable cells per ml in 200ml fresh pre-warmed Expi293 medium. On the day of transfection, cells were diluted to a density of 3 million viable cells per ml before transfection. Pluronic F-68 was added to a final concentration of 0.1% to reduce foaming in the culture. Polyethyleneimine hydrochloride (PEI MAX, MW = 40,000, linear) was used as transfection reagent. 500pl of 1mg / ml PEI MAX was incubated with 12ml Opti-MEM medium at room temperature for 5min. 200p.l of 1 pg / pl Fc plasmid was mixed with 12ml Opti-MEM medium. Then the plasmid-Opti-MEM mixture was incubated with PEI-Opti-MEM mixture at RT for 20min. The Fc plasmid-PEI complex was added to the culture under shaking by dripping. 1620h after transfection, valproic acid and sodium propionate were added into the culture at a final concentration of 3.5mM and 7mM, respectively. 7 days after transfection, the culture was harvested and centrifuged at 15,000g and 4 °C for 30min. The supernatant was filtered with a 0.22um filter to get rid of the cell debris. The Fc was purified in the same way as sortase purification (described in the sortase purification chapter). In the end, Fc was dialyzed twice against Nickel-binding buffer, concentrated with 30k cutoff concentrator (Amicon), aliquoted and stored at -20°C. - Sortase reaction I designed a linker GGGGG-(K-PEG2-alkyne)s (SEQ ID NO: 10) which was synthesized by Genscript for the conjugation of Fc to the p-FTAA-azide. The sortase reaction was performed to label the Fc fragment with the linker. I first performed several trial experiments to find the best reaction condition, including time, temperature, reagent concentration etc. The optimal reaction condition is shown up in the following Table: Table 7: Optimal reaction conditions Fc Fc + sortase Fc + sortase + linker Stock cone. Final cone. Sortase - 1 10 600pM 30pM Fc 1 1 10 100pM 5|iM Linker - - 40 40pM 8pM Buffer 2 2 20 10X 1X h2o 17 16 110 - - In total 20pl 20pl 200pil The reaction was incubated in a shaker set at 30°C and 1000rpm for 1h. Because unreacted Fc has a histidine tag, we mixed the reaction with 2ml Ni-NTA resin washed with the Nickel binding buffer, to purify the labelled Fc from the reaction. The mixture was agitated on an endover-end shaker for 30min at 4°C. After centrifuging, the supernatant, including the labelled protein, was collected. The resin was washed with 2ml Nickel binding buffer and agitated for another 30min at 4°C. The supernatant containing the Fc-linker was collected and concentrated for the click chemistry reaction, using a 500|il 30kDa cutoff concentrator (Amicon). - Click chemistry reaction The copper (I) catalysed alkyne-azide cycloaddition (CuAAC) was performed to couple the p-FTAA-azide, which was kindly provided by Prof. Peter Nilsson, to the Fc-linker. Theoretically, 1 unit of Fc-linker has 2 units of linker on its C-terminal, which is 10 units of alkynes. So, 1 unit of Fc-linker requires a minimum of 10-fold amount of p-FTAA-azide to saturate the alkynes. We used a commercial protein labeling buffer (1,5X, Lumiprobe, Germany) containing copper (II) and a Cu(l) stabilizing ligand tris-hydroxypropyltriazolylmethylamine (THPTA). Copper (II) requires a reductant, which is ascorbic acid to be reduced to copper (I). The reaction was set up as in the following Table and incubated at RT overnight. Table 8: Reaction setup Reagents Volume Final concentration Stock cone. Fc-linker 50|il 4 pM 20 pM Protein labelling buffer 167.5)11 1 X 1.5X LCP-azide 25)11 1000 |iM 10 |iM Ascorbic acid 7.5)11 5 |iM 50 |iM In total 250)11 - Fc-LCP purification Size exclusion chromatography (SEC) was used to purify the Fc-LCP from free unreacted LCP-azide. HiLoad 16 / 600 Superdex 75pg column was connected to an Akta go (Cytiva) protein purification system. Before loading the sample, the system and the column was washed with 500ml milliQ water at a rate of 1.5 ml / min, followed by washing with 500ml PBS as washing buffer. The Fc-LCP was eluted with PBS at a rate of 0.8 ml / min, and fractions were concentrated with a 30kDa cutoff concentrator (Amicon). Aliquoted Fc-LCP was stored at -20°C. - Kinetic measurements between Fc-LCP and Fcyl receptor Surface plasmon resonance (SPR) experiments were conducted by Dr. Jens Sobek at Functional Genomics Center Zurich using Biacore T200 and S200 instruments, respectively. All experiments were performed on a NiHC30M chip (Xantec, Dusseldorf, Germany) in HBS buffer (10 mM HBS, 150 mM NaCI, 50 pM EDTA, 0.05% Tween-20) at 20°C. Surface preparation and kinetic measurements were conducted at a flow rate of 5 pL / min and 30 pL / min, respectively. After conditioning with 0.35 M EDTA for 5 min and a 2 min stabilisation period, the surface in FC2 was activated with 5 mM NiCI2 solution in HBS for 120s. FcyRI at 20 nM in HBS was immobilised at a density of 500 RU. Adjacent flow cell 1 was left empty being used a reference. Due to a slowly decreasing baseline after immobilisation, the surface was allowed to stabilise for a period of 10 hours before quantitative experiments were started. For kinetic experiments, dilution series of 5 concentrations Fc -LCP (cmax= 500 nM) and Fc (20 nM) in HBS-LMNG (10 mM HBS, 150 mM NaCI, 50 pM EDTA, xx% LMNG) were successively injected for 120 s followed by a dissociation period of 600 s (single- cycle kinetics). The surface was regenerated by injection of 0.35 M EDTA for 300s which removes all bound probes. This allows the chip to be used for further experiments. Kinetic data were evaluated using BiaEvaluation v2.03 software. The sensorgrams were fitted using a 1+1 kinetic model (Fc) and a sum of two exponentials model ("heterogeneous ligand model", Fc-LCP). - Binding of Fc-LCP to prion fibrils Immunoprecipitation (IP) was performed to confirm the binding of the Fc-LCP to a Rocky Mountain Laboratory (RML) scrapie strain (passage 6, RML6) infected mouse brain homogenates. 50pl of dynabeads were incubated with 4pd of 1 mg / ml Fc-LCP filled up with PBS to 500pl, on a rotating wheel at RT for 1h. The incubation was inserted into a magnetic rack to collect the beads. After removing the supernatant, the beads were washed in 500pl of PBS, followed by resuspending them in 50pl of PBS. 200pg of RML6 and non-infectious brain homogenates (NBH) were added to the beads, respectively. The mix was filled up to 500pil with PBS and incubated on the wheel at 4°C overnight. On the next day, the beads were collected by going through the magnetic rack, washed with 500ul of PBS for 3 times and resuspended in 20ul of PBS. Proteinase K (PK) was added to the beads at a final concentration of 25ug / ml. The PK added samples were incubated at 37°C and shaking at 700rpm for 30min. 4x loading buffer was added to the PK digested samples right after the incubation finished and samples were boiled at 95°C for 5min. Samples were centrifuged to pellet the beads and the supernatant was loaded into a 4-12% Bis-Tris gel. iBlot2 was used to transfer the protein from SDS-gel to a Nitrocellulose (NC) membrane. The membrane was blocked with 5% Sureblock in PBST for 1 h, followed by overnight incubation with 200ng / ml of anti-PrP in-house produced POM1 antibody at 4°C overnight. After 3 times of washing with PBST for 15min, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibody at RT for 1h. The membrane was washed 3 times with PBST and developed with Immobilon Crescendo HRP substrate. Images were taken with Fuji LAS-3000 Imaging system. - Enzyme-linked immunosorbent assay (ELISA) ELISA was performed to measure the binding between Fc-LCP and Ap fibrils. 1Opg Ap42 fibrils and monomers diluted in 20pl PBS were used to coat a 384well plate and stored at 4°C overnight. The next morning, the coated plate was washed three times with a plate washer. 40jxl 5% sureblock in PBST was used to block the coating at RT for 1h. After removal of the blocking solution without washing the plate, series of diluted Fc-LCP, Fc and LCP in PBS ranging from 0.1 p.M to 6.103515625pM was added to both fibrils and monomers, followed by 2h of incubation at RT. The plate was washed 3 times after incubation. 1:10,000 diluted HRP-conjugated goat anti-mouse IgG antibody was added and incubated for another 1 h at RT. After 3 times of washing with the plate washer, 20ju.l TMB substrate was added and reacted for 5min. Finally, 20,4.1 0.5M H2SO4 was used to stop the reaction. The absorbance at 405nm was recorded with a plate reader. - Immunostaining Paraffin embedded APP / PS1 mouse brain slides were deparaffinized in xylene and gradient ethanol (100%, 90% and 70%), and rehydrated in water. Antigen retrieval was performed by immersing the slides into 10% formic acid for 10min. Permeabilization was done by three times washing with 0.1 M Tris-Buffered Saline (TBS, 50 mM Tris-CI, pH 7.5,150 mM NaCI)for 15min. The slides were blocked with blocking buffering containing 5% donkey serum and 1% bovine serum albumin (BSA) in PBST at RT for 1h. Then, 100pJ of diluted Fc-LCP (10pM), Fc (10p.M) or LCP (1 OOjiM) was dripped on the slide just to cover the tissue, which was circled in by a hydrophobic pen. Incubation was performed at RT for 2h; followed by 3 times of washing with TBS. Alexa647 conjugated goat anti-mouse IgG antibody was added to detect Fc and incubated at RT for 2h. DAPI (1:10,000) was added into TBS during the first washing to stain the nuclei. Slides were mounted in mounting medium, covered with a glass slip and dried overnight in the dark. Images were taken with Leica Stellaris 5 upright microscopy. - Fibril formation assay Recombinant mouse PrP (mPrP) comprising residues 23-231 and a-syn protein were expressed and purified as stocks previously in the lab. To produce fibrils, 50p.M oc-syn was prepared in 10mM 2-(N-morpholino) ethanesulfonic acid (MES, pH5.5) and 50pM mPrP was prepared in 50mM Tris-HCI, pH 7.4, 1M GdHCI and 150mM NaCL Then, the protein solution was mixed with a series of concentration from 0.08pM to 50pM of Fc-LCP and LCP, respectively. Samples mixed with PBS and 10pM ThT were set as controls. 1OOpI of the mixed samples was added into a NUNC plate with optically clear bottom and incubated in Fluostar Omega Microplate reader which was set at 37°C. Absorbance at 350nm was recorded as readout after every 260s of orbital shaking at 600rpm. The Assay was run in triplicate and the scheme is in the following Table. Table 9: Assay protocol Blank 10 pM ThT 50 pM LCP 50 pM Fc-LCP 10 pM LCP 10 pM Fc-LCP 2 pM LCP 2 pM Fc-LCP 0.4 pM LCP 0.4 pM Fc-LCP 0.08 pM LCP 0.08 pM Fc-LCP - BV2 Phagocytosis assay Murine BV2 cells were cultured in T75 flasks in Opti-MEM medium supplemented with 5% FBS and 1% Penicillin-Streptomycin. Cells were passed every 2 days until the phagocytosis assay. One day before the phagocytosis assay, 0.5 * 106 BV2 cells were seeded in Opti-MEM medium containing 0.5% FBS in wells of 6-well plates and grown overnight. Before phagocytosis assay, protein fibrils (mPrP or Ap42) were sonicated in an ultrasonic bath sonicatorfor 10min with 30 seconds on and 30 seconds off intervals. The sonicated protein fibrils were then incubated with Fc-LCP and other controls (Fc and LCP, respectively) in medium for 30 min in a 37°C water bath. The medium was removed and medium including fibrils was added to the cells, and the cells were incubated for 1h. The medium was then removed, and cells were washed with PBS gently once. To detach the cells from the plate, 0.025% trypsin-EDTA was added and incubated for5min in the incubator for digestion. Opti-MEM medium containing 5% FBS and 1% Penicillin-Streptomycin was used to deactivate the trypsin. Cells were harvested, filtered into 5ml Falcon test tubes with cell strainer snap cap, and centrifuged for 5min at lOOOrpm and 4°C. The pellet was washed 3 times with PBS supplemented with 1% FBS and 2mM EDTA. After washing, cells were diluted in 30ul of FACS buffer, kept on ice, and ready for Imaging flowcytometry analysis. The Image Stream X (ISX) Mark II imaging flow cytometer (Luminex) equipped with a 10 mW 488nm argon-ion laser was used to detect the internalized protein fibrils. IDEAS software (Luminex) was used to analyse the images. - Primary mouse microglia phagocytosis assay The protocol for was adapted from (Lian et al., Bio Protoc. 2016 Nov 5;6(21):e1989). Briefly, 0-2 days old C57BL / 6J pups were decapitated. The whole brain was removed from the skull and placed in ice cold dissection medium. The meninges on the cortex and hippocampi were carefully removed under a microscope, and the tissue was cut into small pieces with fine tweezers. Then, the cut tissue was digested with 0.25% Trypsin supplemented with 1mg / ml DNase I for 15 minutes in a shaking incubator at 37°C. After being inactivated with FBS, the tissue was homogenized mechanically with a 10OOjxl pipette tip and filtered through the 40um mesh cell strainer. After centrifuging, the cells were seeded in a T75 flask preliminarily coated with 10pg / ml Poly-D-lysine and cultured in an incubator with 5% CO2 and 95% humidity at 37°C. Medium was changed the day after dissection to remove the cell debris and afterwards every 5 days. Around 10 days after dissection, the astrocytes were confluent at the bottom of the flask and microglia grew on top of the astrocytes. One day before the phagocytosis assay, the mixed glia culture was seeded into a 6 well plate at a density of 0.5 * 106 cells / ml. The phagocytosis assay in primary glia culture was performed as the one in BV2 cells. - Cultured organotypic cerebellar slices (COCS) The protocol was adapted from (Falsig et al., Nat Neurosci. 2008 Jan; 11 (1): 109-17). 12-13-day-old Tga20 (a PrP overexpressing strain) pups were sacrificed, and the cerebellums were taken out in freezing GBSS buffer (137 mM NaCI, 5 mM KCI, 0.845 mM Na2HPO4, 1.5 mM CaCI2'2H2O, 0.66 mM KH2PO4, 0.28 mM MgSO4-7H2O, 1 mM MgCI2-6H2O, 2.7 mM NaHCO3, pH 7.3) supplemented with 33.33mM glucose and 1 mM kynurenic acid (GBSSK buffer). Then, the cerebellums were adjusted standing in agarose gel (20mg / ml in GBSSK buffer) to get sagittal slices. The agarose gel block was placed on a Leica VT1000 S vibrating blade microtome and cut into 350-p.m slices. The extra agarose surrounding cerebellar slices were cleared out with fine tweezers, and the slices were washed with GBSSK buffer for 3 times before infection. 40-50 slices were collected in a well of 24-well plate with 2-3mm depth of GBSSK above the slices. For one slice, 30pg of 20% RML6 brain homogenate was used to achieve successful prion infection. The slices were incubated after infection at 4°C for 1 h, followed by 1-2 times of washing with GBSSK. Slices were randomly seeded into inserts in a 6-well plate with final numbers of slices of 6-8 per well. 1 ml COCS medium at RT was added outside the insert. The medium was changed 3 times per week. Treatment including PBS, Fc, LCP, and Fc-LCP, respectively, were added into the medium on two time points which are 14- and 30-days post infection. Around 50 days post infection, slices were harvested for analysis including, WB and immunostaining. - Proteinase K (PK) digestion and western blotting Slices were briefly washed with PBS once and then scratched down and transferred into an Eppendorf tube containing 140p.l radioimmunoprecipitation assay buffer (RIPA, 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCI) supplemented with proteinase inhibitors. To break the tissue, 20 homogenizer beads were added to the tube to homogenize the slices using a homogenizer. The homogenized tissue was centrifuged at a speed of 1000g for 10min. The supernatant was diluted 20 times in PBS and a bicinchoninic acid assay (BCA) assay was performed to measure the protein concentration in the supernatant. PK digestion was performed before western blotting to detect Proteinase K resistant PrPSc. In total, 25pg / ml PK was used to digest 40pg of total protein in a 40p.l reaction volume for 30min at 37°C. 13pd of 4x NuPAGE LDS loading buffer were added to the digested sample before boiling them at 95°C for 5min. After a short spin down, boiled samples were loaded onto a 412% Bis-Tris gradient gel and run under 150Volt and 120mA for 1 h. The gel was blotted onto a Nitrocellulose membrane (for prion) or a PVDF membrane (for non-prion antigens). Membranes were then blocked with 5% sureblock buffer in PBST for 1h at RT. Then the membrane was incubated with the primary antibody (in-house produced POM-1 mouse IgG 1 200ng / ml, rabbit anti-mouse neuronal marker (NeuN) (Abeam, ab177487) 800ng / ml) diluted in 5% sureblock buffer in PBST overnight at 4°C. Membranes were incubated in secondary antibodies (HRP conjugated goat anti-mouse or goat anti-rabbit) the next day for 1h at RT after 3 times washing with PBST. Afterwards, membranes of nondigested samples were incubated in stripping buffer (50 mM Tris-HCI, pH 7, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol (DTT)) for 15min at 37°C to strip the primary antibody. Membranes were blocked with 5% sureblock in PBST for 1h at RT and incubated in diluted HRP conjugated actin mouse antibody for 1h at RT. Immobilon Crescendo western HRP substrate was used to detect the HRP conjugated secondary antibodies. The images were acquired with a Fuji LAS-3000 Imaging system. - Immunostaining Slices were washed with PBS once and fixed with 4% PFA (1ml in the insert and 1ml in the well) for 30min at room temperature. After fixation, slices were washed with PBS for 3 times (1 ml in the insert and 1 ml in the well). The membrane containing slices were cut and placed in 24-well plate with slices on top. The slices were blocked with 5% goat serum in PBST overnight at 4°C. Without washing, the blocking buffer was removed, and 500,4.1 of diluted antibodies (1:1000 Alexa Fluor 647 anti-NeuN antibody (Abeam, ab177487), 1:1000 Alexa Fluor 488 anti Iba1 antibody (Abeam, ab225260)) in PBST containing 5% goat serum were added into the wells and incubated for 3 days at 4°C, shaking. Slices were washed with 1 ml PBST containing 1:10,000 diluted DAPI for30min at RT to stain the nuclei. Then, after3 times of 30min washing with PBST, slices were mounted on glass slides with mounting medium (DAKO) with slices on top, covered with glass coverslip. Slides were sealed with nail polish. Images were acquired with a Fluoview confocal microscope (Olympus). - Statistical analysis GraphPad Prism software was used for statistical analysis. Data were assumed normally distributed. For ELISA results, a sigmoid fitting was selected to visualize the binding between the Fc-LCP and Ap fibrils. For comparison of more than two groups in phagocytosis assays and COCS treatment, one-way analysis of variance (ANOVA) was used, and multiple comparison tests were performed to acquire the p-values. P-values < 0.05 were considered statistically significant. In graphs, data were plotted with standard error of mean (SEM) and * was used to indicated p-values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns means not significant. Cited references: All scientific publications and patent documents cited in the present specification are incorporated by reference herein. Chen I, Dorr BM, Liu DR. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul 12; 108(28):11399-404. doi: 10.1073 / pnas.1101046108. Epub 2011 Jun 22. PMID: 21697512; PMCID: PMC3136257. Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AE, Ploegh HL. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc. 2013 Sep;8(9):1787-99. doi: 10.1038 / nprot.2013.101. Epub 2013 Aug 29. PMID: 23989673; PMCID: PMC3943461. Brelstaff J, Spillantini MG, Tolkovsky AM. pFTAA: a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons. Neural Regen Res. 2015 Nov; 10(11):1746-7. doi: 10.4103 / 1673-5374.165298. PMID: 26807101; PMCID: PMC4705778. Margalith I, Suter C, Ballmer B, Schwarz P, Tiberi C, Sonati T, Falsig J, Nystrom S, Hammarstrbm P, Aslund A, Nilsson KP, Yam A, Whitters E, Hornemann S, Aguzzi A. Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates. J Biol Chern. 2012 Jun 1;287(23): 18872-87. doi: 10.1074 / jbc.M112.355958. Epub 2012 Apr6. PMID: 22493452; PMCID: PMC3365923. Park et aL, Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals. Front. Immunol., 18 February 2020, Sec. Comparative Immunology Volume 11 -2020 | https: / / doi.org / 10.3389 / fimmu.2020.00203 Polymenidou M, Moos R, Scott M, Sigurdson C, Shi Yz, et al. (2008) The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes. PLOS ONE 3(12): e3872. https: / / doi.org / 10.1371 / journal.pone.0003872 Lian H, Roy E, Zheng H. Protocol for Primary Microglial Culture Preparation. Bio Protoc. 2016 Nov 5;6(21):e1989. doi: 10.21769 / BioProtoc.1989. PMID: 29104890; PMCID: PMC5669279. Falsig J, Julius C, Margalith I, Schwarz P, Heppner FL, Aguzzi A. A versatile prion replication assay in organotypic brain slices. Nat Neurosci. 2008 Jan;11(1):109-17. doi: 10.1038 / nn2028. Epub 2007 Dec 9. PMID: 18066056; PMCID: PMC2754795. SEQUENCES: In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail. mlgG1-Fc (SEQ ID NO: 1) GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPRE EQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKD KVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK hlgG1-Fc (SEQ ID NO: 2) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK Amino acid inker (SEQ ID NO: 3) GGGGS Amino acid linker + Sortase A recognition site + His6-Tag (SEQ ID NO: 4) GGGGSLPETGGHHHHHH Sortase binding motif (SEQ ID NO: 5) LPETG Sortase recognition site (SEQ ID NO: 6) LPETGG His6-Tag (SEQ ID NO: 7) HHHHHH Sortase binding motif portion (SEQ ID NO: 8) LPET Sortase binding motif portion (SEQ ID NO: 9) LPAT Manifold linker (SEQ ID NO: 10) ggggg-(k-PEG2) Internal forward (SEQ ID NO: 11) AACCCCGGGAGGAGCAGTTCgctAGCACTTTCCGCTCAGTCAG Internal reverse: (SEQ ID NO: 12) gtccaccagtttcaggaagagaacctccacctccACCAGGAGAGTGGGAGAGG vector forward (SEQ ID NO: 13): ctcttcctgaaactggtggacatcaccatcaecatcactaaTGATCCCAGTGTCGCTAG vector reverse (SEQ ID NO: 14): GAACTGCTCCTCCCGGGGTTG Signal sequence insertion forward (SEQ ID NO: 15) GCACTAAGTCTTGCACTTGTCACGAATTCGGGTTGTAAGCCTTGCATATG Signal sequence insertion reverse (SEQ ID NO: 16) AATGCAAGACAGGAGTTGCATCCTGTACATAGATCTAACCATGGTGCTCG EF1a Fwd sequencing (SEQ ID NO: 17) CGCAACGGGTTTGCCGCCAG Fc Rev sequencing (SEQ ID NO: 18) CCATACCACATTTGTAGAGG Fig. 3b (SEQ ID NO: 19) GGHHHHHH Linker Fig. 3b (SEQ ID NO: 20) GGGGG Linker + Sortase A recognition site Fig. 3b (SEQ ID NO: 21) GGGGSLPET Sortase A recognition site + Linker Fig. 3b (SEQ ID NO: 22) TEPLSGGGG 5 Fig. 18 (SEQ ID NO: 23) GGGGGKKKKK Fig. 3b (SEQ ID NO: 24) HHHHHHGGTEPLSGGGG

Claims

1. A conjugate comprisinga. an immunoglobulin Fc polypeptide, andb. an oligothiophene moiety.

2. The conjugate according to claim 1, wherein the immunoglobulin Fc polypeptide and the oligothiophene moiety are linked covalently through a linker that is not cleavable under physiological conditions.

3. The conjugate according to claim 1 or 2, wherein the oligothiophene moiety is conjugated to a C-terminus of the immunoglobulin Fc polypeptide.

4. The conjugate according to any one of the preceding claims, wherein the immunoglobulin Fc polypeptide comprises an immunoglobulin G fragment crystallizable region (IgG-Fc).

5. The conjugate according to claim 4, wherein the IgG-Fc is a human IgG-Fc.

6. The conjugate according to any one of the preceding claims 4 or 5, wherein the IgG-Fc is an lgG1-Fc.

7. The conjugate according to any one of the preceding claims 4 or 5, wherein the IgG-Fc is an lgG4-Fc.

8. The conjugate according to any one of the preceding claims, wherein the immunoglobulin Fc polypeptide consists of an amino acid sequence >85% identical, particularly >90% identical, more particularly >95% identical, even more particularly >97% identical, yet even more particularly >98% identical, most particularly >99% identical to (SEQ ID NO: 2).

9. The conjugate according to any one of the preceding claims, wherein the immunoglobulin Fc polypeptide is a homodimeric Ig-Fc.

10. The conjugate according to any one of the preceding claims, wherein each monomer of the homodimeric Ig-Fc is conjugated to an oligothiophene moiety.

11. The conjugate according to any one of the preceding claims, wherein the oligothiophene moiety comprises a sub-moiety of a general formula (I):n whereinn is an integer denoting a number of thiophene monomer subunits selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, wherein- R1 is independently selected from: H, C1-C4 carboxyl;- R2 is independently selected from: H, C1-C4 carboxyl;- Rl designates the bond to the immunoglobulin Fc polypeptide, optionally through a manifold linker;- Re is a terminal moiety.

12. The conjugate according to claim 11, wherein the terminal moiety RE is selected fromH, Ci to C4 alkyl or Ci to C4 carboxyalkyl or a salt thereof or             R .wherein0 Rw, Rx, Ry and Rz are independently selected from CH, N, NRS, CRT;0 Ru is selected from CH2, S, NH, NRS, CRT,wherein■ Rs and RT are independently selected from C1-C4 alkyl or C2-C6 alkene;and wherein0 only one of CH, N, NRS or CRT is covalently bound to a thiophene directly preceding the terminal moiety RE.

13. The conjugate according to any one of the preceding claims 11 to 12, wherein RE is selected from:- C1-C6 carboxyl, particularly COOH;- an azaindole, particularly 6-azaindole;- benzothiazole.

14. The conjugate according to any one of the preceding claims 11 to 13, wherein the oligothiophene moiety is selected from a group consisting of:

15. The conjugate according to any one of the preceding claims 11 to 14, wherein the16. The conjugate according to any one of the preceding claims 11 to 15, wherein RL comprises an oligo(ethyleneglycol) conjugating the oligothiophene moiety to the immunoglobulin Fc polypeptide.

17. The conjugate according to any one of the preceding claims, wherein more than one oligothiophene moiety is present in the conjugate per immunoglobulin Fc polypeptide.

18. The conjugate according to claim 17, wherein 1 to 25 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide.

19. The conjugate according to claim 18, wherein 2 to 20, particularly 5 to 15 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide.

20. The conjugate according to claim 17, wherein 5 oligothiophene moieties are attached to an immunoglobulin Fc polypeptide.

21. The conjugate according to any one of the preceding claims, wherein the conjugate further comprises a manifold linker, connecting the immunoglobulin Fc polypeptide and the oligothiophene moiety.

22. The conjugate according to claim 21, wherein the manifold linker allows attachment of more than one oligothiophene moiety to one immunoglobulin Fc polypeptide.

23. The conjugate according to claim 21 or 22, wherein the manifold linker comprises an oligopeptide, and an N-terminus of the linker binds the C-terminus of the immunoglobulin Fc polypeptide.

24. The conjugate according to any one of the preceding claims 21 to 23, wherein the manifold linker comprises an oligopeptide moiety constituted of amino acids bearing an oligothiophene moiety on their side chain.

25. The conjugate according to claim 24, wherein the of amino acid bearing an oligothiophene moiety on their side chain is a lysine bearing an oligothiophene moiety connected to the s-amino group of the lysine through a bridging moiety consisting of 2 to 50 atoms selected from C, O, N and S.

26. The conjugate according to any one of the preceding claims 21 to 25, wherein an immunoglobulin Fc polypeptide to linker ratio Fc / L is 1 / 1.

27. The conjugate according to any one of the preceding claims 21 to 25, wherein a homodimeric Ig-Fc to linker ratio Ig-Fc / L is 1 / 2.

28. The conjugate according to any one of the preceding claims, wherein the conjugate comprises a first affinity to an Fc receptor, wherein the affinity is characterized by the values in the following table:Probe Analyte ko„[M-1s1] Aoff[s-1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 1*E+04 to 1+E06 3*E-03 to 3*E-05 3*E-07 to 3*E-11 50 to 60 0.1 to 0.

529. The conjugate according to claim 28, wherein the first affinity to a Fc receptor is characterized by the values in the following table:Probe Analyte ko„[M-1s1] Aoff[s-1] Kd[M] RU (max) Chi2 Fcyl receptor conjuga te 9.072*E04 2.65*E-04 2.92*E-09 56.4 0.2130. The conjugate according to any one of the preceding claims, wherein the conjugate comprises a second affinity to a prion fibril.

31. The conjugate according to any one of the preceding claims, wherein the conjugate is characterized by a phagocytic index (PIconjugate), characterized in that Plconjugate > PhgGi-Fc and / or PI conjugate > Plp-FTAA-32. The conjugate according to any one of the preceding claims, wherein Plconjugate > (PhgGi-Fc + Plp-FTAA).

33. The conjugate according to any one of the preceding claims, for use in treatment and / or prevention of a disease.

34. The conjugate for use according to claim 33, wherein the disease is a neurodegenerative disease.

35. The conjugate for according to claim 33 or 34, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer’s disease and a prion disease.

36. The conjugate for use according to any one of claims 33 to 35, wherein the prion disease is selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS).

37. The conjugate for use according to claim 33, wherein the disease is amyloidosis.

38. Use of the conjugate according any one of claims 1-32 in a method of manufacture of a medicament for treatment and / or prevention of a neurodegenerative disease or amyloidosis.

39. The use according to claim 38, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer’s disease and a prion disease.

40. The use according to claim 38 or 39, wherein the prion disease is selected from a group consisting of: Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Sporadic Creutzfeldt-Jakob Disease (sCJD), Familial Creutzfeldt-Jakob Disease (fCJD), Iatrogenic Creutzfeldt-Jakob Disease (iCJD), Kuru, Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker Syndrome (GSS).

41. A composition comprising a conjugate according to any one of claims 1-32 and a pharmaceutically acceptable excipient.

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