General amyloid-interacting motifs (GAIM)

文档序号：722534 发布日期：2021-04-16 浏览：11次中文

阅读说明：本技术 通用淀粉样蛋白相互作用基序(gaim) (General amyloid-interacting motifs (GAIM) ) 是由 R.克里什南 E.艾斯普 M.普罗奇斯基 R.费希尔于 2019-06-14 设计创作，主要内容包括：本发明涉及噬菌体基因3蛋白(g3p)的通用淀粉样蛋白相互作用基序(GAIM)的变体及其融合蛋白。本发明的GAIM变体和融合蛋白被部分或完全去免疫化,并显示出对多种多样的淀粉样蛋白的优异结合和特异性,以及表现出增强的淀粉样蛋白重构和对淀粉样蛋白聚集的抑制。本发明进一步涉及核酸、载体、宿主细胞以及制备GAIM变体及其融合蛋白的方法。本发明还涉及药物组合物和提高噬菌体感染性的方法,检测淀粉样蛋白聚集体的方法,以及诊断和/或治疗与错误折叠和/或聚集的淀粉样蛋白相关的疾病的方法。(The present invention relates to variants of the universal amyloid-interacting motif (GAIM) of the bacteriophage gene 3 protein (g3p) and fusion proteins thereof. The GAIM variants and fusion proteins of the invention are partially or fully deimmunized and exhibit excellent binding and specificity for a wide variety of amyloid proteins, as well as exhibit enhanced amyloid remodeling and inhibition of amyloid aggregation. The invention further relates to nucleic acids, vectors, host cells and methods for making GAIM variants and fusion proteins thereof. The invention also relates to pharmaceutical compositions and methods of increasing bacteriophage infectivity, methods of detecting amyloid aggregates, and methods of diagnosing and/or treating diseases associated with misfolded and/or aggregated amyloid proteins.)

1. A polypeptide comprising a variant of the starting amino acid sequence SEQ ID NO:16, wherein said variant differs from SEQ ID NO:16 by one or more sets of amino acid changes selected from the group consisting of:

a) substitution of any amino acid for T50 and H55T;

b)N137G；

c)N142A；

d) R143V and Q144N; or

R143V, Q144N and a 146V; or

R143V, Q144N and a 146T; or

R143V, Q144N and a 146K; and

e) Q156V, G157N, Δ T158, Δ D159, Δ P160, and V161G; or

Q156Y, G157N, Δ T158, Δ D159, Δ P160, and V161G; or

G157N, Δ T158, Δ D159, Δ P160, and V161G; or

Δ T158, Δ D159, Δ P160, and V161G;

optionally, wherein the variant further differs from SEQ ID No. 16 by one or more sets of amino acid changes selected from:

f) Δ M1; or

Δ M1 and Δ a 2; and

g) substitution of N38 with any amino acid other than cysteine; or

N38 with any amino acid other than cysteine, and G40 with any amino acid other than cysteine; or

G40 was substituted with any amino acid except cysteine, threonine or serine.

2. The polypeptide of claim 1, wherein the T50 substitution is selected from the group consisting of: T50G, T50H, T50K and T50R.

3. The polypeptide of claim 2 wherein the T50 substitution is T50H.

4. The polypeptide of claim 1, wherein the variant differs from SEQ ID NO 16 by at least Δ M1 and Δ A2.

5. The polypeptide of any one of claims 1 to 4, wherein the variant differs from SEQ ID NO 16 by at least N137G and/or N142A.

6. The polypeptide of claim 4, wherein the variant further differs from SEQ ID NO 16 by one or more sets of amino acid changes selected from the group consisting of:

a) substitution of any amino acid for T50 and H55T; and

b) R143V and Q144N; or

R143V, Q144N and a 146V; or

R143V, Q144N and a 146T; or

R143V, Q144N and a 146K.

7. The polypeptide of claim 6, wherein said T50 substitution is selected from the group consisting of: T50G, T50H, T50K and T50R.

8. The polypeptide of claim 7 wherein the T50 substitution is T50H.

9. The polypeptide of claim 1, wherein the variant of SEQ ID NO 16 is selected from the group consisting of: 19, 20, 21, 22, 23, 24, 25 and 26.

10. The polypeptide of any one of claims 1 to 9, further comprising an immunoglobulin constant region.

11. The polypeptide of claim 10, wherein the immunoglobulin constant region sequence is the Fc portion of a human IgG.

12. The polypeptide of claim 11, consisting essentially of the polypeptide of any one of claims 1 to 9 and the Fc portion of a human IgG.

13. The polypeptide of claim 12, wherein the human IgG is human IgG 1.

14. A polypeptide consisting essentially of an amino acid sequence selected from the group consisting of:

a)SEQ ID NO:29；

b)SEQ ID NO:30；

c)SEQ ID NO:31；

d)SEQ ID NO:32；

e)SEQ ID NO:33；

f)SEQ ID NO:34；

g) 35 in SEQ ID NO; and

h)SEQ ID NO:36；

optionally, wherein the variant has one or more sets of amino acid changes selected from:

i) Δ M1; or

Δ M1 and Δ a 2; and

j)ΔK485。

15. a pharmaceutical composition comprising the polypeptide of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A method of reducing amyloid, inhibiting amyloid formation, inhibiting amyloid aggregation, or removing and/or preventing toxic oligomer formation in a subject in need thereof, comprising the step of administering to the subject the polypeptide of any one of claims 1 to 14 or the pharmaceutical composition of claim 15.

17. The method of claim 16, wherein the amyloid protein or oligomer comprises a protein selected from the group consisting of: an androgen receptor; apolipoprotein AI; apolipoprotein AII; apolipoprotein AIV; apo serum (aposerum) amyloid a; a beta; ABri; ADan; atrophin-1; atrial natriuretic peptides; ataxin; a calcitonin; gamma-crystalline protein; cystatin C; fibrinogen; gelsolin; huntingtin; insulin; an amylin polypeptide; an immunoglobulin kappa light chain; an immunoglobulin lambda light chain; corneal-epithelin; a keratin protein; a milk mucin; lactoferrin; lysozyme; pulmonary surfactant protein C; medin; odontogenic ameloblast-associated protein; a prion protein; procalcitonin; prolactin; protamine I; serum amyloid a; superoxide dismutase I; beta 2-microglobulin; a TATA box binding protein; tau; transthyretin; and alpha-synuclein.

18. A method of treating a disease selected from the group consisting of: alzheimer's disease; early-onset alzheimer's disease; late-onset alzheimer's disease; pre-symptomatic alzheimer's disease; AL amyloidosis; amyotrophic Lateral Sclerosis (ALS); amyotrophic lateral sclerosis/parkinson-dementia syndrome; dementia with silvery particles; aortic medial amyloidosis; ApoAI amyloidosis; ApoAII amyloidosis; ApoAIV amyloidosis; atrial amyloidosis; dementia of the british/denmark type; cataract; degeneration of the cortical basal lamina; corneal amyloidosis associated with trichiasis; cystatin C plaque associated diseases; cystatin C plaque associated coronary heart disease; cystatin C plaque associated nephropathy; cutaneous licheniform amyloidosis; dementia pugilistica; globus pallidus with dentate nucleus and red nucleusHypothalamic atrophy; diffuse neurofibrillary tangles with calcification; dementia with lewy bodies; down syndrome; familial Amyloidosis Cardiomyopathy (FAC); familial Amyloid Polyneuropathy (FAP); familial dementia of the british type; dementia of the danish type familiarity; familial encephalopathy; familial mediterranean fever; amyloidosis of fibrinogen; hereditary amyloidosis of the finnish type; frontotemporal dementia with parkinson's disease; frontotemporal lobar degeneration (FTLD); frontotemporal dementia; Hallervorden-Spatz disease; hemodialysis-related amyloidosis; hereditary cerebral amyloid angiopathy; hereditary cerebral hemorrhage with amyloidosis; hereditary lattice corneal dystrophy; huntington's disease; icelandic hereditary cerebral amyloid angiopathy; inclusion body myositis; injecting local amyloidosis; amyloidosis of islet amyloid polypeptide; lysozyme amyloidosis; multiple myeloma; myotonic dystrophy; niemann-pick disease type C; non-synaptonemal motor neuron disease with neurofibrillary tangles; parkinson's disease; peripheral amyloidosis; pick disease; pituitary prolactin tumors; postencephalitic parkinsonism; prion protein cerebral amyloid angiopathy; prion-mediated diseases; kuru disease; creutzfeldt-jakob disease (CJD); gerstmann--Scheinker disease (GSS); fatal Familial Insomnia (FFI); scrapie of sheep; spongiform encephalopathy; pulmonary alveolar proteinosis; progressive subcortical gliosis; progressive supranuclear palsy; senile systemic amyloidosis; serum AA amyloidosis; spinal bulbar muscular atrophy; spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA 7); subacute sclerosing panencephalitis; systemic amyloidosis; familial amyloidosis; wild-type amyloidosis; tangle dementia; and tauopathies.

19. The method of claim 18, wherein the disease is selected from the group consisting of parkinson's disease, alzheimer's disease, and huntington's disease.

20. The method of claim 19, wherein the disease is alzheimer's disease.

21. The method of claim 18, wherein the disease is a prion-mediated disease selected from the group consisting of: Creutzfeldt-Jakob disease, Kuru, fatal familial insomnia and Gerstmann--Scheinker syndrome.

22. An oligonucleotide comprising a nucleic acid encoding the polypeptide of any one of claims 1 to 14.

23. The oligonucleotide of claim 22, wherein the nucleic acid encoding a polypeptide comprises a sequence selected from the group consisting of: 37, 38, 39, 40, 41, 42, 43 and 44.

24. The oligonucleotide of claim 22, wherein the nucleic acid encoding a polypeptide consists of a sequence selected from the group consisting of seq id no: SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 and SEQ ID NO 52.

25. A vector comprising the nucleic acid of any one of claims 22 to 24.

26. A host cell comprising the vector of claim 25.

27. The host cell of claim 26, wherein the host cell is selected from the group consisting of: insect cells, fungal cells, plant cells, bacterial cells, mammalian cells, and transgenic animal cells.

28. The host cell of claim 27, wherein the host cell is selected from the group consisting of: HEK293 cells, HEK 293-derived cells, CHO-derived cells, HeLa cells, and COS cells.

29. A method of making an amyloid-binding protein comprising expressing a protein encoded by the nucleic acid in the vector of claim 25, and isolating the expressed protein.

30. An amyloid-binding protein produced by expressing a protein encoded by the nucleic acid in the vector of claim 25 and isolating the expressed protein.

31. The protein of claim 30, wherein the protein is expressed by culturing the host cell of any one of claims 26 to 28.

Prior art antibody-related therapies may also fail clinically because they fail to block the aggregation of phosphorylated tau and/or fail to block the spread of tau from one region of the brain to another. The open stabilized GAIM-Ig fusions of the present invention address this need. The GAIM-Ig fusions of the present invention cause tau remodeling, thereby preventing tau aggregates from seeding with soluble tau and blocking tau aggregate propagation.

In summary, the GAIM-Ig fusions disclosed herein provide a unique method of preventing or removing pathological amyloid aggregates. The open stabilized GAIM fusions described herein exhibit greater potency, structural stability and specificity for amyloid proteins including both a β and tau fibrils, and are also partially or fully deimmunized relative to prior art alternatives.

Preparation of Polypeptides

Polypeptides of the invention (e.g., polypeptides comprising one or more GAIM variants, including fusion proteins) can be synthesized using techniques well known in the art. For example, the polypeptides of the invention can be recombinantly synthesized in cells (see, e.g., Sambrook et al 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., and Ausubel et al 1989, Current protocols Molecular Biology, Green Publishing Associates and Wiley lnterstance, N.Y.). Alternatively, the polypeptides of the invention may be synthesized using known synthetic methods, such as solid phase synthesis. Synthetic techniques are well known in The art (see, e.g., Merrifield,1973, Chemical polypetides, (eds. Katsoyannis and Panayotis) pp.335-61; Merrifield 1963, J.Am.chem.Soc.85: 2149; Davis et al 1985, biochem.Intl.10: 394; Finn et al 1976, The Proteins (3 rd edition) 2: 105; Erikson et al 1976, The Proteins (3 rd edition) 2: 257; U.S. Pat. No. 3,941,763). Alternatively, the final construct may share essentially the same function as the recombinantly produced fusion protein, but be produced using only non-recombinant techniques (e.g., ligation chemistry). The components of the fusion protein can be prepared using the same general methods as described for g3p expression and the g3p mutation.

In some embodiments, the polypeptide may be fused to a marker sequence, such as a peptide that facilitates purification of the fusion polypeptide (either alone or fused to another protein or incorporated outside of a carrier molecule). The tag amino acid sequence can be a hexa-histidine peptide (SEQ ID NO:53), such as the tag provided in the pQE vector (Qiagen, Mississauga, Ontario, Canada), and the like, many of which are commercially available. For example, hexahistidine (SEQ ID NO:53) provides for convenient purification of the fusion protein as described in Gentz et al, Proc.Natl.Acad.Sci. (1989)86: 821-824. Another peptide tag, Hemagglutinin (HA) tag, which can be used for purification, corresponds to an epitope derived from the influenza HA protein (Wilson et al, (1984) Cell 37: 767).

Pharmaceutical composition

In some embodiments, the present invention provides pharmaceutical compositions comprising any polypeptide comprising a GAIM variant as described herein, optionally together with a pharmaceutically acceptable carrier, diluent or excipient. By "pharmaceutical composition" is meant a therapeutically effective amount of a composition as described herein with physiologically suitable carriers and/or excipients. The pharmaceutical composition does not cause significant irritation to the organism. The phrases "physiologically suitable carrier" and "pharmaceutically acceptable carrier" are used interchangeably and refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered composition. The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples include, but are not limited to, saline, calcium carbonate, calcium phosphate, various sugar and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

The pharmaceutical compositions for use according to the invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions which can be used pharmaceutically. The appropriate formulation depends on the chosen route of administration and the nature of the composition being delivered (e.g., the size and solubility of the polypeptide). In one aspect of these embodiments, the pharmaceutical composition is formulated for injection or infusion into the blood of a patient. In another aspect of these embodiments, the pharmaceutical composition is formulated for direct administration to the brain or central nervous system of a patient, for example, by direct intramedullary, intrathecal, or intraventricular injection.

The compositions described herein may be formulated for parenteral administration, for example by bolus injection or continuous infusion. Pharmaceutical compositions for parenteral administration include aqueous solutions of the compositions in water-soluble form. Additionally, suspensions of the active ingredients can be prepared as oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions (e.g., surfactants such as polysorbates (Tween 20)). Protein-based agents (such as, for example, albumin) can be used to prevent adsorption of the polypeptides of the invention to a delivery surface (i.e., IV bag, catheter, needle, etc.).

For oral administration, the pharmaceutical compositions may be formulated by combining the polypeptides described herein with pharmaceutically acceptable carriers well known in the art.

The formulations may be presented in unit dosage form, for example, in vials, ampoules, or multi-dose containers, optionally with an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Single dosage forms may be in liquid or solid form. The single dosage form may be administered directly to the patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered as a bolus, e.g., a single injection, a single oral dose, including an oral dose comprising a plurality of tablets, capsules, pills, and the like. In alternative embodiments, a single dosage form may be administered over a period of time, such as by infusion, or via an implanted pump, such as an ICV pump. In the latter embodiment, the single dosage form may be an infusion bag or pump reservoir pre-filled with an appropriate amount of polypeptide comprising a GAIM variant. Alternatively, the infusion bag or pump reservoir may be prepared by mixing an appropriate dose of the polypeptide comprising the GAIM variant with the infusion bag or pump reservoir solution just prior to administration to a patient.

Another aspect of the invention includes a method of preparing the pharmaceutical composition of the invention. Techniques for preparing drugs can be found, for example, "Remington's Pharmaceutical Sciences," Mack Publishing co., Easton, Pa, (latest edition), which is incorporated herein by reference in its entirety.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredient is contained in an amount effective to achieve the intended purpose.

Determination of a therapeutically or diagnostically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

Dosage amounts and intervals can be adjusted individually to provide brain levels (minimum effective concentration, MEC) of the phage display vehicle sufficient to treat or diagnose a particular brain disease, disorder or condition. MEC will vary for each formulation, but can be estimated from in vitro data. The dosage necessary to achieve MEC depends on the individual characteristics.

Dosage intervals may also be determined using MEC values. The formulation should be administered using a regimen that maintains brain levels above MEC for 10 to 90% of the time, preferably 30 to 90% of the time, most preferably 50 to 90% of the time.

Depending on the severity and responsiveness of the disease to be treated, administration may be a single or multiple administrations, with the course of treatment lasting from days to weeks or until a cure is effected or a reduction in the disease state is achieved.

The amount of the composition to be administered will, of course, depend on the subject being treated or diagnosed, the severity of the affliction, the judgment of the prescribing physician, and the like.

If desired, the compositions of the present invention may be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The package may for example comprise a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The package or dispenser may also contain a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the composition or form of human or veterinary administration. For example, such a notification may be a label approved by the U.S. food and drug administration for a prescription drug or an approved product insert. Compositions comprising the formulations of the invention formulated in compatible pharmaceutical carriers can also be prepared, placed in an appropriate container, and labeled for treatment of a designated disease, as further detailed herein.

Therapeutic uses

Another aspect of the invention relates to the use of any one of the polypeptides, nucleic acid molecules or compositions of the invention in the treatment of one or more diseases associated with misfolded and/or aggregated amyloid proteins, including but not limited to those involving any one of: transthyretin, immunoglobulin light chain (κ or λ), fAbeta 42, fAlpha syn, or ftau.

In the context of treatment, the terms "patient," "subject," and "recipient" are used interchangeably and include humans as well as other mammals. In some embodiments, the patient is a human that is positive for a biomarker associated with a protein misfolding disease. In one embodiment, the patient exhibits β -amyloid deposits as detected by PET imaging with florbetapir.

The term "treating" and its cognates refer to reducing, slowing, or reversing the progression of a disease in a patient exhibiting one or more clinical symptoms of the disease. "treating" also refers to alleviating, slowing, or reversing the symptoms of a disease in a patient exhibiting one or more clinical symptoms of the disease. In one embodiment, the patient exhibits beta-amyloid deposits as detected by PET imaging with florbetapir, and the amount of beta-amyloid deposits is reduced by treatment. In one embodiment, the patient exhibits beta-amyloid deposits, as detected by a polypeptide or polypeptide composition of the invention, and the amount of beta-amyloid deposits is reduced or maintained by treatment. In another embodiment, the patient exhibits any type of amyloid deposit as detected by PET imaging, and the cognitive function of the patient is improved by the treatment. Improvement in cognitive function can be determined by the methods and tests of McKhann et al, Alzheimer's & Dementia7(3):263-9 (2011).

"prevent" or "prevention" (used interchangeably herein) is distinct from treatment and refers to administration of a polypeptide, nucleic acid, or composition to an individual prior to the onset of any clinical symptoms. Prevention using any of the polypeptides, nucleic acids, or compositions thereof of the invention is contemplated. Prevention may involve an individual known to be at increased risk of or identified as diseased based solely on one or more genetic markers. A number of genetic markers have been identified for various protein misfolding diseases. For example, individuals with one or more of the swedish, indiana, or london type mutations in hAPP are at increased risk of developing early-onset alzheimer's disease and are therefore candidates for prophylaxis. Likewise, individuals with trinucleotide CAG repeats in the huntingtin gene, particularly individuals with 36 or more repeats, will eventually suffer from huntington's disease and are therefore candidates for prophylaxis.

Diseases associated with or characterized by misfolded and/or aggregated amyloid include diseases associated with (e.g., caused at least in part by or associated with) misfolded amyloid, aggregated amyloid, or both misfolded and aggregated amyloid. Peptides or proteins that can form amyloid are as described above. For example, in some embodiments, amyloid is formed from A β, including but not limited to A β 40, A β 42, N-truncated A β 11-42, A β 11-42-Pyro, A β 3-42-Pyro, A β 1-42-E22Q-Dutch mutation, or combinations thereof. In some embodiments, the amyloid protein is derived from a prion protein, such as PrP^ScAnd (4) forming. In some embodiments, the amyloid protein is formed from transthyretin. In some embodiments, the amyloid protein is formed from an immunoglobulin light chain, such as an immunoglobulin kappa light chain and/or an immunoglobulin lambda light chain. In some embodiments, the amyloid is formed from tau. In some embodiments, the amyloid protein is formed from alpha-synuclein.

Diseases associated with or characterized by misfolded and/or aggregated amyloid proteins are described above. Many of the above mentioned misfolded and/or aggregated amyloid diseases occur in the Central Nervous System (CNS). Non-limiting examples of diseases occurring in the CNS are parkinson's disease; alzheimer's disease; frontotemporal dementia (FTD), including patients with the following clinical syndromes: behavioral abnormalities ftd (bvftd), progressive non-fluent aphasia (PNFA), and lewy dementia (SD); frontotemporal lobar degeneration (FTLD); and huntington's disease. The polypeptides, nucleic acids and compositions of the invention are useful for treating diseases characterized by misfolded and/or aggregated amyloid proteins that occur in the Central Nervous System (CNS).

Misfolding and/or aggregation of proteins may also occur outside the CNS. Amyloidosis a (aa) (the precursor protein is serum acute phase apolipoprotein, SAA) and multiple myeloma (the precursor protein immunoglobulin light and/or heavy chains) are two well-known protein diseases that occur outside the CNS and are protein misfolding and/or aggregation. Other examples include diseases involving amyloid proteins formed from: alpha 2-microglobulin, transthyretin (e.g., FAP, FAC, SSA), (apo) serum AA, apolipoprotein AI, AII, and AIV, gelsolin (e.g., finland type of FAP), immunoglobulin light chain (κ or λ), lysozyme, fibrinogen, cystatin C (e.g., cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis, icelandic type), calcitonin, procalcitonin, amylin polypeptide (e.g., IAPP amyloidosis), atrial natriuretic peptide, prolactin, insulin, lactadherin, corneal-epithelium, lactoferrin, odontogenic amelogenin-related protein, and protamin I. The polypeptides, nucleic acids and compositions of the invention may be used to treat diseases involving misfolding and/or aggregation of proteins occurring outside the CNS.

Diseases associated with or characterized by misfolded and/or aggregated amyloid proteins may also involve tauopathies. Reviewed in Lee et al, Annu. Rev. Neurosci.24: 1121-. Tau protein is a microtubule-associated protein expressed in axons of both central and peripheral nervous system neurons. Neurodegenerative tauopathies (sometimes referred to as tauopathies) are contemplated. Examples of tauopathies include Alzheimer's disease, amyotrophic lateral sclerosis/Parkinson-dementia syndrome, dementia with silvery particles, corticobasal degeneration, Creutzfeldt-Jakob disease-Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia (including frontotemporal dementia with parkinsonism linked to chromosome 17), Gerstmann--Scheinker's disease, Hallervorden-Spatz disease, myotonic dystrophy, niemann-pick disease type C, non-synaptonemal motor neuron disease with neurofibrillary tangles, pick's disease, postencephalitic parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, and tangle dementia. Some of these diseases may also comprise deposits of amyloid beta peptide. For example, alzheimer's disease exhibits both amyloid beta deposition and tauopathies. Similarly, prion-mediated diseases such as Creutzfeldt-Jakob disease, prion protein cerebral amyloid angiopathy, and Gerstmann-Scheinker syndrome may also have tauopathies. Thus, reference to a disease as "tauopathy" should not be understood to exclude the disease from other neurodegenerative or misfolded and/or aggregated amyloid disease classifications or groupings, which are provided for convenience only. The polypeptides and compositions of the invention may be used to treat neurodegenerative diseases as well as diseases involving tauopathy.

In some embodiments, the polypeptide, pharmaceutical composition or formulation is a method for reducing amyloid in a patient exhibiting symptoms associated with the presence of amyloid or positive for a biomarker associated with a protein misfolding disease, comprising administering to the patient an effective amount of a pharmaceutical composition or formulation as described herein. In some embodiments, the polypeptide, pharmaceutical composition or formulation is a method for maintaining amyloid levels in a patient exhibiting symptoms associated with the presence of amyloid or positive for a biomarker associated with a protein misfolding disease, comprising administering to the patient an effective amount of a pharmaceutical composition or formulation as described herein. In some aspects of these embodiments, the biomarker is beta-amyloid, which is detectable by the radiopharmaceutical florbetapir (AV-45, Eli Lilly). In some aspects of these embodiments, the route of administration is intrathecal injection or infusion, direct intraventricular injection or infusion, intraparenchymal injection or infusion, or intravenous injection or infusion.

In some embodiments, the polypeptide, pharmaceutical composition, or formulation is a method for disaggregating or reconstituting amyloid in a patient. In some embodiments, the polypeptide, pharmaceutical composition, or formulation is a method for reducing amyloid formation in the brain. In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for promoting amyloid clearance in the brain. In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for inhibiting amyloid aggregation in the brain. In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is used in a method of clearing brain toxic oligomers. In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for preventing the formation of brain toxic oligomers. In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for protecting neurons from amyloid damage. In some embodiments, the polypeptide, pharmaceutical composition, or formulation is a method for reducing intercellular propagation of alpha-synuclein aggregates. In some embodiments, the polypeptide, pharmaceutical composition, or formulation is a method for blocking intercellular propagation of alpha-synuclein aggregates. In some aspects of these embodiments, the polypeptide, pharmaceutical composition or formulation is administered to a patient in need thereof by intrathecal injection or infusion, direct intraventricular injection or infusion, intraparenchymal injection or infusion, or intravenous injection or infusion.

In some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for causing disaggregation of a β -amyloid deposits in the brain, comprising injecting an effective amount of the polypeptide, pharmaceutical composition or formulation directly into the brain of a patient in need thereof, thereby causing a reduction of a β -amyloid deposits in the brain. In other embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for causing disaggregation of a β -amyloid deposits in the brain, comprising intravenously delivering an effective amount of the polypeptide, pharmaceutical composition or formulation for injection into a patient in need thereof, thereby causing a reduction of a β -amyloid deposits in the brain.

In one embodiment, the pharmaceutical composition or formulation of the invention for protecting neurons from amyloid damage is administered prophylactically.

In some embodiments, the patient is positive for a biomarker associated with a protein misfolding and/or aggregation disease. In one embodiment, the biomarker is β -amyloid and the reagent used to detect β -amyloid is florbetapir (AV45, Eli Lilly).

Unlike antibody-based prior art therapies (e.g., 6E10), GAIM-Ig fusions as described herein target the core of amyloid rather than the unstructured or partially structured N-terminal residues and exhibit excellent remodeling activity (fig. 10A). Thus, in some embodiments, the polypeptide, pharmaceutical composition or formulation of the invention is a method for reconstituting amyloid. In some aspects of these embodiments, the route of administration is intrathecal injection or infusion, direct intraventricular injection or infusion, intraparenchymal injection or infusion, or intravenous injection or infusion.

In general, the polypeptides disclosed herein bind amyloid at least as efficiently as variants or fusion proteins of the M13 phage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein bind amyloid more efficiently than variants or fusion proteins of M13 phage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein reconstitute amyloid more efficiently than variants or fusion proteins of M13 phage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein inhibit amyloid aggregation more effectively than variants or fusion proteins of M13 bacteriophage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein scavenge toxic oligomers more efficiently than the M13 phage g3p or g3p variants or fusion proteins disclosed in the prior art. In some embodiments, the polypeptides disclosed herein reduce the intercellular spread of alpha-synuclein aggregates more effectively than the M13 phage g3p or variants or fusion proteins of g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein detect amyloid more efficiently than variants or fusion proteins of the M13 phage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein are more effective in preventing diseases associated with misfolded and/or aggregated amyloid than variants or fusion proteins of the M13 bacteriophage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein are more effective in treating diseases associated with misfolded and/or aggregated amyloid than variants or fusion proteins of the M13 bacteriophage g3p or g3p disclosed in the prior art. In some embodiments, the polypeptides disclosed herein elicit a smaller immune response in a patient compared to variants or fusion proteins of the M13 bacteriophage g3p or g3p disclosed in the prior art. In at least one embodiment, the polypeptides disclosed herein do not elicit an immune response in a patient.

In another embodiment, any of the diseases described above can be treated by direct administration to a patient by any suitable route, such as inhalation and intravenous infusion, of a nucleic acid molecule of the invention (i.e., encoding a polypeptide comprising a GAIM variant that exhibits reduced immunogenicity or no immunogenicity and has the ability to bind amyloid, disaggregate/reconstitute amyloid, and/or inhibit amyloid aggregation), alone or in combination with a suitable carrier, such as a lipid nanoparticle, a polymeric carrier, or a carrier, such as a viral carrier. The nucleic acid molecule encoding a polypeptide comprising a GAIM variant may be DNA or RNA.

Diagnosis of

Diagnostic compositions are encompassed by the invention and can comprise any of the above-described polypeptides of the invention (e.g., a polypeptide comprising a GAIM variant, e.g., a GAIM-Ig fusion-containing polypeptide). Thus, in some embodiments, the polypeptides, pharmaceutical compositions, and formulations described herein are used in diagnostic applications related to various diseases described herein. For example, binding of a polypeptide of the invention to amyloid can be used to detect bound amyloid. Similarly, binding of a polypeptide of the invention may be part of the protein misfolding, protein aggregation or diagnosis of neurodegenerative diseases described herein, when used as an imaging agent in vivo or in vitro.

In some embodiments, the polypeptides described herein are used as amyloid imaging agents, wherein the imaging agents can detect amyloid and diagnose diseases associated with misfolded and/or aggregated amyloid. Since the polypeptides described herein bind amyloid, regardless of fiber type, they can image and detect any amyloid aggregates (Α β, tau, alpha-synuclein, transthyretin, immunoglobulin light chains, etc.), and can diagnose a wide array of amyloid-associated diseases and conditions. In some embodiments, the polypeptide for use as an amyloid imaging agent further comprises a detectable label.

Various labels can be attached to polypeptides comprising GAIM variants as described herein using standard techniques for labeling proteins. Examples of labels include fluorescent labels and radioisotope labels. A variety of radioisotope labels may be used, but in general, the label is typically selected from the group consisting of radioisotope labels, including but not limited to¹⁸F、¹¹C. And¹²³I. these and other radioisotopes can be attached to proteins using well-known chemical methods. In one embodiment, the label is detected using Positron Emission Tomography (PET). However, any other suitable technique for detecting a radioisotope may also be used to detect a radiotracer.

The polypeptides and compositions of the invention can be used as diagnostic imaging agents in combination with imaging agents specific for beta-amyloid, such as, for example, F18-AV-45(Eli Lilly). Since the use of the diagnostic composition of the invention together with a beta-amyloid specific imaging agent results in the detection of non-beta-amyloid aggregates based on differential detection, in one embodiment the diagnostic composition of the invention is used as an imaging agent in combination with a beta-amyloid imaging agent to detect non-beta-amyloid aggregates.

In some embodiments, the polypeptides described herein or compositions thereof are used to detect beta-amyloid in the CNS, including the brain.

The diagnostic compositions of the invention may be administered using the same routes as described for the therapeutic compositions. In some embodiments, the route of administration is intrathecal injection or infusion, direct intraventricular injection or infusion, intraparenchymal injection or infusion, or intravenous injection or infusion.

Recombination technique

In some aspects, the invention relates to oligonucleotides comprising nucleic acid sequences encoding polypeptides of the invention. For example, as disclosed herein, the invention relates to nucleic acid sequences encoding polypeptides comprising GAIM variants, including polypeptides comprising a GAIM variant attached directly or through a small linker to an immunoglobulin constant region. Typically, nucleic acids encoding polypeptides comprising GAIM variants or GAIM-Ig fusions are prepared using conventional recombinant DNA techniques, such as cloning of mutant GAIM domains, direct DNA synthesis, or by isolating the corresponding DNA from a library using, for example, the M13 sequence as a probe. See, e.g., Sambrook et al 1989, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; ausubel et al 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley lnterscience, N.Y. Nucleic acids encoding polypeptides comprising GAIM variants or GAIM-Ig fusions can also be prepared according to the methods provided in the examples below.

For recombinant production, any of the nucleic acid sequences of the invention may be inserted into a suitable expression vector containing the elements necessary for transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, for replication and translation. The encoding nucleic acid is inserted into the vector in an appropriate reading frame. Thus, the invention provides a vector comprising a nucleic acid of the invention. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. The vector may include, for example, baculovirus, cauliflower mosaic virus, tobacco mosaic virus, Ri plasmid, or Ti plasmid. One skilled in the art can select an appropriate vector in which to clone a nucleic acid of the invention, using common general knowledge of the compatibility of the vector with the host cell of choice for expression. This can be accomplished in any of a mammalian cell, a plant cell, an insect cell, a bacterial cell, a fungal cell, a transgenic animal cell, and the like. Exemplary mammalian cells suitable for producing the polypeptides described herein include, but are not limited to, HEK293 cells, HEK293 derived cells, CHO derived cells, HeLa cells, and COS cells. Exemplary bacterial cells include, but are not limited to, E.coli cells. Exemplary plant cells include, but are not limited to, duckweed cells. See, for example, U.S. patent No. 8,022,270. Suitable vectors for each of these cell types are well known in the art and are generally commercially available. Non-limiting exemplary transfection methods are described in Sambrook et al, Molecular Cloning, Arabidopsis Manual, 3 rd edition, Cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected in the desired host cell according to methods known in the art.

In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO 19. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO 20. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO 21. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 22. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 23. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 24. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 25. In at least one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 26.

In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO 19 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO:20 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO:21 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO:22 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO:23 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO:24 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO 25 and the Fc portion of a human IgG (e.g., human IgG 1). In at least one embodiment, the nucleic acid encodes a polypeptide consisting essentially of SEQ ID NO 26 and the Fc portion of a human IgG (e.g., human IgG 1).

In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:29(PB 108). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:30(PB 122). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO 31(PB 116). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:32(PB 114). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO 33(PB 109). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:34(PB 110). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO 35(PB 105). In some embodiments, the nucleic acid encodes a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:36(PB 127). As described above, these embodiments include nucleic acids encoding variants of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, wherein the variants lack amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2, and 485(Δ M1, Δ A2, and Δ K485).

In some embodiments, the nucleic acid encoding a polypeptide of the invention comprises SEQ ID NO 37. In some embodiments, the nucleic acid comprises SEQ ID NO 38. In some embodiments, the nucleic acid comprises SEQ ID NO 39. In some embodiments, the nucleic acid comprises SEQ ID NO 40. In some embodiments, the nucleic acid comprises SEQ ID NO 41. In some embodiments, the nucleic acid comprises SEQ ID NO 42. In some embodiments, the nucleic acid comprises SEQ ID NO 43. In some embodiments, the nucleic acid comprises SEQ ID NO 44. In some embodiments, the nucleic acid comprising any of SEQ ID NOs 37, 38, 39, 40, 41, 42, 43, or 44 further comprises a nucleic acid encoding an Fc portion of an IgG (e.g., human IgG1 or IgG 2). In at least one embodiment, the nucleic acid encoding the open-stabilized GAIM variant and the nucleic acid encoding the Fc portion of the IgG are linked by a nucleic acid encoding a small linker. In at least one embodiment, the nucleic acid encodes a small linker ARS. In some embodiments, the nucleic acid further encodes a signal sequence. In some embodiments, the nucleic acid further encodes a signal sequence having the 18 amino acid N-terminal sequence of GenBank Ref Seq NP _ 510891.1.

In at least one embodiment, the nucleic acid encoding a polypeptide of the invention is SEQ ID NO 45. In at least one embodiment, the nucleic acid is SEQ ID NO 46. In at least one embodiment, the nucleic acid is SEQ ID NO 47. In at least one embodiment, the nucleic acid is SEQ ID NO 48. In at least one embodiment, the nucleic acid is SEQ ID NO 49. In at least one embodiment, the nucleic acid is SEQ ID NO 50. In at least one embodiment, the nucleic acid is SEQ ID NO 51. In at least one embodiment, the nucleic acid is SEQ ID NO 52.

The vectors used for transformation will typically contain a selectable marker for the identification of transformants. In bacterial systems, this may include antibiotic resistance genes, such as ampicillin or kanamycin. Selectable markers for mammalian cells in culture include genes that confer drug resistance such as neomycin, hygromycin and methotrexate. The selectable marker may be an amplifiable selectable marker. One selectable marker that can be amplified is the DHFR gene. Another amplifiable marker is DHFRr eDNA (Simonsen and Levinson, PNAS (1983) 80: 2495). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA) and selection of selectable markers is well within the capabilities of one of ordinary skill in the art. Expression systems differ in the strength and specificity of the expression elements. Depending on the host/vector system used, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage lambda, etc.; when cloning in an insect cell system, a promoter such as a baculovirus polyhedron promoter; in cloning in plant cell systems, promoters derived from plant cell genomes (e.g., heat shock promoters; promoters of the small subunits of RUBISCO; promoters of the chlorophyll a/b binding protein) or plant viruses (e.g., 35S RNA promoter of CaMV; coat protein promoter of TMV) may be used, in cloning in mammalian cell systems, promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) may be used, when cell lines containing multiple copies of the expression product are produced, SV40, BPV and EBV-based vectors may be used with appropriate selectable markers. Viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al, Nature (1984) 310: 511-514), or the coat protein promoter of TMV (Takamatsu et al, EMBO J (1987) 6: 307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al, EMBO J. (1984) 3: 1671-. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and the like. See, e.g., Weissbach & Weissbach 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463; and Grierson & Corey 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch.7-9. In one insect expression system that can be used to produce the protein of the present invention, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing a foreign gene. The virus grows in Spodoptera frugiperda cells. Coding sequences can be cloned into nonessential regions of the virus (e.g., polyhedral genes) and placed under the control of an AcNPV promoter (e.g., polyhedral promoter). Successful insertion of the coding sequence results in inactivation of the polyhedral gene and the production of a non-occluded recombinant virus, i.e., a virus lacking the proteinaceous coat encoded by the polyhedral gene. These recombinant viruses were used to infect spodoptera frugiperda cells expressing the inserted gene. See, e.g., Smith et al, j.viral. (1983)46: 584; U.S. patent No. 4,215,051. Other examples of such expression systems are found in Ausubel et al, eds 1989, Current Protocols in Molecular Biology, Vol.2, Greene publishing. Assoc. & Wiley lnterscience.

In mammalian host cells, any of several virus-based expression systems can be used. In the case of an adenovirus used as an expression vector, the coding sequence may be linked to an adenovirus transcription/translation control complex, such as a late promoter and tripartite leader sequence. The fusion gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the peptide in the infected host (see, e.g., Logan & Shenk, PNAS (1984) 81: 3655). Alternatively, the vaccinia 7.5K promoter (Mackett et al, PNAS (1982)79: 7415; Mackett et al, J.Viral. (1984)49: 857; Panicali et al, PNAS (1982)79:4927) may be used. Other viral expression systems include adeno-associated viruses and lentiviruses.

In another embodiment, the invention provides a host cell carrying a vector comprising a nucleic acid of the invention. Methods of transfecting or transforming the vectors of the invention into host cells or otherwise obtaining host cells are known in the art. When cultured under appropriate conditions, the cells carrying the vector will produce the polypeptide of the invention. As mentioned above, suitable host cells include, but are not limited to, mammalian cells, transgenic animal cells, plant cells, insect cells, bacterial cells, and fungal cells. For example, suitable host cells include, but are not limited to, HEK293 cells, HEK293 derived cells, CHO derived cells, HeLa cells, and COS cells.

Host cells comprising the nucleic acid construct (e.g., vector) are grown in a suitable growth medium. As used herein, the term "suitable growth medium" refers to a medium having nutrients required for cell growth. Recombinantly produced polypeptides of the invention can be isolated from the culture medium using techniques known in the art.

Specific examples of vectors and cells for recombinant production of the polypeptides of the invention are set forth in the examples below.

Brief Description of Drawings

FIG. 1A is a graphical representation of the tertiary structure of GAIM N1 and N2 domains (shown using PDB structure 2G 3P). The beta strands subjected to site-directed mutagenesis are shown in dark grey. Arrows indicate the position of polar residues in N1 and hydrophobic residues in N2. FIG. 1B is a graphical representation of GAIM-Ig fusions of the present invention. FIG. 1C is a graphical representation of GAIM dimer.

FIG. 2 depicts the results of a hydrogen/deuterium (H/D) exchange study showing GAIM interaction sequences in fA β 42.

FIG. 3A compares the amino acid sequence of the N1 portion of g3p from fd phage (SEQ ID NO:1) with the corresponding portion of the amino acid sequence of the corresponding N1 portion of g3p from IF1 phage (SEQ ID NO: 2). Open boxes, depicted at the top are amino acids 23-28 (DDKTLD; SEQ ID NO:3) of PB106 and GAIM scaffold PB120 (derived from PB106), and at the bottom are EGDS (SEQ ID NO:4) substitutions present in the open-stabilized GAIM-Ig fusions of the present invention (EGDS ═ SEQ ID NO: 4). Fig. 3B is a graphical representation of the tertiary structure of GAIM N1 and N2 domains (shown using PDB structure 2G 3P). The open box in FIG. 3B shows the position of SEQ ID NO 3 in fd g3 p. Fig. 3C is a graphical representation of three slow-folding loops (also referred to as "corners") involved in N2 domain stabilization. T1 ═ turn 1 (FQNN; SEQ ID NO: 5); t2 ═ turn 2 (RQGA; SEQ ID NO: 6); t3-corner 3 (QGTDPVK; SEQ ID NO: 7). As shown below, N2 domain was stabilized by removal of one or more of T1, T2, or T3 by mutagenesis. Figure 3D depicts changes in binding and stability based on selected amino acid substitutions of T50.

Fig. 4A and 4B show representative data from thermal fusion experiments of GAIM-Ig fusions compared to GAIM dimers or monomers. By passingOrange incorporates monitoring of thermal melting. The first transition Tm1 is calculated by non-linear fitting from the normalized fluorescence intensity.

FIGS. 5A and 5B depict fluorescence emission spectra of GAIM at 0M guanidine hydrochloride (GuHCl) (open line), 2M GuHCl (solid line) and 5M GuHCl (dashed line) excited at 295nm (FIG. 5A) and 280nm (FIG. 5B). Figure 5C depicts equilibrium unfolding of GAIM dimer by GuHCl. Fig. 5C shows two transitions, the first between 1M and 2M GuHCl and the second between 2M and 4M GuHCl. The relative fluorescence intensity at 340nm (excitation 280nm) was plotted at various concentrations of GuHCl. FIG. 5D depicts domain unfolding of N2 under 1.5M GuHCl. FIG. 5E depicts domain unfolding of N1 under 2.6M GuHCl.

Fig. 6A and 6B depict the positioning of GAIM residues that modulate amyloid binding. Fig. 6A is a scatter plot showing a β 42 fiber binding potency of GAIM variants associated with Tm1 (Spearman correlation coefficient 0.703, p < 0.0001). Fig. 6B is a scatter plot showing ftau fiber binding potency of GAIM variants associated with Tm1 (spearman correlation coefficient 0.878, p < 0.0001). Variants with poor ftau binding, EC, were presented due to inaccurate curve fitting of variants that did not reach saturation in ELISA₅₀1000 nM. For fig. 6A and 6B, GAIM stent PB120 is represented by gray triangles and variants are represented by circles. A decrease in Tm1 indicates a more open GAIM conformation, resulting in increased binding, while a stabilized variant with a higher Tm1 tends to lose binding activity.

FIGS. 7A and 7B show amyloid binding of selected GAIM-Ig fusion proteins. Figure 7A compares amyloid binding of GAIM scaffolds (filled circles) and their stabilizing variants. FQGN, VNGV and QGGK are SEQ ID NO 8-10, respectively. FIG. 7B shows excellent binding of open-stabilized (but not ultra-stabilized) GAIM-Ig fusion proteins. Open stabilized polypeptides (except for the hyperstabilized polypeptide PB113 shown at the far right) are indicated in open boxes.

Fig. 8A-8D show the binding of representative open-stabilized GAIM-Ig fusions (circles) to various a β fibers, compared to the binding of control scaffolds to those a β fibers (squares). FIG. 8A shows the binding to A β 3-42-Pyro fibers; FIG. 8B shows binding to A β 1-42E22Q fibers; FIG. 8C shows binding to A β 11-42 fibers; FIG. 8D shows the binding to A β 11-42-Pyro fibers. The aggregates used in these experiments showed a very diverse morphology, ranging from unbranched long fibers (A β 1-42E22Q fibers) to highly zigzag (zig-zag) conformers (A β 3-42-Pyro fibers). Pyro ═ pyroglutamic acid.

Figure 9 shows the effect of N2 stabilizing mutations on off-target binding to collagen. FQGN ═ SEQ ID NO: 8; VNGV ═ SEQ ID NO 9; QGGK-SEQ ID NO: 10.

FIGS. 10A to 10D relate to the reconstitution efficiency of different GAIM-Ig fusion proteins incubated with A β 42 fibers. Fig. 10A further shows a comparison with the reconstruction achieved by 6E10 Mab. Circle is the average; bar-standard deviation. FIG. 10B further demonstrates the correlation between A β 42 binding and reconstitution of various GAIM-Ig fusion proteins. The open stabilized GAIM-Ig fusion is represented by a dark gray inverted triangle. The GAIM scaffold is depicted by a light gray front-up triangle. Circles represent GAIM-Ig fusions tested elsewhere. Figure 10C compares the efficiency of reconstitution of representative open-stabilized polypeptides (circles) versus GAIM scaffold (triangles) or fibers alone (squares). Bar-standard deviation. Greater solubility of the fibers in urea (e.g., lower ThT fluorescence) indicates greater reconstitution efficiency. Fig. 10D shows Transmission Electron Microscopy (TEM) images showing a β 42 fibers before incubation (left) and after 6 days of incubation with 0.8 μ M PB108 at 37 ℃ (right).

FIGS. 11A-11C relate to the reconstitution efficiency of different GAIM-Ig fusion proteins incubated with tau fibers. FIG. 11A compares the reconstitution efficiencies of a representative open-stabilized GAIM-Ig fusion protein PB108 and a hyperstabilized fusion protein PB 113. Reconstitution is indicated by the presence of tauKL monomers and dimers (middle panel) in the treated aggregates. Figure 11B compares the reconstitution efficiency of two representative open-stabilized GAIM-Ig fusion proteins and a GAIM scaffold at different concentrations of the fusion protein. Error bars represent standard deviations from three independent experiments. Figure 11C shows TEM images showing tauKL fibers before incubation (left) and after 3 days incubation with 100nM PB108 at 37 ℃ (right).

Fig. 12A-12D depict that GAIM-Ig fusion proteins of the invention inhibit amyloid assembly. Error bars represent standard deviations from three or more independent experiments. Fig. 12A shows concentration-dependent inhibition of Α β 42 fiber assembly. PB120 (control stent) is represented by a circle, PB108 by a square, and PB116 by a triangle. Figure 12B compares inhibition of Α β 42 fiber assembly under 250nM GAIM fusion. Figure 12C shows concentration-dependent inhibition of tauKL fiber assembly. PB120 (control stent) is represented by a circle, PB108 by a square, and PB116 by a triangle. FIG. 12D compares inhibition of tauKL fiber assembly under 250nM GAIM fusion.

Brief description of the sequences

SEQ ID NO:1＝AETVESCLAKPHTENSFTNVWKDDKTLDRYAN

SEQ ID NO:2＝ATTDAECLSKPAFDGTLSNVWKEGDSRYAN

SEQ ID NO:3＝DDKTLD；SEQ ID NO:4＝EGDS

SEQ ID NO:5＝FQNN；SEQ ID NO:6＝RQGA；SEQ ID NO:7＝QGTDPVK；

SEQ ID NO:8＝FQGN；SEQ ID NO:9＝VNGV；SEQ ID NO:10＝QGGK

SEQ ID NO:11

MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:12＝

SEQ ID NO:13＝

MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:14＝

SEQ ID NO:15＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:16＝

SEQ ID NO:17＝

MAETVESSLAKPHIEGSFTNVWKDDKTLDWYANYEGILWKATGVVVITGDETQVYAIWVPVGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYIYINPLDGTYPPGTEQNPANPNPSLEESHPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDVAFHSGFNEDPLVAEYQGQLSYLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:18＝

SEQ ID NO:19＝

SEQ ID NO:20＝

SEQ ID NO:21＝

SEQ ID NO:22＝

SEQ ID NO:23＝

SEQ ID NO:24＝

SEQ ID NO:25＝

SEQ ID NO:26＝

SEQ ID NO:27＝GGGGS；SEQ ID NO:28＝GGGS

SEQ ID NO:29＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:30＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:31＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:32＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:33＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:34＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:35＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:36＝

MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWAAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:37＝

SEQ ID NO:38＝

SEQ ID NO:39＝

SEQ ID NO:40＝

SEQ ID NO:41=

SEQ ID NO:42=

SEQ ID NO:43=

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCATCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGA

SEQ ID NO:44＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGGCCGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCATCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGA

SEQ ID NO:45＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:46＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGAACGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:47＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:48＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:49＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA\

SEQ ID NO:50＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:51＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACTGGGGATGAGCACCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO:52＝

ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGGCCGCCGGTGGAGTGGTCGTCTGCACTGGGGATGAGCACCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG A

SEQ ID NO:53＝HHHHHH；SEQ ID NO:54＝EDGS

Definition of

The terms used in this specification generally have their ordinary meanings in the art, within the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner describing the compositions and methods of the invention and how to make and use them. The articles "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. The term "or" means and is used interchangeably with the term "and/or" unless the context clearly dictates otherwise. In this application, the use of the singular includes the plural unless specifically stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "including," is not limiting. Any range recited herein is to be understood as including the endpoints and all values between the endpoints.

The term "g 3 p" used alone or in terms such as "g 3 p-derived" or "g 3p fusion" refers to any wild-type or recombinant filamentous bacteriophage g3p protein, including fragments, variants, and mutants of g3p that retain the ability to bind amyloid. These terms should not be construed as being limited to any particular filamentous bacteriophage g3 p.

The terms "filamentous bacteriophage" and "phage" are used interchangeably herein and include both wild-type and recombinant filamentous bacteriophage.

As used herein, the term "wild-type filamentous bacteriophage" refers to a filamentous bacteriophage found in nature, a filamentous bacteriophage that has been designated as "wild-type" in any nucleotide or amino acid sequence database, a filamentous bacteriophage that is commercially available and characterized as "wild-type", and a filamentous bacteriophage that has been passaged to obtain a non-recombinant mutation relative to any of the foregoing.

As used herein, the term "domain" means a region of a polypeptide or protein that has some unique physical characteristic or unique role, including, for example, an independently folded structure consisting of portions of a polypeptide chain. The domain may comprise a sequence of a unique physical feature of the polypeptide, or it may comprise a fragment of the physical feature that retains its binding properties (e.g., it retains the ability to bind a second domain). A domain may be associated with another domain. For example, the g3p N2 domain binds F-pili, while the g3p N1 domain binds TolA.

As used herein, "universal amyloid-interacting motif" or "GAIM" refers to a two-domain polypeptide (N1 and N2 domains of g3p) that mediates amyloid binding using a combination of both hydrophobic and polar residues lining the inner surface of the molecule. The N1 and N2 domains of GAIM monomers have an asymmetric distribution of aromatic amino acids. The GAIM N2 domain contains 11 tyrosine (Tyr) residues and 1 exposed tryptophan (Trp) residue; the N1 domain contains 3 Trp and 3 Tyr residues. The N1 and N2 domains adopt an inverted horseshoe conformation and are held together in a closed conformation (locked conformation) by a complex network of hydrogen bonds (Weininger et al, 2009). Cis-trans isomerisation of proline in the interdomain linker results in the gradual cleavage of hydrogen bonds and partial opening of both domains. The "open" rearrangement of the N1 and N2 domains of GAIM exposes β -strands 4 and 5 of N1 (containing polar residues) and 9 and 10 of N2 (containing aromatic/hydrophobic residues) and allows binding to amyloid. See fig. 1A.

As used herein, the "control scaffold" or "GAIM scaffold" corresponds to the GAIM-Ig fusion protein PB120, having the amino acid sequence of SEQ ID NO: 11. The GAIM portion of the GAIM scaffold has the amino acid sequence of SEQ ID NO 12. PB120 is derived from PB106 having the amino acid sequence of SEQ ID NO 13. The GAIM portion of PB106 has the amino acid sequence of SEQ ID NO. 14.

As used herein, "PB 106+ EDGS" (published as "EDGS" as SEQ ID NO: 54) refers to an open conformation polypeptide having the amino acid sequence provided as SEQ ID NO: 15. The GAIM portion of "PB 106+ EDGS" (published as "EDGS" as SEQ ID NO: 54) has the amino acid sequence of SEQ ID NO: 16.

As used herein, "hyperstabilized" GAIM or GAIM fusion refers to the GAIM-Ig fusion PB113 having the amino acid sequence of SEQ ID NO: 17. The GAIM portion of PB113 has the amino acid sequence of SEQ ID NO 18.

The terms "GAIM-Ig fusion," "GAIM-Ig fusion protein," and "GAIM fusion" are used interchangeably herein and refer to a polypeptide comprising the g3p GAIM domain linked directly or via a small linker to an immunoglobulin constant region. As shown in fig. 1B, the Fc region of the GAIM-Ig fusions of the present invention dimerize, resulting in a complex comprising two copies of GAIM linked to an immunoglobulin constant region, either directly or through a small linker. GAIM fusions of the invention can further comprise a signal sequence. GAIM fusions with any immunoglobulin constant region are contemplated by the invention, such as immunoglobulin constant regions of IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgD, IgA, IgE, or IgM. In some aspects, the GAIM-Ig fusion is an open-stabilized GAIM-Ig fusion. In some aspects, the GAIM-Ig fusion is partially or fully deimmunized.

As used herein, "GAIM dimer" refers to the two GAIM domains of a GAIM-Ig fusion described herein. Figure 1C graphically represents GAIM dimer.

The terms "open-stabilized fusion," "open-stabilized GAIM-Ig fusion," "open-stabilized variant," and "open-stabilized GAIM-Ig variant" are used interchangeably herein and refer to a GAIM-Ig fusion that comprises at least one open conformation mutation and at least one stabilizing mutation in the GAIM portion of the fusion. The open conformation mutation of the open stabilized variants described herein is the substitution of SEQ ID NO:3 (DDKTLD; amino acids 24-29 relative to SEQ ID NO:13 and SEQ ID NO: 15) with SEQ ID NO:4 (EGDS). The stabilizing mutation may be an N2 stabilizing mutation, for example selected from the group consisting of the substitution of SEQ ID NO:5 (FQNN; amino acids 137 and 140 relative to SEQ ID NO: 13; amino acids 135 and 138 relative to SEQ ID NO: 15) with SEQ ID NO:8(FQGN), the substitution of SEQ ID NO:6 (RQGA; amino acids 145 and 148 relative to SEQ ID NO: 13; amino acids 143 and 146 relative to SEQ ID NO: 15), the substitution of SEQ ID NO:7 (QGTDPVK; amino acids 158 and 164 relative to SEQ ID NO: 13; amino acids 156 and 162 relative to SEQ ID NO: 15) with SEQ ID NO:9(VNGV), or a combination thereof. Other N2 stabilizing mutations are described below. An open stabilized fusion as described herein may further comprise one or more substitutions, insertions or deletions. For example, the open stabilized GAIM-Ig fusion may be further modified to reduce or eliminate immunogenicity, remove potential glycosylation sites, or further modulate binding activity or specificity for amyloid.

As used herein, a GAIM-Ig fusion protein of the invention "consisting essentially of" a given amino acid sequence may further comprise a small linker linking the GAIM domain and the Fc domain of the fusion, an N-terminal signal sequence or fragment thereof, a deletion of the N-terminal methionine (Δ M1) or both the N-terminal methionine and alanine (Δ M1 and Δ a2), and/or a deletion of the C-terminal lysine (K) of the Fc domain of the fusion.

As used herein, "small linker" refers to a peptide linker of up to 25 amino acids in length that links the GAIM domain and Fc domain of a GAIM-Ig fusion. As described further below, exemplary "small linkers" that join the GAIM and Fc domains of a GAIM-Ig fusion may comprise a GS-rich sequence or may comprise the amino acid sequence ARS.

As used herein, "signal sequence" refers to a short peptide of about 16 to 30 amino acids present at the N-terminus of a polypeptide of the present invention. For example, the signal sequence may comprise the 18 amino acid N-terminal sequence of GenBank Ref Seq NP _ 518911.1. The signal sequence is used by eukaryotic cells to secrete the polypeptide of the invention. It is usually cleaved from the polypeptide prior to secretion and is therefore not normally present in the secreted polypeptide.

The term "amyloid" or "amyloid fibrils" is used herein as a generic term for tertiary structures formed by misfolding or aggregation of any of several different proteins and including an ordered arrangement of stacked β -sheets perpendicular to the fiber axis. Sunde et al, J.mol Biol. (1997)273: 729-39.

As used herein, amyloid may be formed from any one of the following proteins: an androgen receptor; apolipoprotein AI; apolipoprotein AII; apolipoprotein AIV; apo serum (aposerum) amyloid a; a beta; ABri; ADan; atrophin-1; atrial natriuretic peptides; ataxin; a calcitonin; gamma-crystalline protein; cystatin C; fibrinogen; gelsolin; huntingtin; insulin; an amylin polypeptide; an immunoglobulin kappa light chain; an immunoglobulin lambda light chain; corneal-epithelin (kerato-epithelin); a keratin protein; mucin (lactahedrin); lactoferrin; lysozyme; pulmonary surfactant protein C; medin; odontogenic ameloblast-associated protein; a prion protein; procalcitonin; prolactin; protamine I; serum amyloid a; superoxide dismutase I; beta 2-microglobulin; a TATA box binding protein; tau; transthyretin; and alpha-synuclein, or a combination thereof. As used herein, amyloid may also be formed from truncated or post-translationally modified forms of any of the above proteins. "amyloid" or "amyloid fibrils" include different or multiple conformations or morphologies of amyloid.

As used herein, "toxic oligomer" refers to a small assembly or aggregate of monomers in the open-pathway (on-pathway) that typically forms amyloid.

Exemplary amyloid proteins include amyloid- β aggregates formed in alzheimer's disease, which comprise the β -amyloid peptide "a β 0," an internal fragment of 39 to 43 amino acids that is cleaved from human amyloid precursor protein (hAPP). A β 1 includes truncated and post-translationally modified forms. For example, a β 40 is a short form of a β, while the more fibrilligenic isoform a β 42 is a long form. Other examples of A.beta.include, but are not limited to, N-truncated A.beta.11-42, A.beta.11-42-Pyro, A.beta.3-42-Pyro, and A.beta.1-42-E22Q-Dutch mutations. See Levy et al, 1990; van Broeckhoven et al, 1990. Other exemplary amyloid proteins include beta 2-synuclein (associated with Parkinson's disease), Huntington's protein (associated with Huntington's disease), tau (associated with Alzheimer's disease), abnormal conformation of prion protein, PrP^ScAnd various amyloidosisDisease-associated amyloid proteins, including but not limited to: immunoglobulin light chains (kappa or lambda), transthyretin, gelsolin, and amylin. Additional examples are provided throughout the specification and are known to those skilled in the art (see, e.g., Aguzzi (2010), and Eichner and Radford, mol. cell (2011) 43: 8-18). The use of the terms "amyloid" or "amyloid fibril" should not be construed as being limited to any particular protein, morphology, disease or condition unless a protein or peptide is specified.

The term "beta amyloid peptide" is synonymous with "beta amyloid peptide", "beta AP", "beta A", and "A beta". All these terms refer to amyloidogenic peptides derived from human amyloid precursor protein (hAPP).

As used herein, "PrP protein," "PrP," and "prion" refer to polypeptides that are capable of inducing, under appropriate conditions, the formation of aggregates that lead to protein misfolding diseases. For example, normal cellular prion protein (PrP)^c) Is converted under appropriate conditions into the corresponding scrapie isoform (PrP) that causes the disease^Sc) Such as, but not limited to, Bovine Spongiform Encephalopathy (BSE) (mad cow disease), feline spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (CJD)Scheinker's disease (GSS) and Fatal Familial Insomnia (FFI).

As used herein, "diseases associated with misfolded and/or aggregated amyloid" include, but are not limited to, alzheimer's disease; early-onset alzheimer's disease; late-onset alzheimer's disease; pre-symptomatic alzheimer's disease; AL amyloidosis; amyotrophic Lateral Sclerosis (ALS); amyotrophic lateral sclerosis/parkinsonism-dementia syndrome (parkinsonism-dementia complex); dementia with silvery particles (Argyrophilic grain dementia); aortic medial amyloidosis (aotic medial amyloidosis); ApoAI amyloidosis; ApoAII amyloidosis; ApoAIV amyloidosis; atrial amyloidosis; UK/Denmark type dementiaStaying; cataract; corticobasal degeneration (Corticobasal degeneration); corneal amyloidosis associated with trichiasis; cystatin C plaque-related disease (cystatin C plaque-related disease); cystatin C plaque-associated coronary heart disease (cystatin C plaque-related coronary disease); cystatin C plaque associated renal disease; licheniform amyloidosis (cutinous lichen amyloidosis); dementia pugilistica (Dementia pugilistica); dentatorubral-pallidoluysian atrophia rostratus (dentatorubral-pallidoluysian atrophia); diffuse neurofibrillary tangles with calcification (neurofibrillary tangles with calcification); dementia with Lewy bodies (dementias); down syndrome; familial Amyloidotic Cardiomyopathy (FAC); familial Amyloid Polyneuropathy (FAP); familial dementia British (family British dementia); dementia of the family Danish type (family Danish dementias); familial encephalopathy; familial Mediterranean fever (family Mediterranean farm); fibrinogen amyloidosis (Fibrinogen amyloidosis); hereditary amyloidosis of the Finnish type (Finnish hereditary amyloidosis); frontotemporal dementia with parkinson's disease (Frontotemporal dementia with Parkinsonism); frontotemporal lobar degeneration (FTLD); frontotemporal dementia; Hallervorden-Spatz disease; hemodialysis-related amyloidosis; hereditary cerebral amyloid angiopathy (hereditary cerebral amyloid angiopathy); hereditary cerebral hemorrhage with amyloidosis (heredity cereral hemorrhage with amyloidosis); hereditary lattice corneal dystrophy (hereditary lattice corneal dystrophy); huntington's disease; icelandic hereditary cerebral amyloid angiopathy (Icelandic cardiac amyloid angiopathy); inclusion body myositis (Inclusion-body myositis); injecting local amyloidosis (Injection-localized amyloidosis); amylin amyloidosis (islet amyloid polypeptide amyloidosis); lysozyme amyloidosis; multiple myeloma; myotonic dystrophy; Niemann-Pick disease type C (Niemann-Pick disease type C); accompany spiritNon-synaptophytic motor neuron diseases (Non-Guamanian motor neuron diseases with neurofibrilary tandles); parkinson's disease; peripheral amyloidosis (periphytol amyloidosis); pick's disease; pituitary prolactin tumors; postencephalitic parkinsonism; prion protein cerebral amyloid angiopathy; prion-mediated diseases; kuru (kuru); Creutzfeldt-Jakob disease (CJD); gerstmann--Scheinker disease (GSS); fatal Familial Insomnia (FFI); scrapie (scrapie); spongiform encephalopathy (spongiform encephalopathy); pulmonary alveolar proteinosis; progressive subcortical gliosis; progressive supranuclear palsy; senile systemic amyloidosis; serum AA amyloidosis; spinal and bulbar muscular atrophy (spinal and bulbar muscular atrophy); spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA 7); subacute sclerosing panencephalitis (Subacute sclerosing panencephalitis); systemic amyloidosis; familial amyloidosis; wild-type amyloidosis; tangle dementia (Tangle only dementia); and Tauopathies (Tauopathies). See, e.g., Chiti&Dobson, Annu Rev Biochem (2006)75: 333-66; and Josephs et al, Acta Neuropodhol (2011)122: 137-. There is an urgent need to prevent and/or reduce amyloid aggregate formation (i.e., misfolded and/or aggregated proteins) to treat or alleviate the symptoms or severity of these diseases.

As used herein, an "amyloid-reducing" polypeptide, composition, formulation, or nucleic acid accomplishes one or more of the following: inhibiting amyloid formation, causing amyloid disaggregation, causing amyloid remodeling, promoting amyloid clearance, inhibiting amyloid aggregation, blocking and/or preventing the formation of toxic oligomers, and/or promoting the clearance of toxic oligomers.

A polypeptide, nucleic acid, or composition of the invention described as "disaggregating" or "mediating disaggregation" reduces aggregates that have formed. Depolymerization can be measured by filter trap assay (Wanker et al, Methods Enzymol (1999)309:375-86) or other Methods known in the art. Filter-trap assays can be used to both detect aggregates and monitor depolymerization mediated by the compositions of the invention. In the presence of increasing concentrations of disaggregation agent, disaggregation was detected as a decrease in amyloid retention on the filter, as indicated by a decrease in staining.

The polypeptide, nucleic acid or composition of the invention described as "protecting neurons from amyloid damage" prevents the accumulation of new amyloid and/or prevents the formation of toxic oligomers. The product or composition of the invention described as "protecting neurons from amyloid damage" may be taken prophylactically. Whether a product or composition protects neurons from amyloid damage can be measured by a neuronal cell culture cytotoxicity assay as described in WO2014/055515 (herein incorporated by reference in its entirety).

The polypeptides, nucleic acids or compositions of the invention described as "remodeling" amyloid protein cause the partial or complete conversion of a fiber conformer to an amorphous aggregate. Reconstitution can be measured by denaturation studies using urea (e.g., for the a β form) or Sarkosyl solubility assays (e.g., for the tau form). Reconstitution can be detected by loss or failure of amyloid to bind thioflavin t (ThT), resulting in an increase in ThT fluorescence decrease. The reconstruction can also be detected using Transmission Electron Microscopy (TEM).

A polypeptide, nucleic acid, or composition of the invention described as "inhibiting amyloid aggregation" prevents amyloid aggregation partially or completely. Inhibition of amyloid aggregation can be measured by ThT fluorimetry (e.g., lower fluorescence indicates a lower percentage of amyloid aggregation).

As used herein in connection with a bacteriophage, protein, polypeptide, or amino acid sequence (e.g., a GAIM variant), the term "variant" refers to a corresponding substance that comprises at least one amino acid difference (at least one mutation that is a substitution, insertion, or deletion) as compared to a reference. In certain embodiments, a "variant" has high amino acid sequence homology and/or conservative amino acid substitutions, deletions, and/or insertions compared to a reference sequence. In some embodiments, a variant has no more than 25, 20, 17, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference compared to a reference sequence. Variants as described herein may retain or increase, as compared to a reference sequence: amyloid binding activity, amyloid binding specificity and/or protein mass. The variants as described herein may be deglycosylated. Variants as described herein may reduce or eliminate immunogenicity.

By "conservative substitution" is meant that a first amino acid is substituted for a second amino acid that does not substantially alter the chemical, physical and/or functional properties of g3p protein or g3p amyloid binding fragment (e.g., g3p protein or amyloid binding fragment retains the same charge, structure, polarity, hydrophobicity/hydrophilicity, and/or retains functions such as the ability to recognize, bind and/or reduce amyloid). Such conservative amino acid modifications are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary sets of amino acids that can be interchanged with conservative substitutions and which take into account the various features described above are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The term "immunogenic" or "immunogenicity" is used herein to refer to the ability of a composition to elicit an immune response in a mammal that has been exposed to the composition. In certain aspects, the invention relates to polypeptides or compositions having reduced immunogenicity or that are completely deimmunized. Complete deimmunization indicates that all 5T cell recognition epitopes present in the native GAIM amino acid sequence are removed by one or more mutations in those epitope sequences. Such de-immunized mutations may constitute substitutions, insertions or deletions of one or more amino acid residues in the epitope, or may constitute partial or complete deletion of the epitopic sequence.

Universal amyloid-interacting motif (GAIM) of G3p and GAIM-Ig fusions thereof

The universal amyloid-interacting motif (GAIM) is a two-domain polypeptide comprising the N1 and N2 domains of g3 p. The GAIM N2 domain is composed of three distinct structural elements: the structure is similar to the globular part of N1 (Holliger et al (1999) J Mol Biol,288:649-57), the alpha-helix and the unordered region that forms an extensive H-bond network with the N1 domain. The N2 hinge region also contains several proline residues, one of which (P213) is involved in maintaining GAIM in an open TolA-binding state. The N1 and N2 domains of GAIM monomers have an asymmetric distribution of aromatic amino acids. The GAIM N2 domain contains 11 tyrosine (Tyr) residues and 1 exposed tryptophan (Trp) residue; the N1 domain contains 3 Trp and 3 Tyr residues. Thus, the intrinsic fluorescence of the tyrosine and tryptophan residues allows the conformational changes of the N2 and N1 domains to be monitored, respectively (Martin and Schmid (2003) J Mol Biol, 405: 989-.

H/D exchange studies have shown that GAIM binds to the central core of A β 42 fibers. As demonstrated in figure 2, H/D exchange studies also show that GAIM engages discontinuous sequences on the fibril core and binds to sequences rich in aromatic residues (e.g., residues 17-25 in a β 42) and aliphatic residues (e.g., residues 31-40 in a β 42). This results in a strong inhibition of amyloid assembly and efficient reconstitution of fibers into amorphous aggregates (Krishnan et al (2014) J Mol Biol,426: 2500-19).

In some aspects, the polypeptide or a composition comprising the polypeptide comprises a GAIM variant. In some embodiments, the GAIM variant has no more than a 25 amino acid difference compared to the reference sequence. In some embodiments, the GAIM variant has no more than a 17 amino acid difference compared to the reference sequence. In some embodiments, the GAIM variant has no more than a 10 amino acid difference compared to the reference sequence. In some embodiments, the variant has no more than 7 amino acid differences compared to the reference sequence. In some embodiments, the reference sequence is SEQ ID NO 12 (GAIM portion of PB 120). In some embodiments, the reference sequence is SEQ ID NO 14 (GAIM portion of PB 106). In some embodiments, the reference sequence is the GAIM portion of SEQ ID NO:16 ("PB 106+ EDGS" (published as "EDGS" as SEQ ID NO: 54)).

Unless otherwise indicated, all GAIM amino acid sequence numbering is based on the amino acid sequence of SEQ ID NO. 16, and all GAIM-Ig amino acid sequence numbering is based on the amino acid sequence of SEQ ID NO. 15, which constitutes SEQ ID NO. 16 fused at the C-terminal end to the human IgG1-Fc amino acid sequence by a short linker ARS.

The polypeptides of the invention comprise any GAIM variant described herein. GAIM variants disclosed herein comprise the substitution of SEQ ID NO:3 (DDKTLD; amino acids 24-29 relative to SEQ ID NO: 13) with SEQ ID NO:4 (EGDS). Such substitutions are present in the reference sequence SEQ ID NO:16 and result in GAIM variants of the invention having an open conformation ("open" or "unlocked") GAIM variant.

The GAIM variants of the invention further comprise at least one set of additional amino acid changes selected from: (i) a surrogate T cell epitope 1-deimmunization change, (ii) a T cell epitope 2-deimmunization change, and (iii) an N2 stabilization change.

In some embodiments, at least one other set of amino acid changes increases amyloid affinity while still being deimmunized in T cell epitope 1. In reference SEQ ID NO 16, deimmunized T cell epitope 1 spans amino acids G47 to H55. H55 in this sequence led to deimmunization; wild type g3p (where T cell epitope 1 was not deimmunized) had threonine at the corresponding position. Although the amyloid affinity was still significant for the g3p polypeptide variant comprising a threonine to histidine change at amino acid 55 of SEQ ID NO:16, the affinity was reduced relative to the wild type. Interestingly, the sequence YGT found in native g3p has been reported to be a TolA binding motif (S Pommier et al, J.Bacteriol. (2005),187(21), pp.7526-34). Without being bound by theory, we believe that GAIM-amyloid binding may require a similar amino acid interaction as that of g3p-TolA binding. Therefore, we explored regenerating the 53YGT55 sequence in SEQ ID NO:16 by making a H55T substitution (e.g., reverting to the wild type T cell epitope 1 sequence) and looking for alternative substitutions in the currently regenerated T cell epitope that would affect deimmunization. We found that the T50 substitution caused deimmunization of T cell epitope 1 without affecting amyloid affinity. Thus, in some embodiments, the GAIM variants of the invention comprise a T50 substitution that is concomitant with a H55T substitution. In some aspects of these embodiments, the T50 substitution is T50R, T50K, T50G, or T50H. In at least one aspect of these embodiments, the T50 substitution is T50H.

In some embodiments, at least one other set of amino acid changes deimmunize T cell epitope 2 without significantly changing amyloid affinity. In reference SEQ ID No. 16, T cell epitope 2 spans from amino acid M134 to N142 (see, U.S. patent No. 9,988,444B2 and U.S. patent publication No. US2018/0207231a1, each incorporated by reference in its entirety), and is unchanged from the corresponding wild-type g3p sequence. Prior to the present invention, we have not been able to de-immunize this epitope without a significant reduction in amyloid binding and/or a significant reduction in the stability of the GAIM produced. We have now found that substitution of N142 and/or N137 with another amino acid deimmunizes T cell epitope 2 without significant effect on amyloid binding or stability. Thus, in some embodiments, the GAIM variants of the invention comprise a substitution of N142. In some aspects of these embodiments, the N142 substitution is N142A. In an alternative embodiment, the GAIM variants of the invention comprise a substitution of N137. In some aspects of these embodiments, the N137 substitution is N137G. In other alternative embodiments, GAIM variants of the invention comprise a substitution of N137 and a substitution of N142. In some aspects of these embodiments, the N137 substitution is N137G and the N142 substitution is N142A.

In some embodiments, at least one other set of amino acid changes increases the stability of N2. These changes target one or more so-called slow-folding loops present in SEQ ID NO:16, spanning amino acids 135-138 (FQNN; SEQ ID NO: 5; corner 1), 143-146 (RQGA; SEQ ID NO: 6; corner 2) and 156-162 (QGTDPVK; SEQ ID NO: 7; corner 3), as depicted in FIG. 3C. We have found that certain amino acid substitutions and/or deletions in one or more of these regions increase the stability of N2 in GAIM. Thus, in some embodiments, at least one other set of amino acid changes is selected from the group consisting of: (i) N137G; (ii) R143V, Q144N, and optionally a146V, a146T, or a 146K; and (iii) deletion of V161G, T158, D159 and P160, optionally Q156V or Q156Y, and optionally G157N. As provided above, substitution of N137G to stabilize N2 by removing the slow-folding loop at corner 1 also deimmunized T cell epitope 2. In some aspects of these embodiments, the GAIM variant comprises an amino acid change in only one of corner 1, corner 2, and corner 3, such as one of: (i) N137G; (ii) R143V, Q144N, and optionally a146V, a146T, or a 146K; and (iii) deletion of V161G, T158, D159 and P160, optionally Q156V or Q156Y, and optionally G157N. In some aspects of these embodiments, the GAIM variant comprises amino acid changes in at least two of the turns, for example two of: (i) N137G; (ii) R143V, Q144N, and optionally a146V, a146T, or a 146K; and (iii) deletion of V161G, T158, D159 and P160, optionally Q156V or Q156Y, and optionally G157N. In some aspects of these embodiments, the amino acid change is N137G, resulting in SEQ ID NO 8 at amino acids 135-138. In some aspects of these embodiments, the amino acid changes are R143V, Q144N, and A146V, resulting in SEQ ID NO 9 at amino acids 143- & 146. In some aspects of these embodiments, the amino acid changes are deletions of T158, D159 and P160 and substitution V161G, resulting in SEQ ID NO 10 as a substitution at amino acids 156-162. In at least one aspect of these embodiments, the GAIM variant does not comprise amino acid changes in all three turns.

In some embodiments, the polypeptides of the invention comprise GAIM variants having at least one set of amino acid changes selected from any of the above-described alternative T cell epitope 1-deimmunization changes and at least one set of amino acid changes selected from any of the above-described T cell epitope 2 deimmunization changes.

In some embodiments, the GAIM variant has at least one set of amino acid changes selected from any of the above-described alternative T cell epitope 1-deimmunization changes and at least one set of amino acid changes selected from any of the above-described N2 stabilization changes.

In some embodiments, the GAIM variant has at least one set of amino acid changes selected from any of the above T cell epitope 2-deimmunization changes and at least one set of amino acid changes selected from any of the above N2-stabilization changes.

In some embodiments, the GAIM variant has at least one set of amino acid changes selected from any of the above-described alternative T cell epitope 1-deimmunization changes, at least one set of amino acid changes selected from any of the above-described T cell epitope 2-deimmunization changes, and at least one set of amino acid changes selected from any of the above-described N2-stabilization changes.

From any of the variations described herein for that given type, a particular set of amino acid variations can be selected for each of the foregoing types of variations. Non-limiting examples of sets of amino acid variations can be found in table 1.

TABLE 1 mutations of the open-stabilized GAIM-Ig fusion relative to SEQ ID NO 16

T cell epitope 1 of SEQ ID NO:16 was deimmunized by substitution of T55H relative to wild type g3 p. Mutations in T-cell epitope 1 in the open stabilized GAIM-Ig fusion represent alternative de-immunizing mutations.

N137G substitutions deimmunized T-cell epitope 2 and stabilized the N2 domain.

16 deglycosylated by T40G mutation relative to wild-type g3 p. Mutations in the potential glycosylation signal in the open stabilized GAIM-Ig fusion represent additional deglycosylation substitutions.

The polypeptides of the invention comprise deglycosylated GAIM variants. 16 of the reference sequence, SEQ ID NO, was deglycosylated as it contained a T40G mutation relative to wild-type g3p, changing the native NAT glycosylation signal to NAG. Examples of other deglycosylated g3p mutants and/or variants can be found in U.S. patent publication US2018/0207231a 1. However, GAIM variants having different and/or additional deglycosylation mutations in the native glycosylation signal are also part of the invention. For example, the three amino acid sequence NX (T/S) (where X is any amino acid) is known glycosylation signal. Substitution of asparagine (N) in the sequence with any amino acid other than cysteine will disrupt the glycosylation signal. Similarly, substitution of threonine (T) or serine (S) in the sequence with any amino acid other than cysteine will disrupt the glycosylation signal.

Thus, in some embodiments, the GAIM variants described herein comprise the substitution of N38 with any amino acid other than cysteine, and further comprise one or more of an alternative T cell epitope 1-deimmunization change, T cell epitope 2 deimmunization change and/or N2 stabilization change, as compared to SEQ ID NO: 16. In some aspects of these embodiments, the substitution of N38 is N38A. In some aspects of these embodiments, the GAIM variant further comprises a substitution of G40 to any amino acid other than cysteine.

In some alternative embodiments, the GAIM variants described herein comprise the substitution of G40 with any amino acid other than cysteine, threonine or serine, and further comprise one or more of a T cell epitope 1 deimmunization change, a T cell epitope 2 deimmunization change and/or an N2 stabilization change, as compared to SEQ ID NO: 16.

16 contains the N-terminal amino acids M1 and A2. Recombinant production of GAIM in animal cell lines can result in polypeptides that lack M1 or both M1 and a2 ("N-terminal truncation"). Such N-terminal truncations do not affect amyloid binding activity. Thus, in some embodiments, the GAIM variant optionally lacks amino acid 1(Δ M1) or both amino acids 1 and 2(Δ M1 and Δ A2) of SEQ ID NO:16, in addition to comprising one or more of a T cell epitope 1-deimmunization change, a T cell epitope 2-deimmunization change, and/or a stabilization change of N2. As used herein, a GAIM variant lacking amino acid 1 or both amino acids 1 or 2 may refer to an N-terminal truncation (i.e., post-translational removal) or deletion mutation.

In some embodiments, the GAIM variant is a variant of SEQ ID No. 16, comprising the substitution N137G. In some aspects of these embodiments, the GAIM variant lacks amino acid 1 (e.g., Δ M1). In some aspects of these embodiments, the GAIM variant lacks amino acids 1 and 2 (e.g., Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In some embodiments, the GAIM variant is a variant of SEQ ID No. 16, comprising R143V, Q144N, and optionally a146V, a146T, or a 146K. In certain embodiments, the GAIM variant is a variant of SEQ ID No. 16, comprising R143V, Q144N, and a 146V. In some aspects of these embodiments, the GAIM variant further lacks amino acid 1 (e.g., Δ M1). In some aspects of these embodiments, the GAIM variant further lacks amino acids 1 and 2 (e.g., Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In some embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the substitution T50, the substitution H55T, and the substitution N137G with any other amino acid. In at least one aspect of these embodiments, the substitution of T50 is selected from the group consisting of T50H, T50G, T50K, and T50R. In at least one aspect of these embodiments, the substitution of T50 is T50H. In some aspects of these embodiments, the GAIM variant further (i) comprises an N142A substitution; (ii) a deglycosylation mutation comprising N38 and/or G40; (iii) lacks amino acid 1 or both amino acids 1 and 2; or (iv) any combination thereof. For example, in some aspects of these embodiments, the GAIM variant lacks amino acid 1 (e.g., Δ M1). In some aspects of these embodiments, the GAIM variant lacks amino acids 1 and 2 (e.g., Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the substitutions N137G and N142A. In some aspects of these embodiments, the GAIM variant further (i) comprises the substitution of T50 with any other amino acid and the substitution of H55T; (ii) a deglycosylation mutation comprising N38 and/or G40; (iii) lacks amino acid 1 or both amino acids 1 and 2; or (iv) any combination thereof. In certain aspects of these embodiments, the substitution of T50 is selected from T50H, T50G, T50K, and T50R. In at least one aspect of these embodiments, the substitution of T50 is T50H. In some aspects of these embodiments, the GAIM variant lacks amino acid 1 (e.g., Δ M1). In some aspects of these embodiments, the GAIM variant lacks amino acids 1 and 2 (e.g., Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the following substitutions: N142A, R143V, Q144N, and optionally a146V, a146T, or a 146K. In certain embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the following substitutions: N142A, R143V, Q144N and a 146V. In some aspects of these embodiments, the GAIM variant further (i) comprises the substitution of T50 with any other amino acid and the substitution of H55T; (ii) a deglycosylation mutation comprising N38 and/or G40; (iii) lacks amino acid 1 or both amino acids 1 and 2; or (iv) any combination thereof. In some aspects of these embodiments, the substitution of T50 is selected from T50H, T50G, T50K, and T50R. In at least one aspect of these embodiments, the substitution of T50 is T50H. In some aspects of these embodiments, the GAIM variant lacks amino acid 1(Δ M1). In some aspects of these embodiments, the GAIM variant lacks amino acids 1 and 2(Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the following substitutions: Δ T158, Δ D159, Δ P160, V161G, and optionally (i) Q156V or Q156Y and/or (ii) G157N. In certain aspects of these embodiments, the GAIM variant is a variant of SEQ ID No. 16 comprising the following substitutions: Δ T158, Δ D159, Δ P160, and V161G. In some aspects of these embodiments, the GAIM variant further (i) comprises the substitution of T50 with any other amino acid and the substitution of H55T; (ii) a deglycosylation mutation comprising N38 and/or G40; (iii) lacks amino acid 1 or both amino acids 1 and 2; or (iv) any combination thereof. In some aspects of these embodiments, the substitution of T50 is selected from T50H, T50G, T50K, and T50R. In at least one aspect of these embodiments, the substitution of T50 is T50H. In some aspects of these embodiments, the GAIM variant lacks amino acid 1(Δ M1). In some aspects of these embodiments, the GAIM variant lacks amino acids 1 and 2(Δ M1 and Δ a 2). In some aspects of these embodiments, the GAIM variant further comprises the substitution of N38 with any amino acid other than cysteine, the substitution of G40 with any amino acid other than cysteine, threonine, or serine, or the substitution of both N38 with any amino acid other than cysteine and the substitution of G40 with any amino acid other than cysteine. In at least one aspect of these embodiments, the N38 substitution is N38A.

In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO 19. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 20. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 21. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 22. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 23. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 24. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 25. In at least one embodiment, the polypeptide of the invention comprises a GAIM variant having the amino acid sequence of SEQ ID NO. 26.

Any of the above GAIM variants can be fused to an immunoglobulin constant region at the C-terminal end, either directly or through a short linker, to produce a GAIM-Ig fusion protein. The immunoglobulin constant region of the GAIM-Ig fusion proteins described herein may be that of IgG (including IgG1, IgG2, IgG3, and IgG4), IgA, IgD, IgE, or IgM. In some aspects, the immunoglobulin constant region is an IgG. In certain aspects, the IgG is IgG 1. In other aspects, the IgG is IgG 2. In some embodiments, the immunoglobulin constant region is a human immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is an Fc portion of a human IgG or a fragment thereof. Suitable Fc portions of human IgG for use in the fusion proteins of the invention include wild-type or modified Fc portions. For example, a suitable modified Fc portion of a human IgG can stabilize and/or increase the half-life of the fusion protein relative to a wild-type Fc. Non-limiting examples of modified Fc include those disclosed in U.S. patent No. 7,083,784, U.S. patent No. 7,217,797, U.S. patent No. 7,217,798, U.S. patent application No. 14/214,146, and WO-1997034631. In at least one embodiment, the immunoglobulin constant region is the Fc portion of human IgG 1. In at least one embodiment, the immunoglobulin constant region is the Fc portion of human IgG 2. In some embodiments, the immunoglobulin constant region of the GAIM-Ig fusion protein comprises a C-terminal lysine (e.g., K485). In other embodiments, the GAIM-Ig fusion lacks a C-terminal lysine (e.g., Δ K485).

In some embodiments, the GAIM-Ig fusion protein consists essentially of a polypeptide comprising any of the GAIM variants disclosed herein and an Fc portion of a human IgG (e.g., human IgGl). In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO 19 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 19 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO 19 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:29(PB 108). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:29, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:20 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 20 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:20 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:30(PB 122). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:30, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:21 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO:21 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:21 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:31(PB 116). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:31, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO. 22 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 22 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:22 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:32(PB 114). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:32, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:23 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 23 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:23 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:33(PB 109). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:33, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:24 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 24 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:24 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:34(PB 110). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:34, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:25 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 25 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:25 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:35(PB 105). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:35, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In at least one embodiment, the GAIM-Ig fusion protein consists essentially of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:26 and an Fc portion of a human IgG (e.g., human IgG 1). In some aspects of this embodiment, the amino acid sequence of the GAIM portion of the GAIM-Ig fusion protein differs from the sequence set forth in SEQ ID NO. 26 by 10-15, 1-10, or 1-5 conservative substitutions. In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of SEQ ID NO:26 and the Fc portion of a human IgG (e.g., human IgG 1). For example, in some aspects, the GAIM-Ig fusion protein consists essentially of the amino acid sequence of SEQ ID NO:36(PB 127). In other aspects of this embodiment, the GAIM-Ig fusion protein consists essentially of a variant of SEQ ID NO:36, wherein the variant lacks amino acid 1(Δ M1), amino acids 1 and 2(Δ M1 and Δ A2), amino acid 485(Δ K485), amino acids 1 and 485(Δ M1 and Δ K485), or amino acids 1, 2 and 485(Δ M1, Δ A2 and Δ K485).

In some aspects of the invention, the GAIM portion and the Ig portion of the GAIM-Ig fusions described herein are linked by a small linker. In some embodiments, the small linker is rich in glycine, serine, and/or threonine, comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% glycine, serine, and/or threonine. In some embodiments, the small linker comprises at least or about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the small linker consists essentially of, or consists of, glycine, serine, and/or threonine. The small linker of the GAIM-Ig fusion protein can be up to 25 amino acids in length, e.g., 1 to 5 amino acids in length, 1 to 20 amino acids in length, 5 to 10 amino acids in length, 5 to 25 amino acids in length, or 10 to 25 amino acids in length. The small linker may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the small linker does not comprise a human T cell epitope or produces a human T cell epitope with a GAIM variant or Fc domain bound thereto. Exemplary small linkers include linkers having various numbers of repeats of the sequence GGGGS (4 GS; SEQ ID NO:27) or GGGS (3 GS; SEQ ID NO:28), e.g., 2, 3, 4 to 5 repeats of the sequence. Exemplary linkers can comprise one or more lysine residues. Other exemplary small linkers include the amino acid sequence ARS.

The GAIM-Ig fusions described herein exhibit several advantages over the prior art. Studies aimed at identifying pathological forms of A β in the brains of Alzheimer's Disease (AD) indicate that both insoluble plaques and soluble A β consist of heterogeneous populations of N-and C-terminally truncated A β peptides (Wildburger et al, (2017) Sci Rep7:9520), forming structurally diverse conformations (Condello et al (2018) PNAS,115: E782-91; Rasmussen et al (2017) PNAS,114: 13018-23; Liu et al (2016) Sci Rep,6: 33079). It was also observed that most of the N-terminally truncated a β fragments constituted the major part of the amyloid plaques (willdberger et al, (2017) Sci Rep7: 9520). However, most antibody-related therapies directed to amyloid aggregation or misfolding have failed clinically, at least in part, because they do not effectively engage N-terminally truncated or modified forms of amyloid. The GAIM-Ig fusions of the present invention address the previously unmet need for compositions that can target a variety of amyloid proteins, as these fusions are capable of engaging a variety of Α β aggregates, even aggregates with different morphologies and aggregation properties.

The GAIM-Ig fusions disclosed herein bind, inter alia, truncated 11-42A β aggregates and/or post-translationally modified pyroglutamic acid A β aggregates, both of which are clinically associated with Alzheimer's disease. As further shown in tables 2 and 3, GAIM-Ig fusions of the present invention target multiple types of amyloid proteins, including but not limited to a β aggregates, N-terminally truncated a β aggregates, tau, various conformers of transthyretin (TTR), and different forms of immunoglobulin Light Chain (LC) aggregates. These targets include amyloid found in patients at risk for or having the diseases described herein.

Examples

Example 1: TauK18P301L expression, purification and fiber assembly

The human TauK18P301L fragment corresponding to Tau-441(2N4R) residue 244-372 having the P213L mutation was expressed and purified as described for Tau-MTBR (Krishnan et al (2014) J Mol Biol,426: 2500-19). Tau-K18P301L fibers were assembled by: to 40. mu.M TauK18P301L monomer in 0.1M sodium acetate pH 7.0 buffer containing 2mM DTT was added 40. mu.M low molecular weight heparin (Fisher Scientific) and incubated for 3 days at 37 ℃. Fiber formation was confirmed by thioflavin t (thioflavin t) (tht).

Example 2: ass fiber assembly

A β 1-42(rPeptide), N-truncated A β 11-42(Bachem), A β 11-42-pyro (AnaPec), A β 3-42-pyro (AnaPec), and A β 1-42-E22Q (AnaPec) were dissolved in Hexafluoroisopropanol (HFIP) and incubated at room temperature for 24 hours until a clear solution formed. The peptide solution was dried under vacuum for 1 hour. The fibers were assembled according to Stine et al, 2003. 100 micrograms of A β peptide was dissolved in 40 μ L DMSO, diluted to 1140 μ L in 10mM HCl solution, and incubated at 37 ℃ for 24 hours with shaking at 500 rpm. Fiber formation was confirmed by ThT.

Example 3: production of GAIM-Ig fusion proteins

Site-specific mutagenesis of control scaffold PB120 was performed in the beta strand facing the internal groove of the GAIM domain (fig. 1A). These beta strands, 4 and 5 in domain N1 and 9 and 10 in domain N2, promoted interdomain interactions in the GAIM closed state and prevented exposure to the TolA binding site (Hoffman-Thoms et al (2013) J Biol Chem, 288: 12979-91), which was previously shown to partially overlap with the amyloid binding motif in GAIM (Krishnan et al (2014) J Mol Biol,426: 2500-19). In addition, the N2-hinge region involved in the assembly of the N1-N2 domain, as well as sites in specific regions in N2 important for F-pilus binding, were mutated (Weininger et al (2009) PNAS,106: 12335-40; Deng and Perham (2002) J Mol Biol,319:603-14) to investigate how GAIM amyloid binding activity was converted into its function during phage infection.

Using Expi293^TMExpression system (Thermo Fisher Scientific) GAIM-Ig fusion protein was expressed according to the manufacturer's instructions. Purification of proteins in 20mM sodium phosphate (pH 7.0)MabSelectTM SureTM column (GE Healthcare Lifesciences) and then using AKTA within 20CV^TMPure FPLC system was gradient eluted in 20mM sodium acetate pH 4.0 to pH 3.6. Dialyzing the fusion protein into D-PBS (pH 7) and filter-sterilizing (Spin column, Millipore). Protein purity by NuPAGE^TM4-12% Bis-Tris gel System Using MES SDS running buffer (Thermo Fisher Scientific), followed by InstantBlue^TMThe staining solution (Expedeon) was analyzed. Further, UltiMate was used^TM3000UHPLC focusing System (ThermoFisher Scientific)G3000SW XL, 7.8mm IDx30cm, 5. mu.M column (TOSOH BIOSCIENCES), and dividing into two fractionsSEC was analyzed to assess the purity of GAIM IgG fusions. For each sample, 7.5. mu.g of protein was injected onto a SEC column and separated in a D-PBS mobile phase at a flow rate of 0.5 mL/min. Using Chromeleon^TM7 software analysis of peak purity. GAIM IgG fusion variants were synthesized by ATUM.

Example 4: production of GAIM dimer

Utilization at 37 ℃The (IdeS) enzyme (Genovis) produced GAIM dimer from the GAIM-Ig fusion for 2 hours, specifically sandwiching the fusion at the immunoglobulin hinge to produce GAIM dimer linked by two disulfide bonds (fig. 1C). Cleavage followed by Capto^TMAdhere was isolated from Fc according to the manufacturer's protocol. NuPAGE isolated in MES SDS running buffer^TMThe purity of GAIM dimer was confirmed on a 4-12% Bis-Tris gel system. GAIM dimer (0.5. mu.M) was incubated for 2 hours at 25 ℃ in 100mM potassium phosphate (pH 7.0) with increasing concentrations of guanidine (Sigma). Fluorescence was measured in a 10mm chamber at 310mm and 340nm after 280nm excitation and at 360nm after 295nm excitation. Data were analyzed using a two-state folding model assuming that fluorescence emission is linearly related to guanidine hydrochloride concentration. After confirmation of the expected molecular size and clearance of the Fc fragment on SDS-PAGE gels, unfolding studies were performed on the protein.

Example 5: by passingOrange binding assay monitors pyrolytic folding of GAIM

To carry outOrange binding assay to monitor GAIM domain separation and stability of N2 domain. In aqueous solutionOrange binds poorly to folded GAIM in the closed conformation. When the two domains of GAIM dissociate and expose hydrophobic residues, the dye binds to the exposed hydrophobic surface and becomes apparentShowing enhanced fluorescence. mu.M GAIM-Ig fusion in PBS with a 20-fold excess in 96-well platesOrange (Invitrogen catalog number S-6650) was mixed and sealed. In RocheIn 480RT-PCR, the unfolding was monitored by continuously increasing the temperature from 20 ℃ to 95 ℃ at a rate of 0.24 ℃/min. Excitation was set at 465nm, emission was set at 580nm, fusion factor was 1, quantitation factor was 10, and maximum integration time was 2 seconds. Arbitrary units of fluorescence signal were recorded and normalized to a scale of 0-100 (Layton and Hellinga, 2011).

By passingThe pyrolytic folding of the GAIM monomer monitored by Orange binding showed a single transition at about 43 ℃, which corresponds to the domain opening and N2 unfolding transition (fig. 4A-4B). The GAIM dimer obtained according to example 4 showed the same melting profile as the monomer in solution, whereas the GAIM-Ig fusion in solution showed three different transitions upon thermal unfolding (fig. 4A to 4B). As seen in GAIM monomers and dimers, the first transition Tm1 occurred at about 44 ℃ (fig. 4B). Two additional transitions at 64 ℃ and 81 ℃ are similar to the Fc domain unfolding transition (Traxlmayr et al, 2012, Biochim Biophys Acta, 1824: 524-529). Comparison of GAIM-specific Tm1 shows that GAIM and Fc domains remain as separate folding domains and no new structural elements are generated in the chimeric molecule.

Example 6: GAIM retains its native conformational stability in IgG fusion dimers

Conformational stability of GAIM in IgG fusions was studied by intrinsic fluorescence, using guanidine hydrochloride (GuHCl) induced unfolding of GAIM dimers. GAIM dimer was generated as described in example 4 and equilibrated at 25 ℃ for 2 hours in 0, 2 and 5M GuHCl solutions. Selective excitation of Trp residues at 295nm showed minimal change in fluorescence intensity of GAIM dimer between 0 and 2M concentration, while emission λ max (345nm) was unchanged (FIG. 5A). At 5M GuHCl, Trp fluorescence was red-shifted by 15nm (λ max360 nm) and the fluorescence intensity was significantly higher than both the 0 and 2M samples. GAIM dimers (Trp and Tyr residues) were then excited at 280nm and fluorescence emission spectra were recorded (FIG. 5B). The fluorescence emission intensity at 340nm decreased between 0 and 2M GuHCl, then increased by a similar margin (margin) at 5M GuHCl. Similar spectral changes were also observed in g3p (Martin and Schmid, 2003, J Mol Biol, 328: 863-75), indicating that GAIM in the GAIM-Ig fusion variant retained the native conformational stability of GAIM in native g3 p.

By recording the fluorescence emission intensity at 310, 340 and 360nm in a range of concentrations of GuHCl, we generated a detailed denaturation profile of GAIM dimers. GAIM dimer showed a biphasic denaturation profile at 340nm upon excitation at 280nm (FIG. 5C). The first transition occurred between 1 to 2M GuHCl, the next between 2 to 3M GuHCl. Then, we fit the denaturation profiles at 310nm (excitation 280nm) and 360nm (excitation 295nm) to the two-state protein unfolding model (FIGS. 5D to 5E) and calculate the denaturation transitions of N2 and N1 domains at 1.5M and 2.6M GuHCl, respectively. The first transition at 1.5M GuHCl represents the separation of the two domains N1 and N2 and the simultaneous unfolding of the less stable N2 domain. The second transition represents unfolding of the N1 domain that is more stable at 2.6M GuHCl. These values correspond to the previously reported denaturation transition in g3p (Martin and Schmid, 2003, J Mol Biol, 328: 863-75). Thus, these data indicate that each GAIM of the GAIM dimer in the GAIM-Ig fusion forms an independent folding unit and adopts a conformation similar to that of the g3p tip protein from filamentous phage.

Example 7: GAIM-Ig fusions bind Ass and Tau fibers

50 microliters of A β fibers (0.8 μ M) or tauK18P301L fibers (1 μ M) in 50mM carbonate buffer (pH 9.6) were added to 96 wellsPlates (Thermo Fisher) were placed in each well and incubated at 4 ℃ for 16 hours. The wells were washed 3 times with DPBS-Tween (0.05%) and 2 times with DPBS, then SuperBlock at room temperature^TM(Thermo Scientific) for 1.5 hours. Wells were washed 3 times with PBS. GAIM-Ig fusion was added at the indicated concentration to high concentration PBS-T (14.7mM KH)₂PO₄,80.6mM Na₂HPO₄-7H₂O,27mM KCl,1.38M NaCl, 0.05% Tween), and incubated at 37 ℃ for 2 hours, followed by 3 washes in DPBS-Tween (0.05%) and 3 washes with DPBS. Human specific Fc-HRP antibody (Jackson ImmunoResearch, Cat. No. 109-. After washing 2 times in DPBS-Tween (0.05%) and 2 times in DPBS, the signal was developed with TMB solution (Thermo Fisher), the reaction was stopped by addition of 0.25N HCl, and usedThe absorbance at 450nm was recorded by an M1000PRO plate reader.

Most mutations in residues N1 and N2 facing the internal groove of GAIM were found to affect Α β 1-42 fiber binding by ELISA (fig. 6A). Binding activity of mutant GAIM variants ranged from 0.7nM to 175nM EC₅₀Represents a change of more than 250-fold in binding affinity to fA β 42. Binding potency (EC)₅₀) A strong correlation (P value 10) with the first melting transition (Tm1)^-4，r_s0.703). A decrease in Tm1 indicates a more open conformation with increased bound GAIM, stabilized variants with higher Tm1 tend to lose their binding activity. This indicates that amyloid fiber binding motifs in GAIM are exposed when the interdomain interaction is attenuated, and is consistent with previous data indicating that GAIM binding is temperature dependent (Krishnan et al (2014) J Mol Biol,426: 2500-19).

To discern whether changes in binding activity to fA β 42 are translated to other amyloids, a subset of variants were tested for binding to amyloid fibrils formed from the microtubule binding region of tau by ELISA. Comparison of GAIM-Ig variants for binding Activity (EC) of fA β 42 and ftau₅₀) (P value 10)^-4，r_s0.878; FIG. 6B) shows the close correlation of the binding activity of GAIM-Ig to two different amyloid proteins (P value 10)^-4，r_s＝0.862)。

Example 8: the binding of the open-stabilized GAIM-Ig fusion protein is superior to that of the stabilized GAIM-Ig fusion protein

Several mutations in GAIM that stabilize the N2 domain and favor stronger interactions with the N1 domain result in reduced amyloid binding. By substituting Q with QGGK (SEQ ID NO:10)₁₅₆GTDPVK₁₆₂Loop (SEQ ID NO:7) to eliminate the proline-containing loop in the N2 domain increased Tm1 by 3.6 ℃ and resulted in an 18-fold loss of binding of fA β 42 (FIG. 7A). Likewise, F₁₃₅QNN₁₃₈(SEQ ID NO:5) to FQGN (SEQ ID NO:8) and R₁₄₃QGA₁₄₆Amino acid substitutions (SEQ ID NO:6) to VNGV (SEQ ID NO:9) stabilized N2(Tm1) at 1.8 ℃ and 2.5 ℃ respectively. These stable variants showed reduced binding activity of fA β 42 compared to GAIM scaffolds (fig. 7A). Similarly, a Q128H mutation was introduced, which stabilizes the interaction of the N2 hinge subdomain and N1, thereby reducing fiber binding (fig. 7A). Substitutions in N2 domain of T1, T2 or T3 all reduced non-specific binding to collagen, but Q128H showed a slight increase of 1.4 fold (fig. 9). Such mutants indicate an inverse relationship between the potency of the amyloid-binding activity of the GAIM variants and their stability.

To generate a more open conformation of GAIM, D in N1 was replaced by the homologous sequence EGDS (SEQ ID NO:4) from filamentous phage IF1₂₄DKTLD₂₉(SEQ ID NO:3) loop (FIG. 3A) and tested in combination with N2 stabilizing mutations (e.g., at one or more of the turns/loops shown in FIG. 3C). The protein quality and amyloid binding activity of the GAIM variants, putatively open and N2 stabilized, were tested. All open-stabilized variants show improved fiber binding activity with EC₅₀<1.5 nM; consistent with a more exposed and accessible amyloid fiber binding site (fig. 7B). One exception was the EGDS (SEQ ID NO:4) variant with hyperstabilized N2 (PB 113; SEQ ID NO: 17; Tm1 ═ 52.7 ℃) which comprised all three N2 stabilizing mutations in combination (F2 stabilizing mutations₁₃₅QGN₁₃₈、V₁₄₃NGV₁₄₆And Q₁₅₆GGK₁₆₂) (SEQ ID NOs: 8, 9, 10, respectively) resulting in a loss of fA β 42 binding activity (FIG. 7B). This may be due toMajor structural changes in N2 mask the amyloid interaction site in GAIM by introducing intra-domain interactions or by over-stabilizing the N2 domain. Open-stabilized variants with increased fiber binding lost the correlation between Tm1 and fA β 42 binding, indicating uncoupling of the amyloid binding site and N2 stability in these variants. All stabilized variants of EGDS-N2 ("EGDS" as disclosed in SEQ ID NO:4) showed good protein quality by SDS-PAGE and were presented as monomers by Size Exclusion Chromatography (SEC). In addition, the retention time of SEC shifts, which further indicates a more open GAIM conformer molecule.

Example 9: GAIM-Ig fusions target multiple amyloids with diverse morphologies

The ability of open stabilized GAIM-Ig fusions to engage different types and conformations of a β aggregates was tested. Various modified a β peptides were fibrillated and the binding affinity of GAIM-Ig fusions to these aggregates was measured. N-truncated A β 11-42, A β 11-42-Pyro, A β 3-42-Pyro and A β 1-42-E22Q-Dutch mutations (Levy et al, 1990; Van Broeckhoven et al,1990) were aggregated under the same conditions as A β 42, and fiber formation was confirmed by ThT (FIGS. 8A-8D) and TEM (data not shown). Aggregates formed using these peptides show a very diverse morphology. For example, pyro-glu 3-42 forms fibers with several bends in their structure, the E22Q variant forms smooth long fibers, and the 11-42 peptide forms several short fibers. Both the open-stabilized variant PB108 and the scaffold PB120 were found to bind to these fibers by ELISA. PB108 showed about a 20-fold improvement in PB120 binding compared to various aggregates, EC₅₀0.9-1.9nM (FIGS. 8A-8D). Similarly, excellent binding was observed for the other tested open-stabilized GAIM-Ig fusions (table 2).

TABLE 2 open-stabilized GAIM-Ig fusions bind and reconstitute amyloid

ND is not collected data

TABLE 3 open-stabilized GAIM-Ig fusions target multiple amyloid proteins

ND ═ data not collected. Unless otherwise indicated, binding was determined by ELISA,

tables 2 and 3 further show the ability of the open-stabilized GAIM-Ig fusions to bind different types and conformations of amyloid. For example, both table 2 and table 3 show that open-stabilized GAIM-Ig fusions bind a β 42 and tauKL with low nanomolar affinities, and table 3 further demonstrates that they bind morphologically diverse immunoglobulin Light Chain (LC) and transthyretin (TTR) aggregates with low nanomolar affinities. These data show superior targeting among a diverse array of amyloid fibers compared to the control scaffold, and are consistent with previous NMR studies showing GAIM joining the central and C-terminal sequences in a β 42 fibers (Krishnan et al (2014) J Mol Biol,426: 2500-19).

Example 10: GAIM-Ig fusions specifically bind amyloid

To exclude non-specific binding, EC was performed at high concentration (1.8. mu.M, higher than that of authentic substrate such as fA. beta.42)₅₀100-fold higher) tested off-target binding of GAIM-Ig fusions to other fiber species, such as collagen. 225 ng/well of human collagen (Sigma Cat. No. C5483) in D-PBS was incubated at 37 deg.CImmobilization in 96-well plates (Thermo Fisher Scientific) for 16 hours followed by SuperBlock^TM(Thermo Fisher Scientific) for 1 hour at room temperature. GAIM-Ig fusions in PBS-Tween (0.05%) were incubated for 1 hour at 37 ℃ followed by 35 minute washes in PBS-Tween (0.05%). Human specific Fc-HRP antibody (Jackson ImmunoResearch, Cat. No. 109. 035. 008) was added at 1:5000 in PBS-Tween (0.05%), followed by washing in PBS-Tween (0.05%) 3 times for 5 minutes and 2 times for 5 minutes in PBS, at 37 ℃ for 45 minutes. With TMB solution (S)igma), the reaction was stopped by adding 0.25N HCl, and Tecan was usedThe absorbance at 450nm was recorded by an M1000PRO plate reader. GAIM-Ig fusions showed minimal binding to non-amyloid substrates by ELISA (Krishnan et al (2014) J Mol Biol,426: 2500-19).

Example 11: open-stabilized GAIM-Ig fusions exhibit enhanced A β fiber remodeling

Reconstitution assays were performed in low retention microcentrifuge tubes (Fisher Scientific 02-681-) -320. The buffer used in these assays contained 0.05% sodium azide to prevent microbial growth. To ensure that there was no protease contamination in any of the samples, all of the reconstituted complexes were run on SDS-PAGE gels and examined for any degradation. For assays using <1 μ M fibers, protein quality was confirmed using western blot analysis instead.

A β 42 fibers (2.5 μ M) were co-incubated for 3 days at 37 ℃ with or without GAIM-Ig fusion variant. Aliquots of the complexes were then incubated with different concentrations of urea. ThT fluorescence of the complex in urea was plotted against urea concentration. The efficiency of reconstitution at fixed urea concentrations was plotted as percent ThT fluorescence loss compared to fibers without any GAIM-Ig fusion treatment.

GAIM-Ig fusions with different Α β 42 fiber binding potency were selected to determine whether reconstitution efficiency was dependent on binding potency and open conformational state. Figure 10A shows the reconstitution efficiency of different GAIM fusions incubated with a β 42 fibers under the same conditions and concentrations. Open stabilized variants binding to low nanomolar fA β 42 showed 2-fold to 3-fold increase in remodeling activity, with an average remodeling activity of 83% compared to control scaffolds (35%). Figure 10B reveals a positive correlation between altered fA β 42 binding and remodeling activity, such that GAIM-Ig fusions with excellent a β binding also show excellent remodeling activity.

FIGS. 10A-10C, as well as the transmission electron microscopy data (see example 13; FIG. 10D), further demonstrate that the open-stabilized GAIM-Ig fusion reconstitutes amyloid fibers to cause loss of fibrillar architecture, as opposed to merely adhering and masking fibrillar structures. For example, when incubated in increasing concentrations of urea but not exposed to the GAIM-Ig fusion, Α β 42 fibrils resist denaturation and show less than a 10% structural change in 1M urea as measured by ThT fluorescence (fig. 10C). At higher urea concentrations, ThT fluorescence dropped dramatically, indicating a loss of fibrous structure. In contrast, fibers treated with substoichiometric amounts of GAIM-Ig fusion initially showed a 30-90% reduction in ThT binding under 1M urea. This finding indicates that GAIM-Ig fusions bind and alter fibrillar structures to a state incapable of binding to ThT and that GAIM remodeling activity varies between different GAIM Ig fusions, with open stabilized GAIM-Ig fusions exhibiting high remodeling activity.

Example 12: the open-stabilized GAIM-Ig fusion showed enhanced fiber reconstruction of TauK18P301L

Reconstitution assays were also performed by co-incubation of Tau-K18P301L fibers with GAIM-Ig fusions to demonstrate that the reconstitution of aggregates is universal for amyloid proteins.

Unlike fA β 42 fibers, Tau-k18P301L fibers are readily soluble in low concentration urea solutions. Therefore, the reconstitution efficiency of GAIM-Ig fusion against Tau-K18P301L was investigated using the sarkosyl solubility assay. TauK18P301L fibers (1. mu.M) were incubated for 5 days at 37 ℃ with or without GAIM-Ig fusion variants. Fibers and composites were incubated with or without 1% sarkosyl for 15 minutes and then spun down at 100,000g for 30 minutes. Carefully remove the supernatant from each sample and load it to 4-12%On a gel (Invitrogen). Proteins were transferred to nitrocellulose membranes and probed with TauK18P 301L. Percent reconstitution by use of Biorad Chemicoc^TMThe system quantifies gel bands for calculation.

Fibers assembled in vitro using Tau-K18P310L (not exposed to GAIM-Ig fusions) showed lytic resistance when incubated with 1% sarkosyl. Tau-K18P301L fibers treated with the GAIM-Ig fusion were more soluble in 1% sarkosyl compared to untreated fibers (fig. 11A), indicating that these fibers were also reconstituted like fA β 42. When incubated with different concentrations of GAIM-Ig fusion, these fibers dissolved in a concentration-dependent manner (FIG. 11B).

The reconstitution efficiency of PB120 was compared to that of the open-stabilized GAIM IgG fusions. PB113, a hyperstabilized, disulfide-free GAIM with reduced infectivity (Kather et al, 2005, J Mol Biol, 354: 666-78), had no binding activity to A β 1-42 or Tau-K18P301L fibers (data not shown) and was added as a negative control. The open-stabilized GAIM-Ig fusion protein showed enhanced reconstitution activity compared to PB120 (fig. 11B), while PB113 had no reconstitution activity (fig. 11A).

Upon co-incubation of the samples, we investigated whether the GAIM fusion would reconstitute Tau-K18P301L fibers and release soluble TauK18P301L material. No soluble material was seen in the supernatant of complexes that had not been subjected to sarkosyl treatment at concentrations that resulted in effective reconstitution of GAIM fusion (e.g., 10-250nM) (fig. 11A), indicating that the reconstituted material did not release soluble TauK18P301L material or monomer upon binding to the GAIM fusion.

Example 13: open-stabilized GAIM-Ig fusions cause amyloid fibrils to lose fibrillar architecture

A β 1-42 fibers (15 μ l of a 20 μ M sample) were coated on a carbon-coated copper grid (TedPella catalog No. 01844-F). The sample was then gently washed with 0.5ml of water, inverted and floated on a drop of 2% uranyl acetate solution. After 30 seconds, the grid was removed and dried by using filter paper to suck excess liquid from the edges of the grid. Using FEI Tecnai^TMSpirit TEM images the fibers.Figure 10D shows representative TEM images of a β 42 fibers incubated with a substoichiometric open-stabilized GAIM-Ig fusion. When exposed to the open-stabilized GAIM-Ig fusion, a β 42 fibers lost their fiber architecture (fig. 10D). Similarly, TEM analysis showed that Tau-K18P301L fibers lost their characteristic fiber conformation when incubated with open-stabilized GAIM-Ig fusion (FIG. 11C).

Example 14: open-stabilized GAIM-Ig fusions exhibit increased inhibition of amyloid aggregation

The assembly inhibitory activity of the open-stabilized GAIM-Ig fusion protein was tested by co-incubation with A β 42 monomer at 37 ℃ for 10 hours. Amyloid fibril formation was followed by ThT fluorescence and compared to fibril formation in the absence of GAIM and in the presence of the negative control PB 113.

100 μ g HFIP-treated A β 1-42(rPeptide) monomer samples were dissolved in 80 μ l DMSO, mixed thoroughly by pipetting, vortexing, and diluted in 5.4mL D-PBS to a final A β 1-42 concentration of 4.04 μ M. GAIM-Ig fusion samples were diluted in PBS to intermediate stock solutions of 10, 2.5, 0.63, and 0.16. mu.M. 80 microliters of a β 1-42 monomer solution was dispensed into each well of a black round bottom 96-well plate (LVL, catalog No. 225. ls.pp). Mu.l of each GAIM-Ig fusion stock solution was added to wells containing A.beta.1-42, followed by 10. mu.l ThT (33. mu.M in PBS), at a final concentration of 3.2. mu. M A. beta.1-42 and 3.3. mu.M ThT per well. The plates were sealed with a transparent film and placed at TecanThT fluorescence at 430/485nm (Ex/Em) was recorded every 20 minutes in an M1000PRO plate reader for 14 hours during incubation at 37 ℃ with 3 seconds of vertical shaking every 20 minutes. The percentage of Α β 42 aggregation was calculated for each GAIM-Ig fusion concentration relative to untreated Α β 42 wells (positive control wells).

At the indicated concentrations, 10 hours of addition, the open stabilized GAIM-Ig fusions showed dose-dependent inhibition of assembly (fig. 12A). The open-stabilized fusions further showed increased assembly inhibitory activity compared to the control scaffold PB120 (fig. 12A-12B). For example, representative open-stabilized GAIM-Ig fusions PB108 and PB116 at 250nM showed a 40-20% increase in blocking fibril formation compared to PB120 (fig. 12B). The ability of a substoichiometric amount of an open-stabilized GAIM-Ig fusion to effectively block amyloid fibril formation suggests that the fusion blocks amyloid fibril formation by binding to the core beta-chain in growing fibers or in seeds involved in nucleation-dependent fiber assembly (Krishnan et al (2014) J Mol Biol,426: 2500-19).

Similarly, the open-stabilized GAIM-Ig fusions displayed dose-dependent assembly inhibition against tau fibers (fig. 12C). The TauK18P301L assembly reaction was set as follows: mu.M tau monomer was incubated in 0.1M sodium acetate buffer (pH 7.0) with 2. mu.M low molecular weight heparin (Fisher Scientific) for 3 days at 37 ℃ in the presence of various concentrations of GAIM fusion (0-500 nM). ThT fluorescence of the assembly reaction was recorded by diluting the sample to 1 μ M in 5 μ M ThT solution. Inhibition of TauK18P301L assembly by GAIM was calculated by comparing the assembly of TauK18P301L without GAIM fusion.

In vitro fiber assembly of full-length tau or truncated sequences such as MTBR or K18 requires the presence of heparin to facilitate nucleation and subsequent assembly. The GAIM fusions tested inhibited ftauKL assembly in the presence of heparin. Furthermore, the open-stabilized GAIM fusions blocked nucleation 3-fold to 5-fold better than PB120 (fig. 12C-12D). Together, these results indicate that open-stabilized GAIM fusions bind both a β and tau open-pathway intermediates and inhibit the assembly of amyloid aggregates.

Sequence ratio

<110> Proklala biosciences GmbH

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Polypeptide "

<400> 14

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala

20 25 30

Asn Tyr Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr

35 40 45

Gly Asp Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala

50 55 60

Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

65 70 75 80

Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr

85 90 95

Pro Ile Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro

100 105 110

Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu

115 120 125

Ser Gln Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn

130 135 140

Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr

145 150 155 160

Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala

165 170 175

Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His

180 185 190

Ser Gly Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser

195 200 205

Ser Asp Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly

210 215 220

Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

225 230 235 240

Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

245 250 255

Gly

<210> 15

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 15

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 16

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 16

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 17

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 17

Met Ala Glu Thr Val Glu Ser Ser Leu Ala Lys Pro His Ile Glu Gly

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Trp Tyr Ala

20 25 30

Asn Tyr Glu Gly Ile Leu Trp Lys Ala Thr Gly Val Val Val Ile Thr

35 40 45

Gly Asp Glu Thr Gln Val Tyr Ala Ile Trp Val Pro Val Gly Leu Ala

50 55 60

Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

65 70 75 80

Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr

85 90 95

Pro Ile Pro Gly Tyr Ile Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro

100 105 110

Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu

115 120 125

Ser His Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn

130 135 140

Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr

145 150 155 160

Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala

165 170 175

Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Val Ala Phe His

180 185 190

Ser Gly Phe Asn Glu Asp Pro Leu Val Ala Glu Tyr Gln Gly Gln Leu

195 200 205

Ser Tyr Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly

210 215 220

Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

225 230 235 240

Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

245 250 255

Gly Ala Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

260 265 270

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

275 280 285

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

290 295 300

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

305 310 315 320

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

325 330 335

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

340 345 350

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

355 360 365

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

370 375 380

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

385 390 395 400

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

405 410 415

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

420 425 430

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

435 440 445

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

450 455 460

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

465 470 475 480

Leu Ser Leu Ser Pro Gly Lys

485

<210> 18

<211> 257

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 18

Met Ala Glu Thr Val Glu Ser Ser Leu Ala Lys Pro His Ile Glu Gly

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Trp Tyr Ala

20 25 30

Asn Tyr Glu Gly Ile Leu Trp Lys Ala Thr Gly Val Val Val Ile Thr

35 40 45

Gly Asp Glu Thr Gln Val Tyr Ala Ile Trp Val Pro Val Gly Leu Ala

50 55 60

Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

65 70 75 80

Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr

85 90 95

Pro Ile Pro Gly Tyr Ile Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro

100 105 110

Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu

115 120 125

Ser His Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn

130 135 140

Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr

145 150 155 160

Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala

165 170 175

Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Val Ala Phe His

180 185 190

Ser Gly Phe Asn Glu Asp Pro Leu Val Ala Glu Tyr Gln Gly Gln Leu

195 200 205

Ser Tyr Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly

210 215 220

Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

225 230 235 240

Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

245 250 255

Gly

<210> 19

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 19

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 20

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 20

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 21

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 21

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Ala Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 22

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 22

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Ala Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 23

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 23

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Ala Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 24

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 24

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Ala Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 25

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 25

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 26

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 26

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Ala Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

245 250 255

<210> 27

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Peptides "

<400> 27

Gly Gly Gly Gly Ser

1 5

<210> 28

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Peptides "

<400> 28

Gly Gly Gly Ser

<210> 29

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 29

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 30

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 30

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 31

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 31

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Ala Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 32

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 32

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu Thr Gln Cys Tyr Gly His Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Ala Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 33

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 33

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Ala Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 34

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 34

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Ala Val Asn

130 135 140

Gly Val Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 35

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 35

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Asn Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 36

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polypeptide "

<400> 36

Met Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn

1 5 10 15

Ser Phe Thr Asn Val Trp Lys Glu Gly Asp Ser Arg Tyr Ala Asn Tyr

20 25 30

Glu Gly Cys Leu Trp Ala Ala Gly Gly Val Val Val Cys Thr Gly Asp

35 40 45

Glu His Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro

50 55 60

Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

65 70 75 80

Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile

85 90 95

Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

100 105 110

Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

115 120 125

Pro Leu Asn Thr Phe Met Phe Gln Gly Asn Arg Phe Arg Asn Arg Gln

130 135 140

Gly Ala Leu Thr Val Tyr Thr Gly Thr Phe Thr Gln Gly Thr Asp Pro

145 150 155 160

Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Arg Ala Met Tyr

165 170 175

Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

180 185 190

Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

195 200 205

Leu Pro Gln Pro Pro Ala Asn Ala Gly Gly Glu Ser Gly Gly Gly Ser

210 215 220

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

225 230 235 240

Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Ala

245 250 255

Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

260 265 270

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

385 390 395 400

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 37

<211> 765

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 37

atggccgaaa ccgtggaatc atgtctggcg aagccccata ccgagaactc cttcaccaac 60

gtctggaaag agggcgacag ccgctacgcc aactacgagg gctgcctgtg gaacgccggt 120

ggagtggtcg tctgcaccgg ggatgagact cagtgctacg gacactgggt gcctatcgga 180

ctggccattc ccgagaacga ggggggtggt agcgaaggcg gcggatcgga aggcggagga 240

tctgagggag ggggaaccaa gcctccggaa tacggcgaca ctccgatccc cgggtatacg 300

tacatcaatc cgctggacgg gacctacccg cctggaactg agcagaaccc ggccaaccca 360

aaccctagcc tcgaggaatc ccagccgttg aacaccttca tgttccaagg gaaccgcttc 420

aggaacagac agggagcgct gaccgtgtac actggcacct tcacacaagg caccgacccc 480

gtcaagacct actaccagta cactcctgtg tcctcgcggg ctatgtacga tgcgtactgg 540

aatgggaagt ttcgggactg cgctttccac tccggcttca acgaggatcc attcgtgtgc 600

gaatatcagg gccagagctc cgacctcccc caaccccctg caaacgccgg cggagaatcc 660

ggagggggat caggaggcgg aagcgaaggg ggtggatccg aaggaggcgg atccgagggt 720

ggaggctccg aagggggagg ctctggtggt ggctccggat cggga 765

<210> 38

<211> 765

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 38

atggccgaaa ccgtggaatc atgtctggcg aagccccata ccgagaactc cttcaccaac 60

gtctggaaag agggcgacag ccgctacgcc aactacgagg gctgcctgtg gaacgccggt 120

ggagtggtcg tctgcaccgg ggatgagact cagtgctacg gacactgggt gcctatcgga 180

ctggccattc ccgagaacga ggggggtggt agcgaaggcg gcggatcgga aggcggagga 240

tctgagggag ggggaaccaa gcctccggaa tacggcgaca ctccgatccc cgggtatacg 300

tacatcaatc cgctggacgg gacctacccg cctggaactg agcagaaccc ggccaaccca 360

aaccctagcc tcgaggaatc ccagccgttg aacaccttca tgttccaaaa caaccgcttc 420

aggaacgtga acggagtgct gaccgtgtac actggcacct tcacacaagg caccgacccc 480

gtcaagacct actaccagta cactcctgtg tcctcgcggg ctatgtacga tgcgtactgg 540

aatgggaagt ttcgggactg cgctttccac tccggcttca acgaggatcc attcgtgtgc 600

gaatatcagg gccagagctc cgacctcccc caaccccctg caaacgccgg cggagaatcc 660

ggagggggat caggaggcgg aagcgaaggg ggtggatccg aaggaggcgg atccgagggt 720

ggaggctccg aagggggagg ctctggtggt ggctccggat cggga 765

<210> 39

<211> 765

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 39

atggccgaaa ccgtggaatc atgtctggcg aagccccata ccgagaactc cttcaccaac 60

gtctggaaag agggcgacag ccgctacgcc aactacgagg gctgcctgtg gaacgccggt 120

ggagtggtcg tctgcaccgg ggatgagact cagtgctacg gacactgggt gcctatcgga 180

ctggccattc ccgagaacga ggggggtggt agcgaaggcg gcggatcgga aggcggagga 240

tctgagggag ggggaaccaa gcctccggaa tacggcgaca ctccgatccc cgggtatacg 300

tacatcaatc cgctggacgg gacctacccg cctggaactg agcagaaccc ggccaaccca 360

aaccctagcc tcgaggaatc ccagccgttg aacaccttca tgttccaagg gaaccgcttc 420

agggctagac agggagcgct gaccgtgtac actggcacct tcacacaagg caccgacccc 480

gtcaagacct actaccagta cactcctgtg tcctcgcggg ctatgtacga tgcgtactgg 540

aatgggaagt ttcgggactg cgctttccac tccggcttca acgaggatcc attcgtgtgc 600

gaatatcagg gccagagctc cgacctcccc caaccccctg caaacgccgg cggagaatcc 660

ggagggggat caggaggcgg aagcgaaggg ggtggatccg aaggaggcgg atccgagggt 720

ggaggctccg aagggggagg ctctggtggt ggctccggat cggga 765

<210> 40

<211> 765

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 40

atggccgaaa ccgtggaatc atgtctggcg aagccccata ccgagaactc cttcaccaac 60

gtctggaaag agggcgacag ccgctacgcc aactacgagg gctgcctgtg gaacgccggt 120

ggagtggtcg tctgcaccgg ggatgagact cagtgctacg gacactgggt gcctatcgga 180

ctggccattc ccgagaacga ggggggtggt agcgaaggcg gcggatcgga aggcggagga 240

tctgagggag ggggaaccaa gcctccggaa tacggcgaca ctccgatccc cgggtatacg 300

tacatcaatc cgctggacgg gacctacccg cctggaactg agcagaaccc ggccaaccca 360

aaccctagcc tcgaggaatc ccagccgttg aacaccttca tgttccaaaa caaccgcttc 420

agggccgtga acggagtgct gaccgtgtac actggcacct tcacacaagg caccgacccc 480

gtcaagacct actaccagta cactcctgtg tcctcgcggg ctatgtacga tgcgtactgg 540

aatgggaagt ttcgggactg cgctttccac tccggcttca acgaggatcc attcgtgtgc 600

gaatatcagg gccagagctc cgacctcccc caaccccctg caaacgccgg cggagaatcc 660

ggagggggat caggaggcgg aagcgaaggg ggtggatccg aaggaggcgg atccgagggt 720

ggaggctccg aagggggagg ctctggtggt ggctccggat cggga 765

<210> 41

<211> 765

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/record = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 41

atggccgaaa ccgtggaatc atgtctggcg aagccccata ccgagaactc cttcaccaac 60

gtctggaaag agggcgacag ccgctacgcc aactacgagg gctgcctgtg gaacgccggt 120

ggagtggtcg tctgcaccgg ggatgagcac cagtgctacg gaacttgggt gcctatcgga 180