CN117881414A - peptide-FC fusions for the treatment of amyloid disorders - Google Patents

peptide-FC fusions for the treatment of amyloid disorders Download PDF

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CN117881414A
CN117881414A CN202280045651.5A CN202280045651A CN117881414A CN 117881414 A CN117881414 A CN 117881414A CN 202280045651 A CN202280045651 A CN 202280045651A CN 117881414 A CN117881414 A CN 117881414A
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amyloid
reactive peptide
fusion protein
human
peptide
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J·S·沃尔
J·S·福斯特
J·庞斯
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ATLAS
University of Tennessee Research Foundation
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ATLAS
University of Tennessee Research Foundation
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Priority claimed from PCT/US2022/072112 external-priority patent/WO2022236286A1/en
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Abstract

Provided herein are amyloid-reactive peptide-Fc fusion proteins comprising an amyloid-reactive peptide linked to a human Fc region. Also provided herein are methods of treating amyloid-based diseases and identifying amyloid deposits by administering an amyloid-reactive peptide-Fc fusion protein comprising an amyloid-reactive peptide linked to a human Fc region.

Description

peptide-FC fusions for the treatment of amyloid disorders
Cross Reference to Related Applications
The present application claims priority from U.S. provisional application Ser. No. 63/184,682, filed 5/2021, and U.S. provisional application Ser. No. 63/186,605, filed 5/10/2021, the contents of each of which are hereby incorporated by reference in their entirety.
Submission of sequence listing on ASCII text file
The following submissions regarding ASCII text files are incorporated herein by reference in their entirety: a Computer Readable Form (CRF) of the sequence listing (file name: 165992000840seqlist. Txt, date of record: 2022, 5 months, 4 days, size: 16,694 bytes).
Technical Field
The present invention relates to amyloid reactive peptide-Fc fusion proteins, methods of treating amyloid-related conditions by administering amyloid reactive peptide-Fc fusion proteins, and methods of detecting amyloid using amyloid reactive peptide-Fc fusion proteins.
Background
Amyloidosis is a broad group of diseases belonging to the group of conformational protein diseases, which includes other diseases such as type II diabetes, alzheimer's disease, down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the netherlands, cerebral β -amyloid angiopathy, spongiform encephalopathy, thyroid tumor, parkinson's disease, dementia with lewy bodies, tauopathies, huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndrome, spongiform encephalopathy, reactive chronic inflammation, thyroid tumor, AA amyloidosis of myeloma or other forms of cancer, AL amyloidosis, AH amyloidosis, aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis and IAPP amyloidosis.
Amyloidosis is a rare disease characterized by the presence of insoluble protein deposits with abnormal fibrous conformations in tissues. In most cases, the reason is a fragment of the serum precursor protein. Many organs can be affected by these extracellular deposits called "amyloid materials". The major organs affected by amyloid deposits are kidneys, heart, digestive tract, liver, skin, peripheral nerves and eyes. Organs affected by this disease typically have a considerable volume. Eventually, amyloidosis can affect all organs and the central nervous system, and thus many very different symptoms can occur.
Thus, there is a need for effective treatments for amyloidosis and amyloid-related diseases.
Disclosure of Invention
In one aspect, provided herein is an amyloid-reactive peptide-Fc fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer.
In some embodiments, the first and/or second amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs 1-13.
In some embodiments, the first and/or second human Fc domain is a human IgG1, igG2, or IgG4 Fc.
In some embodiments, the first and/or second human Fc domain is a human IgG1Fc.
In some embodiments, the first and/or second human Fc domain comprises the amino acid sequence set forth in SEQ ID NO. 18.
In some embodiments, the first and/or second amyloid-reactive peptide is linked to the first and/or second human Fc domain via a spacer.
In some embodiments, the spacer is a peptide spacer.
In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
In some embodiments, the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid-reactive peptide from N-terminus to C-terminus.
In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to rvλ6Wil, aβ (1-40), IAAP, alκ4, alλ1, or ATTR amyloid.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is conjugated to a detectable label.
In another aspect, provided herein is a pharmaceutical composition comprising an amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] to [0017 ].
In another aspect, provided herein are one or more nucleic acids encoding the amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] to [0017 ].
In another aspect, provided herein is a vector comprising one or more nucleic acids of paragraph [0019 ].
In another aspect, provided herein is a host cell comprising the vector of paragraph [0020 ].
In some embodiments, the host cell is a mammalian cell, optionally a Chinese Hamster Ovary (CHO) cell.
In another aspect, provided herein is a method of making an amyloid-reactive peptide-Fc fusion protein comprising culturing a host cell of paragraph [0021] or paragraph [0022] under conditions suitable for expression of a vector encoding the fusion protein.
In some embodiments, the method further comprises recovering the amyloid-reactive peptide-Fc fusion protein.
In another aspect, provided herein is a method of treating an amyloid disease, comprising administering to a subject in need thereof a therapeutically effective amount of an amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] to [0017 ].
In some embodiments, the amyloid-related disease is systemic or local amyloidosis.
In some embodiments, the amyloid-related disease is selected from the group consisting of: AL, AH, aβ2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP or aβ amyloidosis.
In some embodiments, treatment with an amyloid-reactive peptide-Fc fusion protein results in clearance of the amyloid protein.
In another aspect, provided herein is a method of targeting amyloid deposits for clearance, the method comprising contacting the amyloid deposits with the amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] to [0017 ].
In some embodiments, targeting amyloid deposits for clearance results in clearance of amyloid deposits.
In some embodiments, the clearance is caused by conditioning of amyloid deposits.
In some embodiments, the individual is a human.
In another aspect, provided herein is a method of treating an individual having or suspected of having an amyloid-based disease, the method comprising: determining whether the individual has amyloid deposits by: a detectable label of the amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] - [0017], administering the labeled amyloid-reactive peptide-Fc fusion protein to the individual, determining whether a signal associated with the detectable label is detectable from the individual; and if the signal is detected, administering an amyloidosis treatment to the individual.
In some embodiments, if no signal is detected, the individual is monitored for later development of amyloid deposits.
In some embodiments, the method further comprises determining the intensity of the signal and comparing the signal to a threshold above which it is determined that the individual has amyloid deposits.
In some embodiments, the amyloidosis treatment comprises administering to the individual the amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] to [0017 ].
In another aspect, provided herein is a method of identifying amyloid deposits in an individual, the method comprising detectably labeling an amyloid-reactive peptide-Fc fusion protein of any one of paragraphs [0006] - [0017], administering the amyloid-reactive peptide-Fc fusion protein to the individual, and detecting a signal from the fusion protein.
In some embodiments, the individual is determined to be amyloid-free or to have a Monoclonal Gammaglobulinosis (MGUS), multiple Myeloma (MM), or one or more related plasma cell disorders.
Drawings
FIG. 1 shows a schematic of exemplary peptide-Fc constructs and nomenclature used herein to refer to each construct.
FIG. 2 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins produced in Chinese Hamster Ovary (CHO) cells containing 2% FBS. Lanes show from left to right molecular weight standards, igG1 antibody controls ("VH 9/VL4IgG 1"), peptide-antibody fusion proteins (where peptide p5R is fused to the N-terminus of human IgG) ("hFcNV 1"), igG1 Fc controls ("hFc 1"), and peptide-Fc fusions (where peptide p5R is fused to the C-terminus of IgG1 Fc) ("hFc 1CV 1"). These lines represent the electrophoretic mobility of the native immunoglobulin light chain (lane 2) and the control Fc domain (lane 4) for comparison with the modified peptide-fusion light chain (lane 3) and the peptide-Fc fusion (lanes 5 and 6). Asterisks on lane 6 indicate Fc-peptide variants with intact peptides associated with Fc domains.
FIG. 3 shows the results of Size Exclusion Chromatography (SEC) analysis of the Fcp5RCV1 (light grey line, lower panel) and Fcp5RNV1 (black line, upper panel) peptide-Fc fusion proteins. The x-axis shows time (minutes), the left y-axis shows absorbance of Fcp5RCV1 at 280nm, and the right y-axis shows absorbance of Fcp5RNV1 at 280 nm.
FIG. 4 shows the result of radioiodination of the Fcp5R CV1 peptide-Fc construct compared to antibody 11-1F 4. These proteins were produced in CHO cells containing 2% FBS. Fcp5RCV1 and 11-1F4 are shown as reduced ("red") and unreduced ("NR"), respectively. The positions of 11-1F 4IgG, igG heavy chain ("HC"), igG light chain ("LC") and Fcp5R CV1 are indicated.
FIG. 5 shows injection 125 I-hFc1CV1 in AA mice 1 hr (black bars), 4 hr (medium gray bars) or 24 hr (light gray bars) after injection 125 I-hFc1CV1( 125 I-I-CV 1). The x-axis represents measured tissue (including muscle, liver, pancreas, spleen, left kidney, right kidney, stomach, upper intestinal segment, lower intestinal segment, heart, lung, and blood) from left to right, and the y-axis represents biodistribution levels as a percentage of injected dose per gram of tissue (average from three mice per group + SD).
FIG. 6 shows AA mice 1, 4 or 24 hours after 125I-hFc1CV1 injection 125 Single Photon Emission Computed Tomography (SPECT) imaging of I-hFc1CV 1.
FIG. 7 shows injection 125 1 hour after I-hFc1CV1, it was shown in AA mice 125 Microscopic autoradiography (ARG; bottom row) and Congo red staining (top row) of spleen (left), heart (center) and liver (right) tissues of I-hFc1CV 1.
FIG. 8 shows injection 125 24 hours after I-hFc1CV1, was shown in AA mice 125 Microscopic autoradiography (ARG; bottom row) and Congo red staining (top row) of spleen (left), heart (center) and liver (right) tissues of I-hFc1CV 1.
FIG. 9 shows uptake of pHrodo red-labeled rVλ6Wil fibrils by human PMA-activated THP-1 macrophages either alone (control) or in the presence of human (h) Fc1, 1 μg Fc1NV1, 3 μg Fc1NV1, 10 μg Fc1NV1, 1 μg Fc1CV1, 3 μg Fc1CV1 or 10 μg Fc1CV1 for one hour, as shown from left to right on the x-axis. The y-axis shows the level of rvλ6Wil fibril uptake (measured in fluorescence units). Data represent mean + standard deviation (n=4).
FIG. 10 shows uptake of pHrodo red-labeled rVλ6Wil fibrils by human PMA-activated THP-1 macrophages either alone (control) or in the presence of 1 μg human Fc1 (hFc 1 control), 1 μg hFc1CV1, 3 μg hFc1CV1, 10 μg hFc1CV1, 30 μg hFc1 or 30 μg Fcp5R CV1 for one hour as shown from left to right on the x-axis. The y-axis shows the level of rvλ6Wil fibril uptake (measured in fluorescence units) and the error bars represent standard deviation. hFc1 and Fcp5R CV1 were produced by CHO cells. Data represent mean + standard deviation (n=4).
Fig. 11 shows the binding of hFc1CV1 to rvλ6Wil fibrils (light grey) compared to human (h) Fc1 control (dark grey). The x-axis shows the concentration (nM) of Fcp5R CV1 or hFc1, and the y-axis shows the amount of binding reagent expressed in fluorescence arbitrary units (au). EC of hFc1CV1 binding to rV lambda 6Wil fibrils 50 Is 2.5nM.
FIGS. 12A-12B show the binding of hFc1CV1 in mice with systemic amyloid A-related amyloidosis. SPECT/Ct images were obtained by detecting radiolabeled Fcp5RCV1 at 1, 4, 24 and 48 hours after injection of Fcp5RCV 1. Fig. 12A shows the distribution in various organs at various time points in AA mice. Fig. 12B shows the distribution in various organs in AA mice compared to wild-type mice at 48 hours post-injection.
FIGS. 13A-13B show I-125 labeled hFc1CV1 # 125 I Fcp5RCV 1) and amyloid co-localization in various tissues. ARG (autoradiogram) showed localization of labeled hFc1CV1 (and CR (congo red) showed localization of amyloid 1 hours (fig. 13A) and 24 hours (fig. 13B) after injection of hFc1CV 1.
FIGS. 14A-D show the results of an ex vivo phagocytosis assay with a Fcp5RCV1 or human IgG1 control. Phagocytosis was detected by labelling with the pH-sensitive dye succinimidyl-pHrodo red fluorophore.
FIG. 15 shows the results of an in vitro phagocytosis assay of rV lambda WIL, AL kappa and AL lambda fibrils with hFc1CV1 in the presence (+C) or in the absence of 20% human plasma (complement source).
Fig. 16 shows the results of a binding experiment testing the affinity of hFc1CV1 for ATTRV, ATTRwt, rV λwil, alκ, and alλ. Attrtwt is a wild type transthyretin-associated amyloidosis. ATTRv is a variant transthyretin-associated amyloidosis.
FIG. 17 shows the results of a binding experiment testing the affinity of Fcp5RCV1 for synthetic amyloid-like fibrils, tau 441, alpha-synuclein and Abeta (1-40).
Fig. 18A-18C show immunohistochemical staining (upper panel) for detection of hFc1CV1 and congo red fluorescence for detection of amyloid fibrils in human tissue sections from individuals with fibril deposits. Fig. 18A shows the binding of hFc1CV1 to ATTR and alκ fibrils in human brain tissue sections. Fig. 18B shows binding of hFc1CV1 to AL kappa and AL lambda amyloid deposits in human kidney and liver tissue sections. Fig. 18C shows the binding of hFc1CV1 to ATTR and alκ fibrils in human heart tissue sections. Arrows show the binding of the position of amyloid and hFc1CV 1.
Detailed Description
Provided herein are amyloid-reactive peptide-Fc fusion proteins capable of binding to amyloid and inducing phagocytosis.
I. Definition of the definition
As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. The abbreviation "e.g. (e.g.)" originates from latin, e.g. (exempli gratia), and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. (e.g.)" is synonymous with the term "e.g. (for example)". As used herein, the term "include" means "include".
Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value of the range and/or to the other particular value of the range. It will be further understood that each end point of the range is significant with respect to the other end point as well as independent of the other end point. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. In certain exemplary embodiments, the term "about" is understood to be within normal tolerances in the art, for example, within 2 standard deviations of the mean. About is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise apparent from the context, all numbers provided herein may be modified by the term about. Furthermore, terms used herein, such as "instance", "exemplary", or "instantiation", are not meant to indicate a preference, but rather to explain that the aspect discussed hereafter is merely one example of the presented aspect.
The terms amyloid, amyloid deposits, amyloid fibrils and amyloid fibers refer to insoluble fibrous protein aggregates having specific structural features. Protein aggregates have a tertiary structure, for example, formed by aggregation of any of several different proteins and consisting of ordered arrangement of beta sheets stacked perpendicular to the fiber axis. See Sunde et al, J.mol.biol. (1997) 273:729-39. Abnormal accumulation of amyloid in organs can lead to amyloidosis. Despite their different appearance, all amyloid proteins have common morphological properties, as they can be stained with specific dyes (such as congo red) and have a characteristic red-green birefringent appearance in polarized light after staining. Amyloid proteins also share common ultrastructural features and common x-ray diffraction and infrared spectra.
Amyloidosis refers to a pathological condition or disease characterized by the presence of amyloid proteins, such as the presence of amyloid deposits. An "amyloid disease" or "amyloidosis" is a disease associated with the formation, deposition, accumulation, or persistence of amyloid fibrils. Such diseases include, but are not limited to, alzheimer's disease, down's syndrome, dutch hereditary cerebral hemorrhage with amyloidosis and cerebral beta-amyloid angiopathy. Other amyloid diseases such as systemic AA amyloidosis, AL amyloidosis, ATTR amyloidosis, alict 2 amyloidosis, IAPP amyloidosis for type II diabetes are also amyloid diseases.
Amyloid production refers to the production or propensity to produce amyloid deposits. For example, certain soluble monomeric proteins can undergo extensive conformational changes, resulting in their aggregation into well-ordered, unbranched, 8-10-nm wide fibrils, which ultimately form amyloid aggregates. For example, more than thirty proteins have been found to form amyloid deposits (or amyloid proteins) in humans. Not all proteins in different protein classes (such as immunoglobulin light chains) are capable of forming amyloid, i.e. some proteins are non-amyloidogenic, meaning that they do not tend to form amyloid. However, other proteins of this class may form amyloid deposits and are therefore amyloidogenic. Furthermore, within the class of light chain proteins, some proteins may be considered more "amyloidogenic" than others based on their ease of formation of amyloid fibrils. Certain light chain proteins are considered non-or less amyloidogenic because they are not capable of readily forming amyloid fibrils in a patient or in vitro.
Animals: living multicellular vertebrate organisms, including a class of mammals and birds, for example. The term mammal includes both human and non-human mammals. Similarly, the terms "subject" and "individual" include both human and veterinary individuals. In some examples, the animal is an individual with amyloid disease.
And (3) clearing: the term "clear" or "clearance" refers to reducing or removing a measurable degree. For example, the removal of amyloid deposits as described herein involves reducing or removing the deposit to a measurable or discernable extent. The removal may result in 100% removal, but is not required. Conversely, the removal may result in less than 100% removal, such as about 10%, 20%, 30%, 40%, 50%, 60% or more removal.
Conjugate: as used herein, the term "conjugate" refers to a coupled or linked product of two or more materials, the resulting product having at least two different elements, such as at least two domains. The coupling materials may be the same or may be different. Such coupling may be via one or more linking groups. For example, a "protein conjugate" results from the coupling of two or more amino acid sequences. For example, a conjugate of two proteins produces a single protein having a domain corresponding to each of the separately linked proteins.
An effective amount or therapeutically effective amount of: an amount of an agent sufficient to prevent, treat (including prevent), alleviate and/or ameliorate symptoms and/or root causes of any disorder or disease, e.g., prevent, inhibit and/or amyloidosis. In some embodiments, an "effective amount" is sufficient to reduce or eliminate symptoms of the disease. The effective amount may be administered one or more times.
Inhibition: reducing the measurable extent. For example, inhibiting aspects that do not require complete loss of function or complete cessation of the measured. For example, inhibiting plaque formation may mean stopping further growth of plaque, slowing further growth of plaque, or reducing the size of plaque.
Inhibiting or treating a disease: inhibiting the overall development of the disease or condition, such as inhibiting amyloidosis. "treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it begins to develop. The term "ameliorating" in relation to a disease or pathological condition refers to any observable beneficial effect of a treatment. The beneficial effect may be demonstrated, for example, by a delayed onset of clinical symptoms of the disease in a susceptible individual, a reduced severity of some or all of the clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the individual, or other parameters characteristic of a particular disease well known in the art. "prophylactic" treatment is the administration of a treatment to an individual that does not show signs of disease or shows only early signs, with the aim of reducing the risk of developing a pathology.
With respect to amyloid deposit formation, "inhibiting" refers to preventing the formation of amyloid deposits from decreasing, such as when compared to a control. For example, inhibition may result in an approximately 10%, 20%, 30%, 40%, 50%, 60% or more reduction in amyloid deposits as compared to a control.
A label refers to any detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of the molecule. Specific non-limiting examples of labels include fluorescent labels, chemiluminescent labels, haptens, enzyme linkages, and radioisotopes. For example, a "detectably labeled" protein means that the presence of the protein can be determined by a label associated with the protein.
Separating: an "isolated" biological component, such as a peptide (e.g., one or more of the peptides disclosed herein), a cell, a nucleic acid, or a serum sample, has been substantially separated from, produced in addition to, or purified from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA and proteins) in the cells of the organism in which the component naturally resides. Thus, nucleic acids, peptides and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in cells, and chemically synthesized peptides and nucleic acids. The terms "isolated" or "purified" do not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein in its natural environment within the cell. Preferably, the formulation is purified such that the protein or peptide comprises at least 50% of the total peptide or protein content of the formulation, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 99% of the peptide or protein concentration.
And (3) connection: as used herein, the terms "ligate", "linked" or "linked" refer to any method known in the art for functionally linking proteins and/or protein domains. For example, one protein domain may be linked to another protein domain by a covalent bond, such as in a recombinant fusion protein, with or without an intervening sequence or domain. Ligation also includes, for example, integrating two sequences together, such as placing two nucleic acid sequences together in the same nucleic acid strand, such that the sequences are expressed together.
Nucleic acid: polymers composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and synthetic non-naturally occurring analogs), related naturally occurring structural variants and synthetic non-naturally occurring analogs thereof linked by phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and linkages between them include synthetic analogues that do not occur naturally, such as, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide Nucleic Acids (PNAs), and the like. Such polynucleotides may be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, typically no more than about 50 nucleotides. It will be appreciated that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).
Nucleotides include, but are not limited to, monomers comprising bases linked to a sugar, such as a pyrimidine, a purine, or a synthetic analogue thereof, or bases linked to an amino acid, as in Peptide Nucleic Acid (PNA). A nucleotide is a monomer in a polynucleotide. Nucleotide sequence refers to the sequence of bases in a polynucleotide.
The nucleotide sequence is described herein using conventional symbols: the left end of the single-stranded nucleotide sequence is the 5' -end; the left-hand direction of the double-stranded nucleotide sequence is referred to as the 5' -direction. The direction of addition of nucleotides 5 'to 3' to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as mRNA is called "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from the DNA and located 5 'to 5' of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as RNA and encoding the 3 'to 3' end of the RNA transcript is referred to as the "downstream sequence".
cDNA refers to DNA that is complementary or identical to mRNA in single-or double-stranded form.
Coding refers to the inherent nature of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) and the biological nature resulting therefrom, as a template for synthesizing other polymers and macromolecules having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences in a biological process. Thus, a gene encodes a protein if transcription and translation of mRNA produced by the gene produces the protein in a cell or other biological system. The coding strand whose nucleotide sequence is identical to the mRNA sequence and is generally provided in the sequence listing, as well as the non-coding strand used as a transcription template for a gene or cDNA, may be referred to as a protein or other product encoding the gene or cDNA. Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may comprise introns.
A pharmaceutically acceptable carrier: the pharmaceutically acceptable carriers used are conventional. Compositions and formulations suitable for drug delivery of the fusion proteins disclosed herein are described by Remington's Pharmaceutical Sciences, mack Publishing co., easton, PA, 19 th edition (1995), by e.w. martin.
In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise an injectable fluid comprising a pharmaceutically and physiologically acceptable fluid, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, and the like as a vehicle. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the bio-neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Polypeptide: wherein the monomers are polymers of amino acid residues linked together by amide linkages. When the amino acid is an alpha-amino acid, an L-isomer or a D-isomer may be used, with L-isomer being preferred. As used herein, the term "polypeptide" or "protein" is intended to encompass any amino acid sequence and includes modified sequences, such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins, as well as those proteins produced recombinantly or synthetically. In some examples, the peptide is one or more of the peptides disclosed herein.
And (3) purifying: the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein in question is purer than the protein in its natural environment, either in the cell or in the production reaction chamber (where appropriate).
Sequence identity: similarity between two nucleic acid sequences or two amino acid sequences is expressed as similarity between the sequences, also known as sequence identity. Sequence identity is typically measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences.
Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: smith and Waterman adv. Appl. Math.2:482,1981; needleman and Wunsch j.mol.biol.48:443,1970; pearson and Lipman Proc.Natl. Acad.Sci.USA 85:2444,1988; higgins and Sharp Gene 73:237-244,1988; higgins and Sharp CABIOS 5:151-153,1989; corpet et al Nuc.acids Res.16,10881-90,1988; huang et al Computer appls. In the Biosciences 8,155-65,1992; pearson et al Meth.mol.Bio.24,307-31,1994.Altschul et al (J.mol. Biol.215:403-410, 1990) propose detailed considerations for sequence alignment methods and homology calculations.
NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al J.mol. Biol.215:403-410, 1990) is available from several sources, including the national center for biological information (NCBI, bethesda, md.) and on the Internet for use in connection with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
Operatively connected to: when a first nucleic acid sequence is in functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, join two protein coding regions in the same reading frame.
Medicament: a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to an individual or cell.
And (3) a carrier: a nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. The recombinant DNA vector is a vector having recombinant DNA. The vector may comprise a nucleic acid sequence, such as an origin of replication, that allows it to replicate in the host cell. The vector may also comprise one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant DNA vector having at least some nucleic acid sequences derived from one or more viruses. The term vector includes plasmids, linear nucleic acid molecules, and as described throughout adenovirus vectors and adenoviruses.
A subject or individual refers to a mammal, such as a human. The individual may be a human patient. An individual may be a patient suffering from or suspected of suffering from a disease or disorder and may need treatment or diagnosis or may need to monitor the progression of the disease or disorder. The patient may also be receiving a therapeutic regimen that requires monitoring efficacy. In some exemplary embodiments, the individual includes an individual having amyloidosis, such as alzheimer's disease, huntington's disease, or prion disease, or peripheral amyloidosis, as seen in patients with light chain (AL) amyloidosis and type 2 diabetes.
The term treatment or treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after the disease or pathological condition has begun to develop. The term "ameliorating" in relation to a disease or pathological condition refers to any observable beneficial effect of a treatment. The beneficial effect may be demonstrated, for example, by a delayed onset of clinical symptoms of the disease in the susceptible individual, a decrease in severity of some or all of the clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the individual, or by other parameters known in the art to be specific for a particular disease. "prophylactic" treatment is a treatment that can be administered to an individual that does not exhibit a symptom of the disease and/or that exhibits only early symptoms for the purpose of reducing the risk of developing a pathology.
Amyloid reactive peptide-Fc fusion proteins
Provided herein are amyloid reactive peptide-Fc fusion proteins. In some embodiments, the amyloid-reactive peptide-Fc fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide linked to a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is a homodimer. The amyloid-reactive peptide-Fc fusion proteins can be used to treat a subject suffering from amyloidosis, e.g., as by administering the amyloid-reactive peptide-Fc fusion proteins of the present disclosure to an individual.
In some embodiments, the first and/or second amyloid-reactive peptide comprises an amino acid sequence as set forth in table 1 below. In some embodiments, one or more of the peptides shown in table 1 below may be linked to the human Fc region through the N-terminus of the human Fc region or the C-terminus of the human Fc region, thereby forming an amyloid-reactive peptide-Fc fusion protein. In some embodiments, the first and/or second amyloid-reactive peptide comprises two or more of the peptides shown in table 1, which may be linked to a single human Fc region. For example, two of the amyloid-reactive peptides may be linked to a single human Fc region.
TABLE 1 exemplary amyloid reactive peptide sequences
Without wishing to be bound by any particular theory, it is believed that the peptide domain of the amyloid reactive peptide-Fc fusion protein targets the amyloid reactive peptide-Fc fusion protein to amyloid deposits when administered to an individual. The Fc domain then triggers an immune response at the site of the amyloid protein, resulting in removal of the amyloid protein, such as by opsonization. Furthermore, it is believed that the amyloid reactive peptide-Fc fusion protein has a longer half-life than the amyloid reactive peptide alone. In certain exemplary embodiments, contacting an amyloid deposit with a fusion protein of the present disclosure results in an increase in half-life of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to contacting the amyloid deposit with an amyloid-reactive peptide alone. Thus, when administered to an individual, the amyloid-reactive peptide-Fc fusion protein may exert its immunostimulatory effect at the amyloid deposition site for a longer period of time, thereby increasing the immune response at the amyloid deposition site.
In some embodiments, an amyloid-reactive peptide of an amyloid-reactive peptide-Fc fusion protein described herein comprises an amino acid sequence that is at least 80%, 85%, 90% or more identical to an amino acid sequence set forth as any one of SEQ ID NOs 1-13, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence set forth as any one of SEQ ID NOs 1-13. In some embodiments, the amyloid-reactive peptide linked to the human Fc region may comprise or consist of about 10 to about 55 amino acids. The amyloid-reactive peptide of the present invention may, for example, comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids. Such peptides are described, for example, in international patent application WO2016032949, which is hereby incorporated in its entirety. In some embodiments, the first and/or second amyloid-reactive peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs 1-13. In some embodiments, the first and second amyloid-reactive peptides comprise an amino acid sequence having at least 80%, 85%, 90%, 95% or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs 1-13. In some embodiments, the first and/or second amyloid-reactive peptide comprises an amino acid sequence as set forth in SEQ ID NO 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the first and second amyloid-reactive peptides comprise the amino acid sequences set forth in SEQ ID NO. 1. In some embodiments, the first and second amyloid-reactive peptides comprise the amino acid sequences set forth in SEQ ID NO. 2. In some embodiments, the first and second amyloid-reactive peptides comprise the amino acid sequences set forth in SEQ ID NO. 12. In some embodiments, the first and second amyloid-reactive peptides comprise the amino acid sequence set forth in SEQ ID NO. 13.
The amino acids forming all or part of the amyloid reactive peptide linked to the human Fc region may be naturally occurring amino acids, non-naturally occurring amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, stereoisomers and modifications designed to mimic the structure or structure of an amino acid, and the like. The amino acids forming the peptides of the invention may be one or more of the 20 common amino acids found in naturally occurring proteins, or one or more of the modified and unusual amino acids.
In some embodiments, the first amyloid-reactive peptide is linked to the N-terminus of the first human Fc domain and the second amyloid-reactive peptide is linked to the N-terminus of the second human Fc domain. In some embodiments, the first amyloid-reactive peptide is linked to the C-terminus of the first human Fc domain and the second amyloid-reactive peptide is linked to the C-terminus of the second human Fc domain. An exemplary structure of an amyloid reactive peptide-Fc fusion protein is provided in fig. 1.
In some embodiments, the first and/or second human Fc domain is a human IgG1, igG2, or IgG4 Fc. In some embodiments, the first and/or second human Fc domain is a human IgG1Fc. In some embodiments, the first and second human Fc domains are human IgG1Fc. In some embodiments, the first and/or second human Fc domain comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the first and second human Fc domains comprise the amino acid sequences set forth in SEQ ID NO. 18. The amino acid sequence of SEQ ID NO. 18 is provided below.
Human IgG1 Fc (SEQ ID NO: 18)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
In some embodiments, the first and/or second human Fc domain is an Fc variant with enhanced effector function. In some embodiments, the first and/or second human F c domain is an Fc variant with enhanced ability to promote phagocytosis. In some embodiments, the first and/or second human Fc domain is an Fc variant having enhanced binding to fcγr. In some embodiments, the first and/or second human Fc domain is an Fc variant having an enhanced ability to recruit complement. In some embodiments, the first and/or second human Fc domain comprises one or more amino acid substitutions that confer enhanced effector function and/or enhanced binding to fcγr. Such amino acid substitutions have been described, for example, in international publication nos. WO2004/099249, WO2005/063815, WO2006/019447, WO2006/020114, WO 2007/04635, WO2009/058492, WO2009/086320, and US publication nos. US20070224192and US20080161541, each of which is incorporated herein by reference. In some embodiments, a human Fc domain with one or more amino acid substitutions has an enhanced ability to promote phagocytosis. In some embodiments, the first and/or second human Fc domain is glycoengineered. In some embodiments, the glycoengineered human Fc domain is one that has enhanced ability to promote phagocytosis.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein comprises an amino acid spacer sequence between a human Fc region and an amyloid-reactive peptide. In some embodiments, the first and/or second amyloid-reactive peptide is linked to the first and/or second human Fc domain via a spacer. In some embodiments, the spacer is a peptide spacer. In some embodiments, the first and/or second amyloid-reactive peptide is fused to the first and/or second human Fc domain via a peptide spacer. In some embodiments, the spacer is a flexible spacer peptide. In some embodiments, the spacer comprises glycine and serine residues. In some embodiments, the spacer comprises a GGGGS motif. In some embodiments, the spacer consists of glycine and serine residues. In some embodiments, the spacer is a rigid spacer peptide. In some embodiments, the spacer is uncharged. In some embodiments, the spacer is a glycine serine linker. In some embodiments, the spacer comprises a glycine serine linker. In some embodiments, the spacer comprises or consists of about 3 to about 55 amino acids. The spacer peptide of the invention may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acids. In some embodiments, the spacer peptide is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, or 155 amino acids in length, including any value or range between these values. In some embodiments, the spacer peptide comprises 15 amino acids. In some embodiments, the spacer peptide comprises an amino acid sequence as set forth in table 2 below. In some embodiments, the spacer comprises the amino acid sequences set forth in SEQ ID NOS.14-17.
TABLE 2 exemplary spacer sequences
Description of the invention Amino acid sequence SEQ ID NO
Rigid short spacer VSPSV SEQ ID NO:14
Rigid long spacer VSPSVVSPSV SEQ ID NO:15
Flexible short spacer GGGSGG SEQ ID NO:16
Flexible long spacer GGGGSGGGGS SEQ ID NO:17
In some embodiments, the first polypeptide comprises a first amyloid-reactive peptide, a first spacer region, and a first human Fc domain from N-terminus to C-terminus, and the second polypeptide comprises a second amyloid-reactive peptide, a second spacer region, and a second human Fc domain from N-terminus to C-terminus. In some embodiments, the first and second polypeptides have the same sequence. In some embodiments, the first and second polypeptides have different sequences. In some embodiments, the first polypeptide comprises the first amyloid-reactive peptide, first spacer region, and first human Fc domain set forth in SEQ ID No. 2 from N-terminus to C-terminus, and the second polypeptide comprises the second amyloid-reactive peptide, second spacer region, and second human Fc domain set forth in SEQ ID No. 2 from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises a first amyloid-reactive peptide, a first spacer region, and a first human IgG1 Fc domain from N-terminus to C-terminus, and the second polypeptide comprises a second amyloid-reactive peptide, a second spacer region, and a second human IgG1 Fc domain from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises a first amyloid-reactive peptide, a first rigid short spacer, and a first human Fc domain from N-terminus to C-terminus, and the second polypeptide comprises a second amyloid-reactive peptide, a second rigid short spacer, and a second human Fc domain from N-terminus to C-terminus. In some embodiments, the rigid short spacer comprises the sequence set forth in SEQ ID NO. 14. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus the first amyloid-reactive peptide, the first rigid short spacer, and the first human IgG1 Fc domain set forth in SEQ ID NO. 2, and the second polypeptide comprises from N-terminus to C-terminus the second amyloid-reactive peptide, the second rigid short spacer, and the second human IgG1 Fc domain set forth in SEQ ID NO. 2. In some embodiments, the first polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 19, and the second polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 19. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 19 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 19. In some embodiments, the amyloid-reactive peptide-Fc fusion protein comprises the structure and/or amino acid sequence of hFc1NV 1.
In some embodiments, the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid-reactive peptide from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid-reactive peptide set forth in SEQ ID NO. 2 from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid-reactive peptide set forth in SEQ ID NO. 2 from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises a first human IgG1 Fc domain, a first spacer region, and a first amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human IgG1 Fc domain, a second spacer region, and a second amyloid-reactive peptide from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises a first human Fc domain, a first rigid short spacer, and a first amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second rigid short spacer, and a second amyloid-reactive peptide from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human IgG1 Fc domain, a first rigid short spacer, and a first amyloid-reactive peptide as set forth in SEQ ID NO. 2, and the second polypeptide comprises from N-terminus to C-terminus a second human IgG1 Fc domain, a second rigid short spacer, and a second amyloid-reactive peptide as set forth in SEQ ID NO. 2. In some embodiments, the rigid short spacer comprises the amino acid sequence set forth in SEQ ID NO. 14. In some embodiments, the first polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 20, and the second polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 20. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20. In some embodiments, the amyloid-reactive peptide-Fc fusion protein comprises the structure and/or amino acid sequence of hFc1CV 1.
In some embodiments, the first polypeptide comprises a first human Fc domain, a first flexible long spacer, and a first amyloid-reactive peptide from the N-terminus to the C-terminus, and the second polypeptide comprises a second human Fc domain, a second flexible long spacer, and a second amyloid-reactive peptide from the N-terminus to the C-terminus. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human Fc domain, a first flexible long spacer, and a first amyloid-reactive peptide set forth in SEQ ID No. 2, and the second polypeptide comprises from N-terminus to C-terminus a second human Fc domain, a second flexible long spacer, and a second amyloid-reactive peptide set forth in SEQ ID No. 2. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human IgG1 Fc domain, a first flexible long spacer, and a first amyloid-reactive peptide set forth in SEQ ID NO. 2, and the second polypeptide comprises from N-terminus to C-terminus a second human IgG1 Fc domain, a second flexible long spacer, and a second amyloid-reactive peptide set forth in SEQ ID NO. 2. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human IgG1 Fc domain, a first spacer region, and a first amyloid-reactive peptide set forth in SEQ ID NO. 13, and the second polypeptide comprises from N-terminus to C-terminus a second human IgG1 Fc domain, a second spacer region, and a second amyloid-reactive peptide set forth in SEQ ID NO. 13. In some embodiments, the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid-reactive peptide set forth in SEQ ID NO. 13 from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid-reactive peptide set forth in SEQ ID NO. 13 from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human Fc domain, a first flexible long spacer and a first amyloid-reactive peptide set forth in SEQ ID NO. 13, and the second polypeptide comprises from N-terminus to C-terminus a second human Fc domain, a second flexible long spacer and a second amyloid-reactive peptide set forth in SEQ ID NO. 13. In some embodiments, the first polypeptide comprises from N-terminus to C-terminus a first human IgG1 Fc domain, a first flexible long spacer, and a first amyloid-reactive peptide set forth in SEQ ID NO. 13, and the second polypeptide comprises from N-terminus to C-terminus a second human IgG1 Fc domain, a second flexible long spacer, and a second amyloid-reactive peptide set forth in SEQ ID NO. 13. In some embodiments, the spacer comprises the amino acid sequence of SEQ ID NO. 17.
In some embodiments, the first polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 21, and the second polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 21. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 21 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 21.
In some embodiments, the first polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 22, and the second polypeptide comprises an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO. 22. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 22 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 22.
In some embodiments, provided herein are fusion proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide and a second amyloid-reactive peptide linked to a first human Fc domain, wherein the second polypeptide comprises a third amyloid-reactive peptide and a fourth amyloid-reactive peptide linked to a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the first and second human Fc domains form dimers through covalent bonding in the antibody hinge region. In some embodiments, the first and second human Fc domains are linked by a disulfide bond. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is a homodimer. In some embodiments, the first polypeptide comprises a first amyloid-reactive peptide, a first spacer, a first human Fc domain, a second spacer, and a second amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a third amyloid-reactive peptide, a third spacer, a second human Fc domain, a fourth spacer, and a fourth amyloid-reactive peptide from N-terminus to C-terminus. In some embodiments, the first, second, third, and fourth amyloid-reactive peptides are any of the amyloid-reactive peptides described herein, e.g., any of the amyloid-reactive peptides described in table 1. In some embodiments, the first, second, third, and fourth are any of the spacers described herein.
In some embodiments, the amyloid-reactive peptide-Fc fusion proteins described herein bind to amyloid deposits or fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to one or more amyloid-producing peptides in an amyloid protein. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to heparin sulfate glycosaminoglycans in the amyloid protein. In some embodiments, the fusion protein binds to human fibrils. In some embodiments, the fusion protein binds to a synthetic fibril. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to rvλ6Wil fibrils, per125 wtATTR extract, KEN hATTR extract, SHI AL λ liver extract, and/or TAL AL κ liver extract. In some embodiments, the amyloid-reactive peptideThe amyloid to which the Fc fusion protein binds comprises an amyloidogenic λ6 variable domain protein (vλ6 Wil) or an amyloidogenic immunoglobulin light chain (AL), an aβ (1-40) amyloid fibril or an amyloid-producing aβ precursor protein or serum Amyloid A (AA). In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to rvλ6Wil, aβ (1-40), IAAP, alκ, alλ, or ATTR amyloid. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to an alκ4, alλ amyloid protein. In other embodiments, the amyloid to which the humanized antibody or antibody-peptide fusion protein binds comprises the following amyloidogenic forms: immunoglobulin heavy chain (AH), beta 2 Microglobulin (aβ) 2 M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect 2), fibrinogen a variant (AFib), cystatin variant (acls), calcitonin ((ACal), lactadherin (AMed), amylin polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), alpha-synuclein (aaasyn), tau (ATau), atrial natriuretic factor (AANF) or IAAP, alκ4, alλ1 other amyloid-producing peptides. The amyloid-producing peptide to which the humanized antibody or antibody-peptide fusion protein binds may be a protein, protein fragment or protein domain. In some embodiments, the amyloid deposit or amyloid fibril comprises a recombinant amyloidogenic protein. In some embodiments, the amyloid protein is part of a disease pathology.
In some embodiments, the amyloid-reactive peptide Fc-fusion protein is pan-amyloid reactive and is capable of binding to different amyloid types in various amyloid tissues. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is capable of binding to an amyloid in the central nervous system. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is capable of binding to amyloid in the brain. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is capable of binding to tau fibrils and/or alpha synuclein aggregates.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is present at a half maximal Effective Concentration (EC) of less than about 1, 10, 100, or 1000nM 50 ) Binds to human amyloid fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is in an EC of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 250, 500, 750, or 1000nM (including any value or range between these values) 50 Binds to human amyloid fibrils. In some embodiments, the amyloid reactive peptide-Fc fusion protein is in an EC of about 1nM, 2nM, 2.5nM, 3nM, 4nM or 5nM 50 Binds to human amyloid fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein has an EC of about 2.5nM 50 Binds to human amyloid fibrils. In some embodiments, the amyloid reactive peptide-Fc fusion protein is expressed in an EC of less than about 3nM, 4nM, 5nM, 10nM, 20nM, 80nM or 100nM 50 Binds to human amyloid fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is expressed in an EC that is less than the binding of the control human Fc region to human amyloid fibrils 50 EC of (2) 50 Binds to human amyloid fibrils. In some embodiments, the amyloid reactive peptide-Fc fusion protein binds to amyloid deposits or fibrils to a greater extent than a human Fc1 control. For calculating EC 50 Is known in the art and includes, for example, surface plasmon resonance. In some embodiments, EC 50 Determined by measuring binding to rvλ6Wil fibrils. An exemplary method of measuring binding to amyloid fibrils is provided in example 4, as shown in fig. 11.
In some embodiments, binding of the amyloid-reactive peptide-Fc fusion protein to human amyloid promotes phagocytosis of human amyloid fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein conditions human amyloid fibrils. In some embodiments, the amyloid-reactive peptide-Fc fusion protein conditions rvλ6Wil fibrils. In some embodiments, contacting human amyloid fibrils with an amyloid-reactive peptide-Fc fusion protein of the present disclosure in the presence of macrophages promotes uptake of human amyloid fibrils by macrophages. In some embodiments, contacting human amyloid fibrils with an amyloid-reactive peptide-Fc fusion protein of the present disclosure in the presence of macrophages promotes conditioning of human amyloid fibrils. In some embodiments, binding of the amyloid-reactive peptide-Fc fusion protein to human amyloid promotes phagocytosis of human amyloid fibrils to an equal or greater extent than a control molecule (e.g., human Fc region). In some embodiments, the amyloid-reactive peptide-Fc fusion protein promotes antibody-dependent cellular phagocytosis.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is conjugated to a detectable label. In some embodiments, the detectable label is selected from the group consisting of: radionuclides (e.g. I- 124 、I- 125 、I- 123 、I- 131 、Zr- 89 、Tc- 99m 、Cu- 64 、Br- 76 、F- 18 ) The method comprises the steps of carrying out a first treatment on the surface of the Enzymes (horseradish peroxidase); biotin; and fluorophores, and the like. Any means known in the art for detectably labeling proteins may be used and/or adapted for use in the methods described herein. For example, the amyloid-reactive peptide-Fc fusion protein may be radiolabeled with a radioisotope or labeled with a fluorescent or chemiluminescent tag. Exemplary radioisotopes include, for example 18 F、 111 In、 99m Tc (Tc) 123 I and 125 I. these and other radioisotopes may be linked to the amyloid-reactive peptide-Fc fusion protein using well-known chemical methods that may or may not involve the use of chelators, such as DTPA or DOTA, for example, covalently linked to the amyloid-reactive peptide-Fc fusion protein. Exemplary fluorescent or chemiluminescent labels includeFluorescein, texas red, rhodamine, alexa dye and luciferase, which can be conjugated to amyloid reactive peptide-Fc fusion proteins by reaction with lysine, cysteine, glutamic acid and aspartic acid side chains. In one exemplary embodiment, a fluorescent microplate reader or fluorometer is used to detect the labels using the appropriate excitation and emission wavelengths for the labels used. For example, a radiolabel may be detected using a gamma or scintillation counter depending on the type of radioactive emission and by using an energy window suitable for accurately detecting a particular radionuclide. However, any other suitable technique for detecting the radioisotope may be used to detect the tag. In some embodiments, the detectable label is 125 I。
Also provided herein are pharmaceutical compositions comprising any of the amyloid-reactive peptide-Fc fusion proteins described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
Nucleic acids, vectors, host cells and methods for preparing fusion proteins
Also provided herein are nucleic acids encoding amyloid-reactive peptide-Fc fusion proteins. In some embodiments, the nucleic acid encodes any of the amyloid-reactive peptide-Fc fusion proteins described herein.
In some embodiments, the nucleic acids provided herein are in one or more vectors. In some embodiments, the vector comprises one or more nucleic acids encoding an amyloid-reactive peptide-Fc fusion protein of the present disclosure.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is a homodimer. In some embodiments, the vector comprises nucleic acids encoding the first and second polypeptides of the amyloid-reactive peptide-Fc fusion protein.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is a heterodimer. In some embodiments, the vector comprises a first nucleic acid encoding a first polypeptide of an amyloid-reactive peptide-Fc fusion protein and a second nucleic acid encoding a second polypeptide of an amyloid-reactive peptide-Fc fusion protein. In some embodiments, the first vector comprises a first nucleic acid encoding a first polypeptide of an amyloid-reactive peptide-Fc fusion protein and the second vector comprises a second nucleic acid encoding a second polypeptide of an amyloid-reactive peptide-Fc fusion protein.
For amyloid reactive peptide-Fc fusion protein production, the amyloid reactive peptide-Fc fusion protein expression vector may be introduced into a suitable production cell line known in the art. The introduction of the expression vector may be accomplished by co-transfection via electroporation or any other suitable transformation technique available in the art. Cell lines producing the amyloid-reactive peptide-Fc fusion protein can then be selected and expanded and the antibodies purified. The purified amyloid reactive peptide-Fc fusion protein can then be analyzed by standard techniques such as SDS-PAGE or SEC.
Also provided is a host cell comprising a nucleic acid encoding an amyloid-reactive peptide-Fc fusion protein described herein. Suitable host cells for cloning or expressing a vector encoding an amyloid-reactive peptide-Fc fusion protein include prokaryotic or eukaryotic cells as described herein. For example, amyloid reactive peptide-Fc fusion proteins can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, methods in Molecular Biology, volume 248 (B.K.C.Lo, editions, humana Press, totowa, NJ, 2003), pages 245-254). After expression, the amyloid reactive peptide-Fc fusion protein may be isolated from the bacterial cell paste in the soluble fraction and may be further purified.
In some embodiments, the host cell comprises one or more nucleic acids encoding an amyloid-reactive peptide-Fc fusion protein of the present disclosure.
Host cells suitable for expressing glycosylated amyloid-reactive peptide-Fc fusion proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in connection with insect cells, particularly for transfecting turf clay (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PLANTIBODIES for antibody production in transgenic plants) TM Technology).
Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CVl line (COS-7) transformed by SV 40; human embryonic kidney lines (293 or 293 cells as described, for example, in Graham et al, J.Gen. Virol.36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli (Sertoli) cells (such as, for example, the TM4 cells described in Mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CVl); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells as described, for example, in Mather et al, annals N.Y. Acad.Sci.383:44-68 (1982), MRC 5 cells, and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc.Natl. Acad.Sci.USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2/0 for reviews of certain mammalian host cell lines suitable for antibody production, see, for example, yazaki and Wu, methods in Molecular Biology, volume 248 (B.K.C.Lo., edit, mana Press, totowa, NJ), pages 2003-268 (1980)).
Also provided herein are methods of making the amyloid-reactive peptide-Fc fusion proteins of the present disclosure. In some embodiments, the methods comprise culturing a host cell of the present disclosure under conditions suitable for expression of a vector encoding an amyloid-reactive peptide-Fc fusion protein. In some embodiments, the method further comprises recovering the amyloid-reactive peptide-Fc fusion protein. In some embodiments, protein recovery involves destroying the host cell, for example, by osmotic shock, sonication, or lysis. Once the cells are destroyed, cell debris is removed by centrifugation or filtration. The amyloid reactive peptide-Fc fusion protein may then be further purified. In some embodiments, the amyloid-reactive peptide-Fc fusion proteins of the present disclosure are purified by various protein purification methods, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. For example, in some embodiments, the amyloid reactive peptide-Fc fusion protein is isolated and purified by appropriately selecting and combining an affinity column, such as a protein a column (e.g., POROS protein a chromatography), with a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the amyloid reactive peptide-Fc fusion protein is conjugated to a labeling sequence (such as a peptide) to facilitate purification. An example of a labeled amino acid sequence is a hexahistidine peptide that can bind with micromolar affinity to a nickel-functionalized agarose affinity column. Alternatively, a hemagglutinin "HA" tag corresponding to an epitope derived from influenza hemagglutinin protein may be used.
The amyloid reactive peptide-Fc fusion protein may be prepared by any technique known to those skilled in the art, including chemical synthesis or recombinant means using standard molecular biology techniques.
IV. method of treatment
Also provided herein are methods of treating an amyloid-related disorder comprising administering to a subject an amyloid-reactive peptide-Fc fusion protein disclosed herein.
In some embodiments, a method of treating an amyloid disease is provided, the method comprising administering to an individual in need thereof a therapeutically effective amount of any one of the amyloid-reactive peptide-Fc fusion proteins described herein.
In some embodiments, amyloid deposits can contribute to the pathology of the disease. In other embodiments, amyloid deposits may be indicative of amyloidosis or amyloid-related disease in an individual. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to an amyloid protein in an individual with amyloidosis. In some embodiments, the amyloidosis is localized to a particular tissue or organ system, such as the liver, heart, or central nervous system.
In other embodiments, the amyloidosis is systemic amyloidosis. In some embodiments, the amyloidosis is familial amyloidosis. In other embodiments, the amyloidosis is sporadic amyloidosis. In some embodiments, the amyloidosis or amyloid-related disease is type II diabetes, alzheimer's disease, down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the netherlands type, cerebral β -amyloid angiopathy, spongiform encephalopathy, thyroid tumor, parkinson's disease, dementia with lewy bodies, tauopathies, huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndrome, spongiform encephalopathy, reactive chronic inflammation, thyroid tumor, myeloma or AA amyloidosis of other forms of cancer, AL amyloidosis, AH amyloidosis, aβ amyloidosis, ATTR amyloidosis, hATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis. In some embodiments, the amyloid-related disease is selected from the group consisting of: AL, AH, aβ2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AApoAIV, AApoCII, AApoCII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP, ASPC, AGal7, ACor, aker, ALac, AOAPP, ASem1, AEnf or aβ amyloidosis. In some embodiments, treatment with an amyloid-reactive peptide-Fc fusion protein results in clearance of the amyloid protein. In some embodiments, the amyloid-reactive peptide-Fc fusion protein binds to amyloid associated with normal aging. In other embodiments, the amyloid-reactive peptide-Fc fusion protein is used for diagnosis, treatment or prognosis of amyloidosis or amyloid-related disease in an individual.
In some embodiments, provided herein are methods of treating an amyloid-related disorder in a subject, the method comprising administering a fusion protein provided herein, wherein the subject has amyloid in the kidney, liver, and/or heart. In some embodiments, the individual has an alλ deposit in the kidney. In some embodiments, the individual has an AL kappa deposit in the kidney. In some embodiments, the individual has an alλ deposit in the liver. In some embodiments, the individual has an AL kappa deposit in the liver. In some embodiments, the individual has ATTR deposits in the heart. In some embodiments, the subject has an AL kappa deposit in the heart. In some embodiments, the subject has alzheimer's disease. In some embodiments, the individual has tau fibrils or alpha synuclein aggregates. In some embodiments, the subject has parkinson's disease. In some embodiments, the individual has fibrils in the spleen. In some embodiments, the fusion protein comprises a first polypeptide comprising a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.1-13. In some embodiments, the first and/or second human Fc domain comprises a peptide spacer. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
In some embodiments, provided herein are methods of treating an amyloid-related disorder in a subject, comprising administering a fusion protein provided herein, wherein the amyloid-related disorder is selected from the group consisting of AA amyloidosis, AL amyloidosis, and ATTR amyloidosis. In some embodiments, the fusion protein comprises a first polypeptide comprising a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.1-13. In some embodiments, the first and/or second human Fc domain comprises a peptide spacer. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17. In some embodiments, the individual has amyloid deposits in the spleen, kidneys, liver, and/or heart. In some embodiments, the fusion protein promotes phagocytosis by fixing complement.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is administered via an intradermal, subcutaneous, intramuscular, intracardiac, intravascular, intravenous, intraocular, intraarterial, epidural, intraspinal, extracorporal, intrathecal, intraperitoneal, intrapleural, intraluminal, intravitreal, intracavernosal, intraventricular, intraosseous, intra-articular, intracellular, or pulmonary route.
In some embodiments, the amyloid-reactive peptide-Fc fusion protein is administered in an amount sufficient to induce phagocytosis of the amyloid by cells of the immune system (e.g., macrophages).
In some embodiments, the subject is a mammal, such as a primate, bovine, rodent, or porcine. In some embodiments, the individual is a human.
Also provided herein are methods of targeting amyloid deposits for clearance, comprising contacting the amyloid deposit with any of the amyloid-reactive peptide-Fc fusion proteins described herein. In some embodiments, targeting amyloid deposits for clearance results in clearance of amyloid deposits. In some embodiments, the clearance is caused by conditioning of amyloid deposits. In some embodiments, the method results in phagocytosis of amyloid. In some embodiments, the individual is a human. In some embodiments, the fusion protein comprises a first polypeptide comprising a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.1-13. In some embodiments, the first and/or second human Fc domain comprises a peptide spacer. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
Also provided herein are methods of treating an individual having or suspected of having an amyloid-based disease, the method comprising: determining whether the individual has amyloid deposits by: a detectable label of any of the amyloid reactive peptide-Fc fusion proteins described herein, administering the labeled amyloid fusion protein to the subject, determining whether a signal associated with the detectable label can be detected from the subject; and if the signal is detected, administering an amyloidosis treatment to the individual. In some embodiments, if no signal is detected, the individual is monitored for later development of amyloid deposits. In some embodiments, the method further comprises determining the intensity of the signal and comparing the signal to a threshold above which it is determined that the individual has amyloid deposits. In some embodiments, the amyloidosis treatment comprises administering to the individual any of the amyloid-reactive peptide-Fc fusion proteins described herein. In some embodiments, the fusion protein comprises a first polypeptide comprising a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.1-13. In some embodiments, the first and/or second human Fc domain comprises a peptide spacer. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
In some embodiments, provided herein are methods of identifying amyloid deposits in an individual comprising administering a detectably labeled fusion protein to the individual and detecting a signal from the fusion protein.
V. detection method
Also provided herein are methods of identifying amyloid deposits in an individual.
The method comprises obtaining a tissue sample from a subject, applying a peptide or fusion peptide to the tissue sample and detecting binding of an amyloid-reactive peptide-Fc fusion to amyloid. Detecting the presence of amyloid may involve visualizing binding of the peptide or fusion peptide to amyloid using fluorescence or standard histochemical techniques. The method may further comprise obtaining a tissue section from a tissue sample and staining the tissue section and detecting the presence of amyloid in the tissue sample by visualizing binding of peptide to amyloid using fluorescence or standard histochemical techniques.
In some embodiments, methods of identifying amyloid deposits in an individual are provided, the methods comprising detectably labeling any of the amyloid-reactive peptide-Fc fusion proteins described herein, administering the fusion protein to the individual, and detecting a signal from the fusion protein. Any of the detectably labeled amyloid-reactive peptide-Fc fusion proteins described herein can be used. In some embodiments, the peptide-Fc fusion protein is radiolabeled. In some embodiments, the peptide-Fc fusion protein is used with I- 125 And (3) radiolabeling. In some embodiments, the amyloid-reactive peptide-Fc fusion protein is detected by SPECT/CT imaging, PET/CT imaging, gamma scintigraphy, or optical imaging. In some embodiments, the peptide-Fc fusion protein is fluorescently labeled. In some casesIn embodiments, the method further comprises determining the signal strength. In some embodiments, the signal intensity is determined by SPECT/CT scanning or radiographs. In some embodiments, the fusion protein comprises a first polypeptide comprising a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.1-13. In some embodiments, the first and/or second human Fc domain comprises a peptide spacer. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
In certain embodiments, the amyloid-reactive peptide-Fc fusion proteins of the invention can be attached to imaging agents useful for imaging amyloid in organs and tissues. For example, the amyloid-reactive peptide-Fc fusion proteins of the invention can be attached to an imaging agent, provided to a subject and the precise location of the amyloid can be determined by standard imaging techniques. Peptides that are not selective for amyloid can be used as controls for comparison. Thus, the biodistribution of the amyloid-reactive peptide-Fc fusion proteins of the invention can be compared to the biodistribution of one or more non-selective peptides or control peptides to provide even greater differentiation in the detection and/or localization of amyloid.
Methods for imaging amyloid include, but are not limited to, magnetic Resonance Imaging (MRI), computer Axial Tomography (CAT) scanning, positron Emission Tomography (PET), ultrasound imaging, x-rays, radionuclide imaging, single Photon Emission Computed Tomography (SPECT), and multiphoton microscopy.
To increase the sensitivity of the scan, various contrast agents may be used. The contrast agent used for scanning may include all molecules that attenuate x-rays. For positron emission tomography and radionuclide imaging, radioisotopes may be used. All positron emitting isotopes are useful for positron emission tomography radionuclide imaging, and all gamma photon emitting isotopes are useful for single photon emission computed tomography radionuclide imaging or scintigraphy imaging.
Contrast agents for ultrasound imaging include positive and negative agents. The positive agents reflect ultrasonic energy so they produce positive (bright) images. Accordingly, the negative agent enhances the transmittance or sound permeability, thus producing a negative (dark) image. Various substances have been studied, gas, liquid, solid and combinations thereof, as potential contrast enhancing agents. Examples of solid particulate contrast agents are disclosed in U.S. patent No. 5,558,854, including but not limited to IDE particles and SHU454. European patent application 0231091 discloses oil-in-water emulsions containing highly fluorinated organic compounds for providing enhanced contrast in ultrasound images. Emulsions containing Perfluorobromooctane (PFOB) were also tested for use as ultrasound imaging agents. Us patent No. 4,900,540 describes the use of phospholipid-based liposomes containing a gas or gas precursor as contrast enhancing agents.
Imaging agents can be attached to the amyloid-reactive peptide-Fc fusion proteins of the invention using known methods. Some attachment methods involve the use of metal chelate complexes employing, for example, organic chelators such as DTPA. Acceptable chelates are known in the art. They include, but are not limited to, 1,4,7, 10-tetraazacyclododecane-N, N' -tetraacetic acid (DOTA); 1,4,7, 10-tetraazacyclododecane-N, N', N "-triacetic acid (DO 3A); 1,4, 7-tris (carboxymethyl) -10- (2-hydroxypropyl) -1,4,7, 10-tetraazacyclododecane (HP-DO 3A); diethylenetriamine pentaacetic acid (DPTA); and many other chelates.
Several classes of compounds have potential as MRI contrast agents. These classes include superparamagnetic iron oxide particles, nitroxides, and paramagnetic metal chelates (Mann et al, 1995). Strong paramagnetic metals are preferred. In general, paramagnetic lanthanoid elements and transition metal ions are toxic in vivo. Therefore, it is necessary to combine these compounds with organic ligands into chelates. The amyloid reactive peptide-Fc fusion proteins of the present invention can be used to enhance targeting of such chelated metals to amyloid, which allows for a reduction in the total dose of imaging composition that would otherwise be required.
A wide range of paramagnetic metals are suitable for chelation. Suitable metals include those having atomic numbers 22-29 (inclusive), 42, 44, and 58-70 (inclusive) and oxidation states of 2 or 3. Examples of such metals include, but are not limited to, chromium (III), manganese (II), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), ytterbium (III), and vanadium (II). In other cases (such as X-ray imaging), usable ions include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).
Among the radioisotopes useful for labelling amyloid reactive peptide-Fc fusion proteins of the present invention suitable for use in localization studies are gamma emitters, positron emitters, X-ray emitters and fluorescent emitters. Suitable radioisotopes for labeling peptides and fusion proteins include astatine 211 Bromine 76 、 14 Carbon (C), 11 Carbon (C), 51 Chromium (Cr), 36 Chlorine (Cl), 57 Cobalt (Co), 58 Cobalt, copper 67 Copper (Cu) 64 、 152 Europium and fluorine 18 Gallium (Ga) 67 Gallium (Ga) 68 、 3 Hydrogen, iodine 123 Iodine 124 Iodine 125 Iodine 126 Iodine 131 Indium (indium) 111 Indium (indium) 113m 、 59 Iron (Fe), 177 Lutetium, mercury 107 Mercury (mercury) 203 、 32 Phosphorus, rhenium 186 Rhenium (Re) 188 Ruthenium (Ru) 95 Ruthenium (Ru) 97 Ruthenium (Ru) 103 Ruthenium (Ru) 105 Rhenium (Re) 99m Rhenium (Re) 105 Rhenium (Re) 101 、 75 Selenium (Se), 35 Sulfur, technetium 99m Tellurium (Te) 121m Tellurium (Te) 122m Tellurium (Te) 125m Thulium (thulium) 165 Thulium (thulium) 167 Thulium (thulium) 168 And yttrium 90 . Halogen can be used more or less interchangeably as a label. Gamma emitter iodides may also be used 123 And technetium 99m Because such radioactive metals can be detected with a gamma camera and have the following characteristicsHalf-life for in vivo imaging is facilitated. Positron emitters suitable for PET imaging and having half-lives suitable for peptide imaging can also be used 18 Fluorine or 124 Iodine. The peptides and fusion peptides of the invention can be purified by conjugation with indium via a conjugated metal chelator such as DTPA (diethylenetriamine pentaacetic acid) 111 Or technetium 99m Labeling, or covalently and directly labeling, flanking peptides containing Cys residues.
The radiolabeled amyloid-reactive peptide-Fc fusion proteins of the invention may be produced according to methods well known in the art. For example, they may be iodinated by contact with sodium or potassium iodide, a chemical oxidant such as sodium hypochlorite or an enzymatic oxidant such as lactoperoxidase. The peptide or fusion peptide according to the invention may be technetium via a ligand exchange process 99m Labelling, e.g. by reduction of pertechnetate (pertechnate) with stannous solution, chelation of reduced technetium onto a Sephadex column and application of peptide to this column or by direct labelling techniques, e.g. by incubation of pertechnetate, reducing agents such as SnCl 2 Buffer solutions such as sodium potassium phthalate solutions and peptides. As previously mentioned, the intermediate functional groups commonly used to bind radioisotopes in the form of metal ions to peptides are Diethylene Triamine Pentaacetic Acid (DTPA) and Ethylene Diamine Tetraacetic Acid (EDTA).
Other useful labels include fluorescent labels, chromogenic labels, and biotin labels. Fluorescent labels include, but are not limited to, rhodamine, fluorescein isothiocyanate, sodium fluorescein, renal contrast media (renograpin), and Texas red sulfonyl chloride. In certain embodiments, the peptides and fusion peptides of the invention can be linked to a secondary binding ligand or enzyme (enzyme tag) that will produce a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase and glucose oxidase. Secondary binding ligands include biotin and avidin or streptavidin compounds. The use of such markers is well known to those skilled in the art and is described, for example, in U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149 and 4,366,241; each of which is incorporated by reference herein.
The amyloid reactive peptide-Fc fusion proteins of the invention may also be reacted with enzymes in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates.
The amyloid reactive peptide-Fc fusion proteins of the invention act as agents that target and bind to amyloid proteins to enable detection of amyloid proteins. The peptides and fusion peptides of the invention can be used to determine whether a subject has amyloid or whether a subject has amyloidosis or an amyloid-mediated condition.
In some embodiments, the invention provides a method for detecting amyloid in a subject. The method comprises administering to a subject a pharmaceutical composition comprising an effective amount of one or more peptides or fusion peptides of the invention, and detecting the peptides or fusion peptides that bind to amyloid. The amyloid-reactive peptide-Fc fusion protein may be labeled with an imaging agent such as a radioisotope. The amyloid reactive peptide-Fc fusion protein has specific binding affinity for the deposit and binding is detectable. Binding of the amyloid-reactive peptide-Fc fusion protein to the amyloid protein can be detected by MRI, CAT scan, PET imaging, ultrasound imaging, SPECT imaging, X-ray imaging, fluorescence imaging, or radionuclide imaging.
In some embodiments, the individual has one or more risk factors associated with an amyloid-related disease. In some embodiments, the individual has one or more symptoms of an amyloid-related disease.
Regarding amyloidosis, for example, such markers can be used to diagnose the presence of amyloid, to determine amyloid burden, to monitor the ability of an amyloid-reactive peptide-Fc fusion protein to bind to amyloid in a particular individual, to monitor the progression of amyloidosis, and/or to monitor the response of an individual to amyloid treatment (including treatments associated with administration of an amyloid-reactive peptide-Fc fusion protein to an individual). For example, an amyloid-reactive peptide-Fc fusion protein is labeled with a detectable label as described herein and then administered to an individual having or suspected of having an amyloid-based disease (e.g., amyloidosis, monoclonal Gammaglobulosis of Unknown Significance (MGUS), multiple Myeloma (MM), or related plasma cell disease). Thereafter, the individual may be imaged, for example, to detect the presence of the detectably labeled amyloid-reactive peptide-Fc fusion protein.
In certain exemplary embodiments, the signal from the detectably labeled amyloid-reactive peptide-Fc fusion protein may be quantified, thereby providing an indication of the level of amyloid deposit in the individual. For example, the signal strength may be compared to a standard signal threshold above which amyloidosis is present, but below which amyloidosis is absent or at a low level. The individual may be diagnosed with amyloid, in which case a treatment such as chemotherapy, a corticosteroid drug (lenalidomide or thalidomide), and/or bortezomib (Velcade) may be administered. Additionally or alternatively, the amyloid-reactive peptide-Fc fusion proteins described herein may be administered to a subject in an effort to treat the subject as described herein. In certain exemplary embodiments, individuals may be stratified into one or more groups, such as low, medium, or high amyloid burden, and then treated accordingly. To monitor treatment progress, the subjects may be reapplied with detectably labeled amyloid-reactive peptide-Fc fusion proteins and their amyloid burden is re-assessed accordingly.
Description of the embodiments
1. An amyloid-reactive peptide-Fc fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide linked to the N-terminus or C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the N-terminus or C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer.
2. The amyloid reactive peptide-Fc fusion protein of embodiment 1, wherein the first and second amyloid reactive peptides are linked to the C-terminus of the first and second human Fc domains.
3. The amyloid reactive peptide-Fc fusion protein of embodiment 1 or 2, wherein the first amyloid reactive peptide and/or the second amyloid reactive peptide comprises an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences set forth in SEQ ID NOs 1-13.
4. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-3, wherein the first and/or second human Fc domain is a human IgG1, igG2, or IgG4Fc.
5. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-4, wherein the first and/or second human Fc domain is a human IgG1 Fc.
6. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-5, wherein the first and/or second human Fc domain comprises the amino acid sequence set forth in SEQ ID No. 18.
7. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-6, wherein the first and/or second amyloid reactive peptide is linked to the first and/or second human Fc domain via a spacer.
8. The amyloid reactive peptide-Fc fusion protein of embodiment 7, wherein the spacer is a peptide spacer.
9. The amyloid-reactive peptide-Fc fusion protein of embodiment 8, wherein the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
10. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-9, wherein the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid reactive peptide from N-terminus to C-terminus.
11. The amyloid reactive peptide-Fc fusion protein of embodiment 10 wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 or SEQ ID No. 13.
12. The amyloid reactive peptide-Fc fusion protein of embodiment 10, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
13. The amyloid reactive peptide-Fc fusion protein of embodiment 10, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 13 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
14. The fusion protein of embodiment 10, wherein the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID NO. 17.
15. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-10, wherein
i) The first polypeptide and/or the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20;
i) The first polypeptide and/or the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 21;
iii) The first polypeptide and/or the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 22.
16. The amyloid reactive peptide-Fc fusion protein of embodiment 15 wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20.
17. The amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-9, wherein the first polypeptide comprises a first amyloid-reactive peptide, a first spacer region, and a first human Fc domain from N-terminus to C-terminus, and the second polypeptide comprises a second amyloid-reactive peptide, a second spacer region, and a second human Fc domain from N-terminus to C-terminus.
18. The amyloid reactive peptide-Fc fusion protein of embodiment 16, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID NO. 2 or SEQ ID NO. 13.
19. The amyloid reactive peptide-Fc fusion protein of embodiment 17 or 18 wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
20. The amyloid reactive peptide-Fc fusion protein of embodiment 17 wherein the first and/or second polypeptide comprises the amino acid sequence set forth in SEQ ID No. 19.
21. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-20, wherein the first polypeptide and the second polypeptide comprise the same amino acid sequence.
22. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-15 and 17-21, wherein the first polypeptide and the second polypeptide comprise different amino acid sequences.
23. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-22, wherein the amyloid reactive peptide-Fc fusion protein binds to rvλ6Wil, aβ (1-40), IAAP, alκ, alλ, or ATTR amyloid.
24. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-23, wherein the fusion protein is conjugated to a detectable label.
25. The amyloid reactive peptide-Fc fusion protein of embodiment 23, wherein the detectable label is selected from the group consisting of a fluorescent label and a radiolabel.
26. A pharmaceutical composition comprising the amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-24.
27. One or more nucleic acids encoding the amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-25.
28. A vector comprising one or more nucleic acids as in embodiment 26.
29. A host cell comprising the vector of embodiment 28.
30. The host cell of embodiment 29, wherein the host cell is a mammalian cell, optionally a Chinese Hamster Ovary (CHO) cell.
31. A method of making an amyloid-reactive peptide-Fc fusion protein, comprising culturing the host cell of embodiment 29 or 30 under conditions suitable for expression of a vector encoding the amyloid-reactive peptide-Fc fusion protein.
32. The method of embodiment 31, wherein the method further comprises recovering the amyloid-reactive peptide-Fc fusion protein.
33. A method of treating an amyloid disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-24.
34. The method of embodiment 33, wherein the amyloid-related disease is systemic or local amyloidosis.
35. The method of embodiment 33, wherein the amyloid-related disease is selected from the group consisting of: AL, AH, aβ2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP, parkinson's disease, alzheimer's disease or aβ amyloidosis.
36. The method of any one of embodiments 33-35, wherein treatment with the amyloid-reactive peptide-Fc fusion protein results in clearance of amyloid.
37. A method of targeting an amyloid deposit for clearance, the method comprising contacting the amyloid deposit with the amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-25.
38. The method of embodiment 37, wherein targeting the amyloid deposit for clearance results in clearance of the amyloid deposit.
39. The method of embodiment 37 or 38, wherein clearing is caused by conditioning of the amyloid deposit.
40. A method of treating an individual having an amyloid-based disease or suspected of having an amyloid-based disease, the method comprising:
Determining whether the individual has amyloid deposits by:
the amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-25,
administering a labeled amyloid-reactive peptide-Fc fusion protein to the subject,
determining whether a signal associated with the detectable label can be detected from the individual; and, in addition, the processing unit,
if the signal is detected, an amyloidosis treatment is administered to the individual.
41. The method of embodiment 40, wherein if no signal is detected, the individual is monitored for later development of amyloid deposits.
42. The method of embodiment 40 or 41, further comprising determining the intensity of the signal and comparing the signal to a threshold above which it is determined that the individual has amyloid deposits.
43. The method of any one of embodiments 40-42, wherein the amyloidosis treatment comprises administering to the individual an amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-25.
44. A method of identifying amyloid deposits in an individual, the method comprising detectably labeling an amyloid-reactive peptide-Fc fusion protein of any one of embodiments 1-25, administering the fusion protein to the individual, and detecting a signal from the fusion protein.
45. The method of any one of embodiments 40-44, wherein the individual is determined to be amyloid-free or to have an unknown Monoclonal Gammaglobulopathy (MGUS), multiple Myeloma (MM), or one or more related plasma cell disorders.
46. The method of embodiment 44 or 45, wherein the amyloid-reactive peptide-Fc fusion protein is detectably labeled.
47. The method of any one of embodiments 40-43 and 46, wherein the amyloid-reactive peptide-Fc fusion protein is detectably labeled with a radionuclide or fluorophore.
48. The method of embodiment 47, wherein the radionuclide is I-123, I-124, F-18, ZR-89, or Tc-99m.
49. The method of any one of embodiments 40-43 and 46-48, wherein the amyloid-reactive peptide-Fc fusion protein is detected by SPECT/CT imaging, PET/CT imaging, gamma scintigraphy, or optical imaging.
50. The method of any one of embodiments 33-49, wherein the individual is a human.
51. The amyloid reactive peptide-Fc fusion protein of any one of embodiments 1-25, wherein the amyloid reactive peptide-Fc first polypeptide and the second polypeptide are covalently linked by a disulfide bond in the Fc domain.
A fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide linked to the C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer.
The fusion protein of embodiment 1A, wherein the first amyloid-reactive peptide and/or the second amyloid-reactive peptide comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs 1-13.
The fusion protein of embodiment 1A or embodiment 2A, wherein the first and/or second human Fc domain is a human IgG1, igG2 or IgG4 Fc.
4A. The fusion protein of any one of embodiments 1A-3A, wherein the first and/or second human Fc domain is a human IgG1 Fc.
The fusion protein of any one of embodiments 1A-4A, wherein the first and/or second human Fc domain comprises the amino acid sequence set forth in SEQ ID No. 18.
The fusion protein of any one of embodiments 1A-5A, wherein the first and/or second amyloid-reactive peptide is linked to the first and/or second human Fc domain via a spacer.
7A. The fusion protein of embodiment 6A, wherein the spacer is a peptide spacer.
The fusion protein of embodiment 7A, wherein the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOS: 14-17.
The fusion protein of any one of embodiments 6A-8A, wherein the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid-reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid-reactive peptide from N-terminus to C-terminus.
The fusion protein of any one of embodiments 1A-9A, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20.
The fusion protein of any one of embodiments 1A-10A, wherein the fusion protein binds to rvλ6Wil, aβ (1-40), IAAP, alκ4, alλ1, or ATTR amyloid.
The fusion protein of any one of embodiments 1A-11A, wherein the fusion protein is conjugated to a detectable label.
A pharmaceutical composition comprising the fusion protein of any one of embodiments 1A-12A.
14A. One or more nucleic acids encoding the fusion protein of any one of embodiments 1A-12A.
15A. A vector comprising one or more nucleic acids as described in embodiment 14A.
A host cell comprising the vector of embodiment 15A.
The host cell of embodiment 16A, wherein the host cell is a mammalian cell, optionally a Chinese Hamster Ovary (CHO) cell.
A method of making a fusion protein comprising culturing the host cell of embodiment 16A or 17A under conditions suitable for expression of a vector encoding the fusion protein.
The method of embodiment 18A, wherein the method further comprises recovering the fusion protein.
A method of treating an amyloid disease, comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein of any one of embodiments 1A-12A.
The method of embodiment 20A, wherein the amyloid-related disease is systemic or local amyloidosis.
The method of embodiment 20A, wherein the amyloid-related disease is selected from the group consisting of: AL, AH, aβ2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP or aβ amyloidosis.
The method of any one of embodiments 20A-22A, wherein treatment with the fusion protein results in clearance of amyloid.
A method of targeting amyloid deposits for clearance, the method comprising contacting amyloid deposits with the fusion protein of any one of claims 1A-12A.
The method of embodiment 24A, wherein targeting the amyloid deposit for clearance results in clearance of the amyloid deposit.
The method of embodiment 24A or 25A, wherein the clearing is caused by conditioning of the amyloid deposit.
The method of any one of embodiments 20A-26A, wherein the subject is a human.
A method of treating an individual having or suspected of having an amyloid-based disease, the method comprising:
determining whether the individual has amyloid deposits by:
the fusion protein of any one of embodiments 1A-12A,
administering the labeled fusion protein to the individual,
determining whether a signal associated with the detectable label can be detected from the individual; and, in addition, the processing unit,
if the signal is detected, an amyloidosis treatment is administered to the individual.
The method of embodiment 28A, wherein if no signal is detected, the individual is monitored for later development of amyloid deposits.
The method of embodiment 29A, further comprising determining the intensity of the signal and comparing the signal to a threshold above which it is determined that the individual has amyloid deposits.
The method of any one of embodiments 28A-30A, wherein the amyloidosis treatment comprises administering to the individual the fusion protein of any one of embodiments 1A-12A.
A method of identifying amyloid deposits in an individual, the method comprising detectably labeling a fusion protein of any one of embodiments 1A-12A, administering the fusion protein to the individual, and detecting a signal from the fusion protein.
The method of any one of embodiments 28A-32A, wherein the individual is determined to be amyloid-free or to have an unknown Monoclonal Gammaglobulopathy (MGUS), multiple Myeloma (MM), or one or more associated plasma cell disorders.
Examples
The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In view of the present disclosure and the general level of skill in the art, those of skill will understand that the following embodiments are intended to be exemplary only and that various changes, modifications and alterations may be employed without departing from the scope of the presently disclosed subject matter. The drawings are intended to be regarded as forming part of the specification and description of the present disclosure.
Example 1. Production of peptide-Fc constructs.
The following examples describe the generation of amyloid reactive peptide-Fc fusion protein constructs. The structure of an exemplary construct is provided in fig. 1. In one construct, termed Fcp5R NV1, the p5R peptide is fused to the N-terminus of the first and second Fc domains by a rigid short spacer (VSPSV, SEQ ID NO: 15), as shown in the top row of fig. 1. In a second construct, called Fcp5R CV1, the p5R peptide is fused to the N-terminus of the first and second Fc domains by a rigid short spacer (VSPSV, SEQ ID NO: 15), as shown in the second row of fig. 1. The amino acid sequences of Fcp5R NV1 and Fcp5R CV1 are provided in table 3 below.
TABLE 3 amino acid sequences of peptide-Fc constructs
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to compare the formulation of the peptide-Fc fusion construct with the peptide-antibody fusion construct. The fusion construct was generated in Chinese Hamster Ovary (CHO) cells containing 2% Fetal Bovine Serum (FBS). The sample was reduced and boiled. 4% -12% bis-tris gel was used with 2- (N-morpholino) ethanesulfonic acid (MES) buffer and protein was detected using Coomassie blue staining. As shown in FIG. 2, the size of the Fcp5R CV1 construct (lane 6) is greater than the Fcp5R NV1 construct (lane 5). Without wishing to be bound by theory, it is believed that this production method results in a construct that is sensitive to cleavage of the amyloid-binding peptide (in the case of Fcp5R NV1, lane 5) or resistant to peptide cleavage (in the case of Fcp5R CV1, lane 6). The data indicate that the position of the peptide on the Fc domain protects it from proteolysis during production.
Size Exclusion Chromatography (SEC) was performed to further analyze Fcp5R NV1 and Fcp5R CV1. As shown in FIG. 3, the Fcp5R NV1 and Fcp5R CV1 eluted from the column at different times, indicating that the two constructs were of different sizes. Fcp5R CV1 eluted before NV1, indicating that it had a higher molecular weight, consistent with the observations from SDS-PAGE analysis (fig. 2), i.e., the peptide was resistant to proteolytic degradation during reagent production. In contrast, fcp5R NV1 is susceptible to proteolytic cleavage.
Example 2 biodistribution of peptide-Fc in mice
The following examples describe radioiodination of Fcp5R CV1, and administration of radiolabeled Fcp5R CV1 to AA amyloidogenic mice.
Fcp5R CV1 was prepared by the method of I- 125 And (3) radiolabeling. Antibody 11-1F4 was used as a control for the radiolabeling reaction. As shown in FIG. 4, fcp5R CV1 produced by HD CHO cells grown in 2% FBS was readily expressed by I- 125 Radiolabeled and eluted with blue dextran. Recovery from the column and tube was very good. SDS-PAGE gels showed that no aggregates were formed during the radiolabeling procedure and the formulations were of high purity and high radiopurity (i.e., no evidence of free radioiodine in the SDS-PAGE gel).
Will be 125 I-Fcp5R CV1 was administered to mice with systemic AA amyloidosis. AA amyloidosis mouse models were generated by intravenous administration of 0.1mg isolated amyloid enhancement factor (AEF, axelrad et al, lab Invest (1982) 47:139-146) in 100 μl sterile Phosphate Buffered Saline (PBS) in H2-Ld-huIL-6Tg Balb/c transgenic mice constitutively expressing human interleukin-6 transgenes. Mice used in these studies were 4-6 weeks after induction of amyloidosis. The AA mouse model is characterized by extensive sinusoidal amyloid deposition in the liver, initial and large perifollicular amyloid deposition in the spleen, and later amyloid deposition in the pancreas, kidney, adrenal gland, intestine and small amounts of interstitial cardiac amyloid deposition.
Administration to mice with AA amyloidosis by tail vein intravenous injection 125 I-Fcp5R CV1 (about 10. Mu.g, about 100. Mu. Ci) and were tested at time points after injection 125 Biodistribution of I-Fcp5R CV 1. Specifically, AA mice groups (n=4 per group) were injected 125 The I-labeled Fcp5R CV1 was then euthanized 1, 4 or 24 hours after injection. Samples of spleen, pancreas, left and right kidneys, liver, heart, muscle, stomach, upper and lower intestines and lung tissue were harvested from AA mice after euthanasia. Each sample was placed in a peeled plastic vial and weighed, and was measured using an automated Wizard 3 gamma counter (1480 Wallac gamma counter, PERKIN) To measure 125 I radioactivity. Biodistribution data are expressed as a percentage of injected dose per gram of tissue (% ID/g). In addition, samples of each tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin for histological and autoradiography. For autoradiography, sections 4 to 6- μm thick were cut from formalin-fixed, paraffin-embedded blocks and placed on Plus microscope slides (FISHER) On top of that, it is immersed in NTB2 emulsion (EASTMAN +.>) Stored in the dark and developed after 96 hours of exposure. Counterstaining was performed on each section with hematoxylin.
As shown in FIGS. 5-8, detection was in mice, particularly in the liver and spleen, which are the major sites of amyloid deposition in this mouse model 125 I-Fcp5R CV1. Additional uptake of the tracer was observed in the kidneys (fig. 5). Binding to amyloid deposits in the liver and spleen was visualized using small animal SPECT/CT imaging, demonstrating liver-spleen uptake of radioiodinated Fcp5R CV1 24 hours or more after injection (fig. 6). Specific binding to amyloid deposits in heart, liver and spleen was demonstrated using microscopic autoradiography (fig. 7-8). In the microautoradiography (ARG), blackThe deposition of the colored silver particles indicates the presence of radiolabeled Fcp5R CV1. The distribution of silver particles and thus 125I-Fcp5R CV1 was correlated with the distribution of amyloid shown in congo red-stained serial tissue sections (congo red). Specific reactivity of 125I-Fcp5R CV1 with amyloid in these tissues was also observed at a later time point (24 hours) after injection (fig. 8).
EXAMPLE 3 binding and phagocytosis of rV.lamda.6 Wil fibrils
The ability of peptide-Fc fusion proteins to promote amyloid fibril phagocytosis was investigated using the pHrodo red-labeled rvλ6Wil fibril system.
Human THP1 cells (10) 6 Individual cells/well) were coated onto wells of a 24-well tissue culture treated plate. Aliquots of 50ng/ml Phorbol Myristate Acetate (PMA) were added and the cells were incubated at 37℃with 5% CO 2 Incubate in incubator for 24 hours. After 24 hours, the PMA-containing medium was carefully removed and replaced with complete DMEM-F12 medium and the cells were allowed to stand for a minimum of 48 hours. For phagocytosis assays, the medium was removed from the wells, rinsed with Dulbecco's PBS, and 500. Mu.L of an aliquot of RPMI was added to each well. Fcp5R variant or control hFc1 was mixed with pHrodo red-labeled fibrils at appropriate concentrations and then started to be added to cells in 24-well plates. After gentle mixing, the plates were cooled at 37 ℃ at 5% CO 2 Incubate for 1 hour in incubator to promote phagocytosis. At the end of the 1 hour incubation, fluorescence emission from the pHrodo red fluorophore was imaged using an epifluorescence microscope equipped with a 4-fold objective and a red fluorescence filter. Four images are captured per well to ensure that all areas of the well are covered and represented without any deviation. Image segmentation and quantification (Image ProPlus) was used to quantify the amount of fluorescence in each Image. The fluorescence units are measured in the form of digital spectral counts.
As shown in fig. 9, fcp5R CV1 promoted Wil fibril uptake to a greater extent than Fcp5R NV 1. Without wishing to be bound by theory, the enhanced phagocytic activity induced by Fcp5R CV1 is thought to be due to the presence of full-length amyloid-reactive peptide in this variant relative to Fcp5R NV 1. Furthermore, fcp5R CV1 showed a dose-dependent response in Wil fibril uptake, as shown in fig. 10.
Furthermore, the ability of Fcp5R CV1 to bind rvλ6Wil fibrils was measured compared to the human Fc1 control. As shown in FIG. 11, the Fcp5R CV1 was expressed as EC of 2.5nM 50 rV.lamda.6Wil fibrils were bound, whereas human Fc1 control did not.
Example 4 design of additional peptide-Fc constructs
Additional peptide-Fc constructs are contemplated. In one construct, the p5R peptide is fused to the C-terminus of the first and second Fc domains by a flexible long spacer (GGGGSGGGGS, SEQ ID NO: 16), as shown in the third row of FIG. 1. In another construct, a p5R+14 peptide (an extended p5R variant with more than 14 amino acids, including an additional four amyloid-binding arginine residues) is fused to the C-terminus of the first and second Fc domains by a rigid short spacer (VSPSV, SEQ ID NO: 15), as shown in the bottom row of FIG. 1. The amino acid sequences of these constructs are provided in table 4 below.
TABLE 4 amino acid sequences of peptide-Fc constructs
Example 5-biodistribution of 125I-Fcp5RCV1 in mice with systemic serum amyloid A-related (AA) amyloidosis.
The hFc1CV1 was expressed by transiently transfected CHO cells. The Fc-peptide fusion was purified by protein a. The hFc1CV1 was radiolabeled with iodine 125 by oxidative incorporation of tyrosine side chains. Free radioiodine was separated by size exclusion chromatography and the radiopurity assessed by SDS-PAGE and autoradiography.
Approximately 100 μCi (10 μg reagent) was injected intravenously into the lateral tail vein of mice with systemic AA amyloidosis or amyloid-free WT mice (as controls). Transgenic AA mice produce systemic amyloid in all organs and tissues, but are characterized by the presence of severe amyloid in the liver, spleen and kidneys, whereas only small amounts of deposits are observed in the heart. Mice (n=4 per group) were euthanized by excess isoflurane at 1, 4, 24 and 48 hours post injection and SPECT/CT images were acquired.
Immediately thereafter, the sample or organ and blood are collected for measuring tissue-related radioactivity as a measure of the biological distribution of the agent in the organ. This analysis revealed a rapid accumulation in amyloid-filled organs, in particular 125I-hFc1CV1 retention in the liver, spleen, kidneys, stomach and heart, with a decrease in radioactivity observed in these organs over time (fig. 12A).
48 hours after injection, radiolabeled hFc1CV1 was significantly retained in the liver, spleen, pancreas, stomach and heart compared to 125I-hFc1CV1 in wild-type mice, indicating specific binding to ligand amyloid in these organs (fig. 12B).
The distribution of radiolabeled hFc1CV1 in AA mouse organs was evaluated using microscopic autoradiography. Tissue samples were fixed in 10% buffered formalin for 24 hours, embedded in paraffin blocks, and 6 μm thick tissue sections were prepared on slides. Slides were exposed to photographic emulsion for three days and then counterstained with H & E. The deposition of black silver particles demonstrated the presence of radioactivity in the tissue. In all organs evaluated, it was observed that the binding of radiolabeled hFc1CV1 was associated with amyloid deposits in tissues, indicating specific binding to pathology (fig. 13A and 13B). 1 hour after injection (FIG. 13A), 125I-hFc1CV1 had accumulated at specific sites in the tissue, which was confirmed in an Autoradiogram (ARG), which correlated with the presence of amyloid, and exhibited green/gold birefringence in Congo Red (CR) -stained tissue sections. The strong and specific amyloid binding of 125I-hFc1CV1 was still evident in ARG 24 hours after injection (fig. 13B).
Example 6-hFc1CV1 enhances phagocytosis of human AL amyloid extract in vitro
hFc1CV1 was expressed from stably transfected CHO cells grown under perfusion culture conditions and purified on day 7. The Fc-peptide fusion was purified by protein a.
Amyloid fibrils (rvλ6 WIL), human AL extracts (alλ or alκ) and human attrtwt amyloid extracts were labeled with the pH-sensitive dye succinimidyl-pHrodo red fluorophore for use in an ex vivo phagocytosis assay. Human THP-1 cells were activated by addition of Phorbol Myristate Acetate (PMA) and inoculated into wells of 24-well tissue culture plates. An amyloid extract of 20- μg mass was added to the wells and the amount of hFc1CV1 or control igg1 antibodies (6 nM, 20nM, 60nM and 200 nM) was increased and the plates incubated for 1 hour at 37C. The wells were observed using an inverted fluorescence microscope (Keyance BZ X800) and four digital images (4-fold objective) were captured per well. Fluorescence in each image was quantified using spectral segmentation and the mean and SD of the four images were determined (fig. 14A-14D).
The results indicate that hFc1CV1 enhances phagocytosis of activated human THP-1 macrophages in a dose-dependent manner against a variety of amyloid extracts with a saturation of action of about 60nM for both AL and attrtwt extracts. The increase in fluorescence emission due to increased phagocytosis of amyloid substrate was significantly greater than control hIgG1 at all concentrations.
These data demonstrate that the opsonization of human amyloid by hFc1CV1 results in significant phagocytosis of amyloid by human macrophages.
Example 7 human plasma as complement source enhances hFc1CV1 mediated in vitro phagocytosis of human AL amyloid extracts
hFc1CV1 was expressed from stably transfected CHO cells grown under perfusion culture conditions and purified on day 7. The Fc-peptide fusion was purified by protein.
Amyloid fibrils (rvλ6wil) and human AL extracts (alλ or alκ) amyloid extracts were labeled with the pH-sensitive dye succinimidyl-borodo red fluorophore. Human THP-1 cells were activated by addition of Phorbol Myristate Acetate (PMA) and inoculated into wells of 24-well tissue culture plates. An amyloid extract of 20- μg mass was added to wells containing 60nm hfc1cv1 in the presence or absence of 20% human plasma (as complement source). The wells were observed using an inverted fluorescence microscope (Keyance BZ X800) and four digital images (4-fold objective) were captured per well. Fluorescence in each image was quantified using spectral segmentation and the mean and SD of the four images were determined. The results demonstrate that plasma/complement significantly enhances the efficacy of hFc1CV1 for inducing phagocytosis of activated human THP-1 macrophages on human amyloid extracts (fig. 15).
These data indicate that the opsonization of human amyloid by hFc1CV1 in the presence of complement components in plasma results in a significant enhancement of phagocytosis of amyloid by human macrophages.
Example 8hFc1CV1 binds to different forms of amyloid with subnanomolar potency
hFc1CV1 was expressed from stably transfected CHO cells grown under perfusion culture conditions and purified on day 7. The Fc-peptide fusion was purified by protein a.
Synthetic amyloid fibrils (rvλ6 WIL) and human AL extracts (alλ or alκ) and human ATTRV and attrtwt amyloid extracts were used as substrates for hFc1CV1 binding. Fc-peptide conjugates were added to wells at 2-fold serial dilutions starting at 400 nM. Detection of bound hFc1CV1 was assessed by measuring time resolved fluorescence after addition of biotinylated goat anti-human Fc reactive secondary antibody and streptavidin-europium conjugate (fig. 16). The mean and SD of three replicates were calculated and potency (EC 50) was determined after fitting with the sigmoid 4PL equation on the logarithmic x-axis (Prism) (table 5).
The estimated potency (EC 50) values for binding of hFc1CV1 to amyloid substrates obtained in ATTRv amyloid extracts ranged from 0.5nM (for synthetic fibrils) to 1.8nM. These data demonstrate the high affinity binding of hFc1CV1 to synthetic fibrils and human AL and ATTR amyloid extracts.
TABLE 5 EC50 s
EC50(M) rV lambda 6Wil fibrils ATTRwt(125) ATTRv(KEN) ALλ(SHI) AL K (TAL)
wxpFcp5RCV1 5.0E-10 7.0E-10 1.8E-09 1.7E-09 5.3E-10
Synthetic amyloid fibrils (Tau 441, alpha-synuclein and aβ (1-40)) were used as substrates for hfcccv 1 binding. hFc1CV1 was added to wells at 2-fold serial dilutions starting at 400nM (50 nM for the aβ fibrils shown). Detection of bound hFcCV1 was assessed by measuring time resolved fluorescence after addition of biotinylated goat anti-human Fc reactive secondary antibody and streptavidin-europium conjugate (fig. 17). The mean and SD of three replicates were calculated and potency (EC 50) was determined after fitting with the sigmoid 4PL equation on the logarithmic x-axis (Prism) (table 5).
The estimated potency (EC 50) values for binding of hFc1CV1 to fibrils for alpha-synuclein, tau441 and Abeta (1-40) were 7.3nM, 7nM and 0.7nM (Table 5).
Example 8-binding of biotinylated hFc1CV1 to amyloid in tissue sections
Formalin-fixed paraffin-embedded sections were prepared from tissues containing AL or ATTR amyloid. Additional brain tissue samples from patients with alzheimer's disease were also evaluated. Tissues were stained with biotinylated hFc1CV1 (2 μg/mL in PBS) using standard immunohistochemical methods and visualized after the addition of diaminobenzidine. After staining the tissue with alkaline congo red solution, the presence of amyloid in the slide from the same tissue was visualized by congo red fluorescence.
hfcvl specifically binds to aβ amyloid in the brain of patients with alzheimer's disease, diffuse plaques and core plaques consisting of aβ amyloid and in the vessel wall (fig. 18A)
Similarly, specific binding to amyloid was observed in the case of AL amyloid deposits in the kidneys and liver (fig. 18B). The specific binding of hFc1CV1 to cardiac amyloid deposits surrounding cardiomyocytes in two samples of ATTR and AL cardiac amyloidosis (fig. 18C).
These data demonstrate the specific reactivity of hFc1CV1 with different types of tissue amyloid deposits in different tissues. Thus, pan-amyloid reactivity of hFc1CV1 mediated by the p5R peptide was demonstrated by immunohistochemical staining using tissues from three of the most common forms of amyloid-related disease.
Sequence listing
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Alplacian Co Ltd
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<211> 263
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 19
Ala Pro Gly Gly Gly Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg
1 5 10 15
Gln Ala Gln Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln Val
20 25 30
Ser Pro Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
35 40 45
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
50 55 60
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
65 70 75 80
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
85 90 95
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
100 105 110
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
115 120 125
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
130 135 140
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
145 150 155 160
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
165 170 175
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
180 185 190
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
195 200 205
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
210 215 220
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
225 230 235 240
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
245 250 255
Leu Ser Leu Ser Pro Gly Lys
260
<210> 20
<211> 266
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 20
Ala Pro Gly Gly Gly Ser Val Ser Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
20 25 30
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
50 55 60
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
65 70 75 80
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
85 90 95
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
100 105 110
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
115 120 125
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
130 135 140
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
145 150 155 160
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
165 170 175
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
180 185 190
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
195 200 205
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
210 215 220
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Ser Pro Ser Val
225 230 235 240
Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln Ala Gln Arg Ala
245 250 255
Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln
260 265
<210> 21
<211> 271
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 21
Ala Pro Gly Gly Gly Ser Val Ser Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
20 25 30
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
50 55 60
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
65 70 75 80
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
85 90 95
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
100 105 110
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
115 120 125
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
130 135 140
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
145 150 155 160
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
165 170 175
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
180 185 190
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
195 200 205
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
210 215 220
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg
245 250 255
Gln Ala Gln Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln
260 265 270
<210> 22
<211> 280
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 22
Ala Pro Gly Gly Gly Ser Val Ser Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
20 25 30
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
50 55 60
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
65 70 75 80
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
85 90 95
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
100 105 110
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
115 120 125
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
130 135 140
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
145 150 155 160
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
165 170 175
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
180 185 190
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
195 200 205
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
210 215 220
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Ser Pro Ser Val
225 230 235 240
Arg Ala Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln Ala Gln Arg Ala
245 250 255
Gln Arg Ala Gln Ala Arg Gln Ala Arg Gln Ala Gln Arg Ala Gln Arg
260 265 270
Ala Gln Ala Arg Gln Ala Arg Gln
275 280

Claims (44)

1. An amyloid-reactive peptide-Fc fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first amyloid-reactive peptide linked to the N-terminus or C-terminus of a first human Fc domain, wherein the second polypeptide comprises a second amyloid-reactive peptide linked to the N-terminus or C-terminus of a second human Fc domain, and wherein the first human Fc domain and the second human Fc domain form a dimer.
2. The amyloid reactive peptide-Fc fusion protein of claim 1, wherein the first and second amyloid reactive peptides are linked to the C-terminus of the first and second human Fc domains.
3. The amyloid reactive peptide-Fc fusion protein of claim 1 or 2, wherein the first amyloid reactive peptide and/or the second amyloid reactive peptide comprises an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences set forth in SEQ ID NOs 1-13.
4. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-3, wherein the first and/or second human Fc domain is a human IgG1, igG2, or IgG4Fc.
5. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-4, wherein the first and/or second human Fc domain is a human IgG1 Fc.
6. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-5, wherein the first and/or second human Fc domain comprises the amino acid sequence set forth in SEQ ID No. 18.
7. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-6, wherein the first and/or second amyloid reactive peptide is linked to the first and/or second human Fc domain via a spacer.
8. The amyloid reactive peptide-Fc fusion protein of claim 7, wherein the spacer is a peptide spacer.
9. The amyloid reactive peptide-Fc fusion protein of claim 8, wherein the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs 14-17.
10. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-9, wherein the first polypeptide comprises a first human Fc domain, a first spacer region, and a first amyloid reactive peptide from N-terminus to C-terminus, and the second polypeptide comprises a second human Fc domain, a second spacer region, and a second amyloid reactive peptide from N-terminus to C-terminus.
11. The amyloid reactive peptide-Fc fusion protein of claim 10, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 or SEQ ID No. 13.
12. The amyloid reactive peptide-Fc fusion protein of claim 10, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
13. The amyloid reactive peptide-Fc fusion protein of claim 10, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 13 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
14. The fusion protein of claim 10, wherein the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 17.
15. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-10, wherein
i) The first polypeptide and/or the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 20;
ii) said first polypeptide and/or said second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 21;
iii) The first polypeptide and/or the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 22.
16. The amyloid reactive peptide-Fc fusion protein of claim 15, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID No. 20.
17. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-9, wherein the first polypeptide comprises a first amyloid reactive peptide, a first spacer region, and a first human Fc domain from N-terminus to C-terminus, and the second polypeptide comprises a second amyloid reactive peptide, a second spacer region, and a second human Fc domain from N-terminus to C-terminus.
18. The amyloid reactive peptide-Fc fusion protein of claim 16, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 or SEQ ID No. 13.
19. The amyloid reactive peptide-Fc fusion protein of claim 17, wherein the amyloid reactive peptide comprises the amino acid sequence set forth in SEQ ID No. 2 and the spacer comprises the amino acid sequence set forth in SEQ ID No. 14.
20. The amyloid reactive peptide-Fc fusion protein of claim 17, wherein the first and/or second polypeptide comprises the amino acid sequence set forth in SEQ ID No. 19.
21. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-20, wherein the first polypeptide and the second polypeptide comprise the same amino acid sequence.
22. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-15 and 17-21, wherein the first polypeptide and the second polypeptide comprise different amino acid sequences.
23. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-22, wherein the amyloid reactive peptide-Fc fusion protein binds to rvλ6Wil, aβ (1-40), IAAP, alκ, alλ, or ATTR amyloid.
24. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-23, wherein the fusion protein is conjugated to a detectable label.
25. The amyloid reactive peptide-Fc fusion protein of claim 23, wherein the detectable label is selected from the group consisting of a fluorescent label and a radiolabel.
26. A pharmaceutical composition comprising the amyloid-reactive peptide-Fc fusion protein of any one of claims 1-24.
27. One or more nucleic acids encoding the amyloid-reactive peptide-Fc fusion protein of any one of claims 1-25.
28. A vector comprising one or more nucleic acids of claim 26.
29. A host cell comprising the vector of claim 28.
30. The host cell of claim 29, wherein the host cell is a mammalian cell, optionally a Chinese Hamster Ovary (CHO) cell.
31. A method of making an amyloid reactive peptide-Fc fusion protein, comprising culturing the host cell of claim 29 or 30 under conditions suitable for expression of a vector encoding the amyloid reactive peptide-Fc fusion protein.
32. The method of claim 31, wherein the method further comprises recovering the amyloid-reactive peptide-Fc fusion protein.
33. A method of treating an amyloid disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the amyloid-reactive peptide-Fc fusion protein of any one of claims 1-24.
34. The method of claim 33, wherein the amyloid-related disease is systemic or local amyloidosis.
35. The method of claim 33, wherein the amyloid-related disease is selected from the group consisting of: AL, AH, aβ2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP, parkinson's disease, alzheimer's disease or aβ amyloidosis.
36. The method of any one of claims 33-35, wherein treatment with the amyloid-reactive peptide-Fc fusion protein results in clearance of amyloid.
37. A method of targeting an amyloid deposit for clearance, the method comprising contacting the amyloid deposit with the amyloid-reactive peptide-Fc fusion protein of any one of claims 1-25.
38. The method of claim 37, wherein targeting the amyloid deposit for clearance results in clearance of the amyloid deposit.
39. The method of claim 37 or 38, wherein clearing is caused by conditioning of the amyloid deposit.
40. A method of treating an individual having an amyloid-based disease or suspected of having an amyloid-based disease, the method comprising:
Determining whether the individual has amyloid deposits by:
the amyloid-reactive peptide-Fc fusion protein of any one of claim 1-25,
administering a labeled amyloid-reactive peptide-Fc fusion protein to the subject,
determining whether a signal associated with the detectable label can be detected from the individual; and, in addition, the processing unit,
if the signal is detected, an amyloidosis treatment is administered to the individual.
41. A method of identifying amyloid deposits in an individual, the method comprising detectably labeling an amyloid-reactive peptide-Fc fusion protein of any one of claims 1-25, administering the fusion protein to the individual, and detecting a signal from the fusion protein.
42. The method of claim 40 or 41, wherein the amyloid-reactive peptide-Fc fusion protein is detectably labeled.
43. The method of any one of claims 31-42, wherein the individual is a human.
44. The amyloid reactive peptide-Fc fusion protein of any one of claims 1-25, wherein the first polypeptide and the second polypeptide are covalently linked by a disulfide bond in the Fc domain.
CN202280045651.5A 2021-05-05 2022-05-04 peptide-FC fusions for the treatment of amyloid disorders Pending CN117881414A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/184,682 2021-05-05
US202163186605P 2021-05-10 2021-05-10
US63/186,605 2021-05-10
PCT/US2022/072112 WO2022236286A1 (en) 2021-05-05 2022-05-04 Peptide-fc fusions for treating amyloid disorders

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