SAP FC fusion protein and method of use

Abstract

The present invention relates to SAP-Fc fusion proteins and methods for treating amyloid-related diseases.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority based on U.S. Provisional Application No. 63 / 108,799, filed on November 2, 2020, and U.S. Provisional Application No. 63 / 153,777, filed on February 25, 2021, and the entire contents of each of them are incorporated herein by reference.

[0002] Submission of Sequence Listing in ASCII Text File By reference, the entire contents of the following submissions in ASCII text file are incorporated herein: Computer - Readable Form (CRF) of the Sequence Listing (File Name: 165992000200SEQLIST.TXT, Date of Record: October 28, 2021, Size: 91,399 bytes).

[0003] The present invention relates to an SAP - Fc fusion protein and a method for treating amyloid - related disorders by administering the SAP - Fc fusion protein.

Background Art

[0004] Amyloidosis is a broad group of diseases belonging to the group of protein conformational diseases, including other diseases such as Alzheimer's disease, transmissible spongiform encephalopathy, Huntington's disease or type II diabetes mellitus.

[0005] [[ID=2'8]]Amyloidosis is a rare disease characterized by the presence of insoluble protein deposits with abnormal fibrous conformations in tissues. Most often, it is caused by fragments of serum precursor proteins. Many organs can be invaded by these extracellular deposits called "amyloid substances". The main organs invaded by amyloid deposits are the kidney, heart, gastrointestinal tract, liver, skin, peripheral nerves and eyes. Organs invaded by this disease generally have a considerable volume. Eventually, amyloidosis can invade all organs and the central nervous system, and for this reason, there are actually a variety of many symptoms.

[0006] Various methods have been attempted to treat amyloidosis. For example, chemotherapy with glucocorticoids (dexamethasone) and antimitotics. The effectiveness of new anti-inflammatory drugs (anti-TNF, anti-IL1) is currently being clinically evaluated. However, for the time being, amyloidosis remains incurable and fatal because there is no effective treatment that can eliminate the deposits more rapidly. DMSO, colchicine, and I-Dox anthracyclines have also been tried.

[0007] Anti-SAP antibodies are another therapeutic agent being developed for amyloidosis. EOD001 is a monoclonal antibody that specifically targets amyloid-amyloid AL or AA. WO2015063728 discloses an SAP-Fc antibody fusion for the treatment of amyloidosis.

[0008] Therefore, effective treatments for amyloidosis and amyloid-related diseases are needed. [Overview of the Initiative]

[0009] In one embodiment, the Specified Reference Indicators provide a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a human serum amyloid-P (SAP) component protein ligated to the N-terminus of a first human Fc domain, and the second polypeptide comprises a second human Fc domain but does not contain a human SAP component protein, the first and second Fc domains form a dimer, one of the two Fc domains comprises a knob mutation, and the other Fc domain comprises a whole mutation. In some embodiments, the first polypeptide comprises a knob mutation and the second polypeptide comprises a whole mutation. In some embodiments, the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 10, and the second polypeptide comprises an amino acid sequence shown in SEQ ID NO: 6, or SEQ ID NO: 9. In some embodiments, the first polypeptide comprises a whole mutation and the second polypeptide comprises a knob mutation. In some embodiments, the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16, and the second polypeptide comprises an amino acid sequence shown in SEQ ID NO: 12, or SEQ ID NO: 15. In some embodiments, the human serum amyloid-P component protein includes an amino acid substitution at position N32 or N110 based on SEQ ID NO: 17. In some embodiments, the human serum amyloid-P component protein includes the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the first and second Fc domains include an amino acid substitution at position C226 or C229 according to EU numbering. In some embodiments, the first and second Fc domains include the amino acid substitution C226S or C229S according to EU numbering. In some embodiments, the first and second Fc domains include an amino acid substitution at amino acid position 11 or 14, numbered based on the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first and second Fc domains include a serine residue at amino acid position 11 or 14, numbered based on the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first and second Fc domains include the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first or second Fc domain includes a mutation that reduces FcRn binding.In some embodiments, the fusion protein forms a pentamer.

[0010] In another embodiment, provided herein are fusion proteins comprising a structure represented by the following formula, SAP-hinge1-Fc1-L1-hinge2-Fc2, from the N-terminus to the C-terminus [wherein SAP is human serum amyloid-P (SAP) component protein, hinge1 is the first hinge sequence, Fc1 is the first Fc domain sequence, L1 is the linker, hinge2 is the second hinge sequence, and Fc2 is the second Fc domain sequence]. In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the human serum amyloid-P component protein comprises an amino acid substitution at position N32 or N110 based on SEQ ID NO: 17. In some embodiments, the human serum amyloid-P component protein comprises the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the first and second Fc domains comprise an amino acid substitution at position C226 or C229 according to EU numbering. In some embodiments, the first and second Fc domains include an amino acid substitution C226S or C229S by EU numbering. In some embodiments, the first and second Fc domains include an amino acid substitution at amino acid position 11 or 14, numbered based on the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first and second Fc domains include a serine residue at amino acid position 11 or 14, numbered based on the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first and second Fc domains include the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first or second Fc domain includes a mutation that reduces FcRn binding. In some embodiments, the fusion protein forms a pentamer.

[0011] In another embodiment, provided herein are pharmaceutical compositions comprising the fusion protein described herein.

[0012] In another embodiment, provided herein are nucleic acids encoding the fusion proteins described herein.

[0013] In another embodiment, provided herein are host cells comprising the nucleic acids described herein.

[0014] In another embodiment, provided herein is a method for producing a fusion protein described herein, comprising culturing a host cell containing a nucleic acid encoding the fusion protein under conditions for expression of the fusion protein. In some embodiments, the host cell is a CHO cell or a 293 cell. In some embodiments, the host cell does not glycosylate the SAP component protein.

[0015] In another embodiment, provided herein is a method for treating amyloid disease, comprising administering a fusion protein described herein to an individual in need thereof. In some embodiments, amyloid disease includes systemic amyloidosis.

[0016] In another embodiment, provided herein is a fusion protein having a structure represented by the following formula from the N-terminus to the C-terminus: hinge1-Fc1-L1-hinge2-Fc2 [wherein hinge1 is the first hinge sequence, Fc1 is the first Fc domain sequence, L1 is the linker, hinge2 is the second hinge sequence, Fc2 is the second Fc domain sequence, the first and second Fc domains include the EU numbered amino acid substitution C226S or C229S, and / or the first and second Fc domains include a serine residue at amino acid position 11 or 14, numbered based on the amino acid sequence of Sequence ID No. 18]. [Brief explanation of the drawing]

[0017] [Figure 1] This shows the SAP-scFc structure. [Figure 2] A shows a deglycosylated SAP variant. B shows the knob and hole Fc region. C shows Fc with an FcRN mutation. [Figure 3]A shows the SAP-scFc construct TNT146. B shows the SAP-scFc construct TNT151. C shows the SAP-scFc construct TNT155. D shows the SAP-scFc construct TNT156. [Figure 4] A shows the SAP-Fc construct TNT148. B shows the SAP-Fc construct TNT152. C shows the SAP-Fc construct TNT157. D shows the SAP-Fc construct TNT158. [Figure 5] A shows the SAP-Fc construct TNT147. B shows the SAP-Fc construct TNT159. C shows the SAP-Fc construct TNT160. D shows the SAP-Fc construct TNT161. [Figure 6] A and B show the SDS-PAGE analysis of reduced and non-reduced SAP-Fc after the purification step. The lane labeled "1" shows the reduced sample of the purified product, the lane labeled "2" shows the non-reduced sample of the purified product, and the lane labeled "M" shows the protein marker (ALL BLUE PRECISION PLUS, BIO-RAD (registered trademark), Cat#161-0373). The arrows indicate the monomer (labeled "

Chemical formula

Chemical formula

[0018] Provided herein are SAP-Fc fusion proteins capable of binding to amyloid with high affinity and inducing phagocytosis. In some embodiments, the SAP-Fc fusion protein has one or more modifications in the Fc region that promote dimerization. In some embodiments, the SAP-Fc fusion protein has improved stability compared to the SAP-Fc fusion. In some embodiments, the SAP-Fc fusion protein forms a stable non-covalent pentamer. In some embodiments, the SAP-Fc fusion protein forms a stable non-covalent decamer. In some embodiments, the SAP-Fc fusion protein provided herein exhibits reduced aggregation.

[0019] SAP-Fc fusion protein [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 3-1] [Table 3-2] [Table 3-3] Provided herein is an SAP-Fc fusion protein comprising, from the N-terminus to the C-terminus, an SAP component protein, a first Fc domain containing a first hinge sequence, a linker peptide, and a second Fc domain containing a second hinge sequence.

[0020] In some embodiments, the hinge sequence is an antibody hinge sequence. In some embodiments, the hinge sequence is a human antibody hinge sequence. In some embodiments, the hinge sequence is an IgG1, IgG2, or IgG4 antibody hinge sequence. In some embodiments, the hinge sequence is a human IgG1 antibody hinge sequence. In some embodiments, the hinge sequence is a human IgG1 antibody hinge sequence having one or more amino acid substitutions. In some embodiments, the hinge sequence is a human IgG1 antibody hinge sequence in which one or more cysteine ​​residues are substituted. In some embodiments, the hinge sequence is a human IgG1 antibody hinge sequence in which one or more cysteine ​​residues are substituted with serine.

[0021] In some embodiments, the SAP component protein is a human SAP protein. In some embodiments, the human SAP protein contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, the human SAP protein includes one or more modifications that reduce the glycosylation of the SAP protein. In some embodiments, the one or more modifications that reduce the glycosylation of the SAP protein are selected from the N32S and N110S amino acid substitutions, numbered starting from the N-terminus of the SAP protein. In some embodiments, the human SAP protein includes the N32S and N110S amino acid substitutions, numbered starting from the N-terminus of the SAP protein. In some embodiments, the human SAP protein contains the amino acid sequence of SEQ ID NO: 20.

[0022] In some embodiments, the first Fc domain, including the first hinge sequence, includes a human IgG1 Fc domain. In some embodiments, the first Fc domain, including the first hinge sequence, includes one or more modifications that remove cysteine ​​residues within the hinge region. In some embodiments, the modification that removes cysteine ​​residues within the hinge region improves the stability of the heterodimeric SAP-Fc fusion protein. In some embodiments, the modification prevents higher-order covalent multimerization.

[0023] Cysteine ​​residues, such as cysteine ​​residues within the hinge region of an Fc domain, can cause protein aggregation due to the formation of intermolecular disulfide bonds between cysteine ​​residues located in different Fc regions. Therefore, in some embodiments, the SAP-Fc fusion proteins provided herein include an Fc domain comprising one or more modifications that remove cysteine ​​residues within the hinge region, thus preventing or mitigating aggregation of the SAP-Fc fusion protein due to intermolecular pairing of cysteine ​​residues. In some embodiments, the one or more modifications that remove cysteine ​​residues within the hinge region are located at EU numbering positions C226 or C229. In some embodiments, the substitution is selected from C226S and C229S amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, the Fc domain comprising the first hinge sequence includes C226S and C229S amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, both the first and second Fc domains include a substitution in the hinge region at position C226 or position C229. In some embodiments, the substitution is selected from a serine residue at amino acid position 11, as numbered in SEQ ID NO: 18, and a serine residue at amino acid position 14, as numbered in SEQ ID NO: 18. In some embodiments, the Fc domain including the first hinge sequence includes a serine residue at amino acid position 11 and a serine residue at amino acid position 14, as numbered in SEQ ID NO: 18. In some embodiments, both the first and second Fc domains include a serine residue at amino acid position 11 and a serine residue at amino acid position 14, as numbered in SEQ ID NO: 18. In some embodiments, the Fc domain including the hinge sequence includes the amino acid sequence of SEQ ID NO: 18.

[0024] In some embodiments, the Fc domain containing a hinge sequence includes one or more modifications that reduce FcRn binding affinity. In some embodiments, the one or more modifications that reduce FcRn binding affinity are located at positions I253, H310, or H435, numbered according to the EU numbering scheme. In some embodiments, the modifications include one or more amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, the Fc domain containing a hinge sequence includes amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, the one or more modifications that reduce FcRn binding affinity are located at amino acid positions 38, 95, or 220, numbered according to Sequence ID No. 9. In some embodiments, the modifications include one or more alanine residues at amino acid positions 38, 95, and 220, numbered according to Sequence ID No. 9. In some embodiments, the Fc domain containing the hinge sequence contains alanine residues at amino acid positions 38, 95, and 220, as numbered in SEQ ID NO: 9. In some embodiments, the first and second Fc domains each contain substitutions at amino acid positions 38, 95, or 220, as numbered in SEQ ID NO: 9. In some embodiments, the Fc domain containing the first hinge sequence contains the amino acid sequence of SEQ ID NO: 9, 15, or 21. In some embodiments, the first and second Fc domains each contain substitutions at positions I253, H310, or H435, as numbered according to the EU numbering scheme.

[0025] In some embodiments, the SAP-Fc fusion protein further comprises a linker. In some embodiments, the linker ligates the C-terminus of the first Fc region to the N-terminus of the second Fc region. In some embodiments, the linker ligates the C-terminus of the first Fc region to the hinge region of the second Fc. In some embodiments, the linker peptide comprises a glycine-serine linker sequence. In some embodiments, the linker peptide comprises (G4S) n The sequence is included. In some embodiments, n=1 to 10. In some embodiments, the linker peptide contains the amino acid sequence of SEQ ID NO: 19.

[0026] In some embodiments, the Fc domain, including a hinge sequence, includes one or more modifications that remove cysteine ​​within the hinge region and one or more modifications that reduce FcRn binding affinity. In some embodiments, the one or more modifications that remove cysteine ​​residues within the hinge region are selected from C226S and C229S amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, the one or more modifications that remove cysteine ​​residues within the hinge region are selected from a serine residue at amino acid position 11, numbered according to Sequence ID No. 18, and a serine residue at amino acid position 14, numbered according to Sequence ID No. 18. In some embodiments, the second Fc domain, including a second hinge sequence, includes one or more modifications that reduce FcRn binding affinity. In some embodiments, the one or more modifications that reduce FcRn binding affinity are selected from I253A, H310A, and H435A amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, the Fc domain includes a hinge sequence and contains amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, one or more modifications that reduce FcRn binding affinity are alanine residues at amino acid positions selected from 38, 95, and 220, according to the numbering scheme of Sequence ID No. 9. In some embodiments, the Fc domain including the hinge sequence contains alanine residues at amino acid positions 38, 95, and 220, according to the numbering scheme of Sequence ID No. 9. In some embodiments, the Fc domain including the hinge sequence contains the amino acid sequence of Sequence ID No. 21.

[0027] In some embodiments, the SAP-Fc fusion protein is a single-chain polypeptide. In some embodiments, the SAP-Fc fusion contains a single-chain Fc. In some embodiments, the SAP-Fc fusion protein comprises, from the N-terminus to the C-terminus, an SAP component protein, a first Fc domain containing a first hinge sequence, a linker peptide, and a second Fc domain containing a second hinge sequence. In some embodiments, the SAP component protein contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, the first Fc domain containing the first hinge sequence contains the amino acid sequence of SEQ ID NO: 18. In some embodiments, the linker peptide contains the amino acid sequence of SEQ ID NO: 19. In some embodiments, the second Fc domain containing the second hinge sequence contains the amino acid sequence of SEQ ID NO: 18. In some embodiments, the SAP-Fc fusion protein contains the amino acid sequence of SEQ ID NO: 1.

[0028] In some embodiments, the SAP-Fc fusion protein comprises an SAP component protein having one or more modifications that reduce the glycosylation of the SAP component protein from the N-terminus to the C-terminus, a first Fc domain including a first hinge sequence, a linker peptide, and a second Fc domain including a second hinge sequence. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first Fc domain including the first hinge sequence comprises the amino acid sequence of SEQ ID NO: 18, the linker peptide comprises the amino acid sequence of SEQ ID NO: 19, and the second Fc domain including the second hinge sequence comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the SAP-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

[0029] In some embodiments, the SAP-Fc fusion protein comprises, from the N-terminus to the C-terminus, an SAP component protein, a first Fc domain including a first hinge sequence and one or more modifications that reduce FcRn binding affinity, a linker peptide, and a second Fc domain including a second hinge sequence. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain including a first hinge sequence and one or more modifications that reduce FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21, the linker peptide comprises the amino acid sequence of SEQ ID NO: 19, and the second Fc domain including a second hinge sequence comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the SAP-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 23.

[0030] In some embodiments, the SAP-Fc fusion protein comprises, from the N-terminus to the C-terminus, an SAP component protein, a first Fc domain including a first hinge sequence, a linker peptide, and a second Fc domain including a second hinge sequence and one or more modifications that reduce FcRn binding affinity. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain including the first hinge sequence comprises the amino acid sequence of SEQ ID NO: 18, the linker peptide comprises the amino acid sequence of SEQ ID NO: 19, and the second Fc domain including the second hinge sequence and one or more modifications that reduce FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the SAP-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 24.

[0031] In some embodiments, the SAP-Fc fusion protein comprises, from the N-terminus to the C-terminus, an SAP component protein, a first hinge sequence, and a first Fc domain containing one or more modifications that reduce FcRn binding affinity, a linker peptide, and a second Fc domain containing a second hinge sequence and one or more modifications that reduce FcRn binding affinity. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain containing the first hinge sequence and one or more modifications that reduce FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21, the linker peptide comprises the amino acid sequence of SEQ ID NO: 19, and the second Fc domain containing the second hinge sequence and one or more modifications that reduce FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the SAP-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 3.

[0032] In some embodiments, the SAP-Fc fusion protein comprises, from N-terminus to C-terminus, an SAP component protein containing one or more modifications that reduce the glycosylation of the SAP component protein, a first Fc domain containing one or more modifications that reduce the first hinge sequence and FcRn binding affinity, a linker peptide, and a second Fc domain containing one or more modifications that reduce the second hinge sequence and FcRn binding affinity. In some embodiments, the SAP component protein containing one or more modifications that reduce the glycosylation of the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first Fc domain containing one or more modifications that reduce the first hinge sequence and FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21, the linker peptide comprises the amino acid sequence of SEQ ID NO: 19, and the second Fc domain containing one or more modifications that reduce the second hinge sequence and FcRn binding affinity comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the SAP-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 4.

[0033] Further provided herein is an SAP-Fc fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a human SAP component protein ligated to the N-terminus of a first human Fc domain, and the second polypeptide comprises a second human Fc domain but does not contain a human SAP component protein, the first and second Fc domains form a dimer, one of the two Fc domains contains one or more knob mutations, and the other Fc domain contains one or more hole mutations.

[0034] In some embodiments, the human SAP component protein includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the human SAP component protein includes one or more modifications that reduce the glycosylation of the SAP component protein. In some embodiments, one or more modifications that reduce the glycosylation of the SAP component protein are selected from the N32S and N110S amino acid substitutions, numbered starting from the N-terminus of the SAP component protein. In some embodiments, the human SAP component protein includes the amino acid substitutions N32S and N110S, numbered starting from the N-terminus of the SAP component protein. In some embodiments, the human SAP component protein includes the amino acid sequence of SEQ ID NO: 20.

[0035] In some embodiments, the SAP-Fc fusion protein includes one or more modifications that promote heterodimerization of the Fc domain. In some embodiments, one of the two Fc domains includes one or more knob mutations, and the other Fc domain includes one or more hole mutations, and the knob and hole mutations promote the association of the first and second Fc domains, for example, to form a dimer. In some embodiments, the Fc domain containing one or more knob mutations includes the T366W amino acid substitution, and the Fc domain containing one or more hole mutations includes the amino acid substitutions T366S, L368A, and Y407V, and the numbering is based on the EU numbering system. In some embodiments, the Fc domain containing one or more knob mutations includes the T366W and Y349C amino acid substitutions, and the Fc domain containing one or more hole mutations includes the amino acid substitutions S354C, T366S, L368A, and Y407V, and the numbering is based on the EU numbering system. In some embodiments, an Fc domain containing one or more knob mutations includes the amino acid substitutions T366W and Y349C, and an Fc domain containing one or more hole mutations includes the amino acid substitutions E356C, T366S, L368A and Y407V, with numbering based on the EU numbering system. In some embodiments, an Fc domain containing one or more knob mutations includes the amino acid substitutions R409D and K370E, and an Fc domain containing one or more hole mutations includes the amino acid substitutions D399K and E357K, with numbering based on the EU numbering system. In some embodiments, an Fc domain containing one or more knob mutations includes the amino acid substitutions T366W, R409D and K370E, and an Fc domain containing one or more hole mutations includes the amino acid substitutions T366S, L368A, Y407V, D399K and E357K, with numbering based on the EU numbering system. In some embodiments, one of the two Fc domains contains the amino acid substitutions Y349C and T366W, and the other Fc domain contains the amino acid substitutions S354C, T366S, L368A, and Y407V, and the numbering is based on the EU numbering system. In some embodiments, one of the two Fc domains contains the T366W amino acid substitution, and the other Fc domain contains the amino acid substitutions T366S, L368A, and Y407V, and the numbering is based on the EU numbering system.In some embodiments, one of the two Fc domains comprises amino acid substitutions Y349C, T366W, R409D, and K370E, and the other Fc domain comprises amino acid substitutions S354C, T366S, L368A, Y407V, D399K, and E357K, with numbering based on the EU numbering scheme. Further or alternative “knob-in-hole” techniques known in the art, such as the technique described in EP1870459A1, may be used in the SAP-Fc fusion protein of this disclosure. In some embodiments, the Fc domain comprising one or more knob mutations comprises a tryptophan residue at amino acid position 151, as numbered in SEQ ID NO: 12, and the Fc domain comprising one or more hole mutations comprises a serine residue at amino acid position 151, an alanine residue at amino acid position 153, and a valine residue at amino acid position 192, with numbering based on SEQ ID NO: 6.

[0036] In some embodiments, the SAP-fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising a human serum amyloid-P (SAP) protein ligated to the N-terminus of a first human Fc domain, and the second polypeptide comprising a second human Fc domain but not a human SAP component protein, the first and second Fc domains forming a dimer, one of the two Fc domains comprising a knob mutation and the other Fc domain comprising a whole mutation. In some embodiments, the first human Fc domain comprises a knob mutation. In some embodiments, the knob mutation is a T366W amino acid substitution numbered according to the EU numbering scheme. In some embodiments, the knob mutation is a tryptophan residue at amino acid position 151 according to the numbering of SEQ ID NO: 12. In some embodiments, the first human Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first human Fc domain further comprises one or more modifications that reduce FcRn binding affinity. In some embodiments, one or more modifications that reduce FcRn binding affinity are selected from the I253A, H310A, and H435A amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, the first human Fc domain includes the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, one or more modifications that reduce FcRn binding affinity are alanine residues at amino acid positions 38, 95, and 220, numbered according to Sequence ID No. 15. In some embodiments, the first human Fc domain includes alanine residues at amino acid positions 38, 95, and 220, numbered according to Sequence ID No. 15. In some embodiments, the first human Fc domain includes the amino acid sequence of Sequence ID No. 15.

[0037] In some embodiments, if the first Fc domain contains a knob mutation, the second human Fc domain contains one or more hole mutations. In some embodiments, the one or more hole mutations include the amino acid substitutions T366S, L368A, and Y407V, numbered according to the EU numbering scheme. In some embodiments, the one or more hole mutations include a serine residue at amino acid position 151, an alanine residue at amino acid position 153, and a valine residue at amino acid position 192, numbered according to SEQ ID NO: 6. In some embodiments, the second human Fc domain contains the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second human Fc domain further includes one or more modifications that reduce FcRn binding affinity. In some embodiments, the one or more modifications that reduce FcRn binding affinity are selected from the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, the second human Fc domain contains the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, one or more modifications that reduce FcRn binding are alanine residues at amino acid positions selected from 38, 95, and 220, as numbered in Sequence ID No. 9. In some embodiments, the second human Fc domain contains alanine residues at amino acid positions 38, 95, and 220, as numbered in Sequence ID No. 9. In some embodiments, the second human Fc domain contains the amino acid sequence of Sequence ID No. 9.

[0038] In some embodiments, the first human Fc domain includes one or more hole mutations. In some embodiments, the one or more hole mutations include the amino acid substitutions T366S, L368A, and Y407V, numbered according to the EU numbering scheme. In some embodiments, the one or more hole mutations include a serine residue at amino acid position 151, an alanine residue at amino acid position 153, and a valine residue at amino acid position 192, numbered according to SEQ ID NO: 6. In some embodiments, the first human Fc domain includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first human Fc domain further includes one or more modifications that reduce FcRn binding affinity. In some embodiments, the one or more modifications that reduce FcRn binding affinity are selected from the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, the first human Fc domain includes the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, one or more modifications that reduce FcRn binding are alanine residues at amino acid positions selected from 38, 95, and 220, as numbered in Sequence ID No. 9. In some embodiments, the first human Fc domain contains alanine residues at amino acid positions 38, 95, and 220, as numbered in Sequence ID No. 9. In some embodiments, the first human Fc domain contains the amino acid sequence of Sequence ID No. 9.

[0039] In some embodiments, if the first Fc domain contains a whole mutation, the second human Fc domain contains a knob mutation. In some embodiments, the knob mutation is the T366W amino acid substitution, numbered according to the EU numbering scheme. In some embodiments, the knob mutation is the tryptophan residue at amino acid position 151, numbered according to SEQ ID NO: 12. In some embodiments, the second human Fc domain contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second human Fc domain further includes one or more modifications that reduce FcRn binding affinity. In some embodiments, one or more modifications that reduce FcRn binding affinity are selected from the I253A, H310A, and H435A amino acid substitutions, numbered according to the EU numbering scheme. In some embodiments, the second human Fc domain contains the amino acid substitutions I253A, H310A, and H435A, numbered according to the EU numbering scheme. In some embodiments, one or more modifications that reduce FcRn binding affinity are alanine residues at amino acid positions selected from 38, 95, and 220, numbered according to SEQ ID NO: 15. In some embodiments, the second human Fc domain contains alanine residues at amino acid positions 38, 95, and 220, as numbered in Sequence ID No. 15. In some embodiments, the second human Fc domain contains the amino acid sequence of Sequence ID No. 15.

[0040] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein ligated to the N-terminus of a first human Fc domain, and the second polypeptide comprising a second human Fc domain but not a human SAP component protein, the first and second Fc domains forming a dimer, the first Fc domain comprising one or more knob mutations, and the second Fc domain comprising one or more hole mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 12, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0041] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein having one or more modifications that reduce the glycosylation of the SAP component protein, the SAP component protein being ligated to the N-terminus of the first human Fc domain, the second polypeptide comprising a second human Fc domain but not the human SAP component protein, the first and second Fc domains forming a dimer, the first Fc domain comprising one or more knob mutations, and the second Fc domain comprising one or more hole mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 12, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 7, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0042] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein ligated to the N-terminus of a first human Fc domain containing one or more modifications that reduce FcRn binding affinity, and the second polypeptide comprising a second human Fc domain containing one or more modifications that reduce FcRn binding affinity but not a human SAP component protein, the first and second Fc domains form a dimer, the first Fc domain contains one or more knob mutations, and the second Fc domain contains one or more hole mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 15, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 9.

[0043] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein having one or more modifications that reduce the glycosylation of the SAP component protein, the SAP component protein being linked to the N-terminus of a first human Fc domain having one or more modifications that reduce FcRn binding affinity, the second polypeptide comprising a second human Fc domain having one or more modifications that reduce FcRn binding affinity but not the human SAP component protein, the first and second Fc domains form a dimer, the first Fc domain comprising one or more knob mutations, and the second Fc domain comprising one or more hole mutations. In some embodiments, the SAP component protein comprises one or more modifications that remove a glycosylation site. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first human Fc domain comprises the amino acid sequence of SEQ ID NO: 15, and the second human Fc domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 9.

[0044] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein ligated to the N-terminus of a first human Fc domain, and the second polypeptide comprising a second human Fc domain but not a human SAP component protein, the first and second Fc domains forming a dimer, the first Fc domain comprising one or more hole mutations, and the second Fc domain comprising one or more knob mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 6, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

[0045] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein having one or more modifications that reduce the glycosylation of the SAP component protein, the SAP component protein being ligated to the N-terminus of the first human Fc domain, the second polypeptide comprising a second human Fc domain but not the human SAP component protein, the first and second Fc domains forming a dimer, the first Fc domain comprising one or more hole mutations, and the second Fc domain comprising one or more knob mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 6, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

[0046] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein ligated to the N-terminus of a first human Fc domain containing one or more modifications that reduce FcRn binding affinity, and the second polypeptide comprising a second human Fc domain containing one or more modifications that reduce FcRn binding affinity but not a human SAP component protein, the first and second Fc domains form a dimer, the first Fc domain contains one or more hole mutations, and the second Fc domain contains one or more knob mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 17, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 9, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 14, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 15.

[0047] In some embodiments, the SAP-Fc fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide comprising an SAP component protein having one or more modifications that reduce the glycosylation of the SAP component protein, the SAP component protein being ligated to the N-terminus of a first human Fc domain having one or more modifications that reduce FcRn binding affinity, the second polypeptide comprising a second human Fc domain having one or more modifications that reduce FcRn binding affinity but not the human SAP component protein, the first and second Fc domains form a dimer, the first Fc domain comprises one or more hole mutations, and the second Fc domain comprises one or more knob mutations. In some embodiments, the SAP component protein comprises the amino acid sequence of SEQ ID NO: 20, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 9, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 16, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 15.

[0048] In some embodiments, the SAP-Fc fusion protein described herein binds to amyloid deposits or fibrils. In some embodiments, the SAP-Fc fusion protein binds to one or more amyloidogenic peptides in the amyloid. In some embodiments, the amyloid bound to the SAP-Fc fusion protein includes amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid bound to the SAP-Fc fusion protein includes amyloidogenic forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. The amyloidogenic peptide bound to the SAP-Fc fusion protein 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, amyloid is part of the pathology of the disease.

[0049] In some embodiments, the SAP-Fc fusion protein promotes phagocytosis of amyloid deposits. In some embodiments, the SAP-Fc fusion protein promotes macrophage-mediated phagocytosis.

[0050] In some embodiments, the SAP-Fc fusion protein described herein is nonglycosylated and binds to amyloid deposits or fibrils. In some embodiments, the nonglycosylated SAP-Fc fusion protein binds to one or more amyloidogenic peptides in amyloid. In some embodiments, the amyloid bound to the nonglycosylated SAP-Fc fusion protein includes amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, amyloid bound to a non-glycosylated SAP-Fc fusion protein includes amyloidogenic forms such as immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), and prion protein (prior The amyloidogenic peptides include protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. The amyloidogenic peptides bound to the non-glycosylated SAP-Fc fusion protein may be proteins, protein fragments, or protein domains. In some embodiments, the amyloid deposit or amyloid fibril contains recombinant amyloidogenic protein. In some embodiments, amyloid is part of the pathology of the disease. In some embodiments, the non-glycosylated SAP-Fc fusion protein promotes phagocytosis of amyloid deposits. In some embodiments, the non-glycosylated SAP-Fc fusion protein promotes macrophage-mediated phagocytosis.

[0051] In some embodiments, the SAP-Fc fusion protein described herein is glycosylated and binds to amyloid deposits or fibrils. In some embodiments, the glycosylated SAP-Fc fusion protein binds to one or more amyloidogenic peptides in amyloid. In some embodiments, the amyloid bound to the glycosylated SAP-Fc fusion protein is amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, amyloid bound to glycosylated SAP-Fc fusion proteins includes amyloidogenic forms such as immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), and prion protein (prior The amyloidogenic peptides include protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. The amyloidogenic peptides bound to the glycosylated SAP-Fc fusion protein may be proteins, protein fragments, or protein domains. In some embodiments, the amyloid deposit or amyloid fibril contains recombinant amyloidogenic protein. In some embodiments, amyloid is part of the pathology of the disease. In some embodiments, the glycosylated SAP-Fc fusion protein promotes phagocytosis of amyloid deposits. In some embodiments, the glycosylated SAP-Fc fusion protein promotes macrophage-mediated phagocytosis.

[0052] It is known that the glycosylation patterns differ between proteins produced in non-human cells (e.g., CHO cells) and proteins produced in human cells (e.g., HEK293 cells). In some embodiments, the SAP-Fc fusion protein described herein is produced in human cells, e.g., HEK293 cells, and binds to amyloid deposits or fibrils. In some embodiments, the SAP-Fc fusion protein is produced in human cells, e.g., HEK293 cells, and binds to one or more amyloidogenic peptides in amyloid. In some embodiments, the amyloid to which the SAP-Fc fusion protein produced in human cells, e.g., HEK293 cells, binds includes amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, amyloid bound to SAP-Fc fusion protein produced in human cells, such as HEK293 cells, is amyloidogenic immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), and prion protein (prior This includes protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. The amyloidogenic peptides bound to the SAP-Fc fusion protein produced in human cells, e.g., HEK293 cells, may be proteins, protein fragments, or protein domains. In some embodiments, the amyloid deposit or amyloid fibril contains recombinant amyloidogenic proteins.In some embodiments, amyloid is part of the pathology of the disease. In some embodiments, the SAP-Fc fusion protein produced in human cells, e.g., HEK293 cells, promotes phagocytosis of amyloid deposits. In some embodiments, the SAP-Fc fusion protein produced in human cells, e.g., HEK293 cells, promotes macrophage-mediated phagocytosis. In some embodiments, the SAP-Fc fusion protein described herein is produced in non-human cells, e.g., CHO cells, and binds to amyloid deposits or fibrils. In some embodiments, the SAP-Fc fusion protein produced in non-human cells, e.g., CHO cells, binds to one or more amyloidogenic peptides in amyloid. In some embodiments, the amyloid bound to the SAP-Fc fusion protein produced in non-human cells, such as CHO cells, includes amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, amyloid bound to SAP-Fc fusion proteins produced in non-human cells, such as CHO cells, is amyloidogenic immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), and prion protein (prior This includes protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. Amyloidogenic peptides bound to SAP-Fc fusion proteins produced in non-human cells, such as CHO cells, may be proteins, protein fragments, or protein domains.In some embodiments, amyloid deposits or amyloid fibrils contain recombinant amyloidogenic proteins. In some embodiments, amyloid is part of the pathology of the disease. In some embodiments, SAP-Fc fusion proteins produced in non-human cells, e.g., CHO cells, promote phagocytosis of amyloid deposits. In some embodiments, SAP-Fc fusion proteins produced in non-human cells, e.g., CHO cells, promote macrophage-mediated phagocytosis.

[0053] In some embodiments, the binding affinity of the SAP-Fc fusion protein described herein to amyloid deposits or fibrils is not affected by the glycosylation state of the SAP-Fc fusion protein. In some embodiments, the binding affinity of the SAP-Fc fusion protein to one or more amyloidogenic peptides in amyloid is not affected by the glycosylation state of the SAP-Fc fusion protein. In some embodiments, the amyloid bound to the SAP-Fc fusion protein includes amyloidogenic λ6 variable domain protein (Vλ6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloidogenic Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid bound to the SAP-Fc fusion protein includes amyloidogenic forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, Alλ1, or other amyloidogenic peptides. The amyloidogenic peptide bound to the SAP-Fc fusion protein 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, amyloid is part of the pathology of the disease. In some embodiments, the activity of the SAP-Fc fusion protein described herein in promoting phagocytosis of amyloid deposits is not affected by the glycosylation state of the SAP-Fc fusion protein.In some embodiments, the activity of the SAP-Fc fusion protein described herein in promoting macrophage-mediated phagocytosis is not affected by the glycosylation state of the SAP-Fc fusion protein.

[0054] In some embodiments, the SAP-Fc fusion protein provided herein comprises two Fc polypeptides and one SAP polypeptide. In some embodiments, the SAP-Fc fusion protein forms a stable non-covalent heterodimer. In some embodiments, the SAP-Fc fusion protein does not contain one or more cysteine ​​molecules located in the Fc hinge region. In some embodiments, the SAP-Fc fusion protein exhibits reduced aggregation.

[0055] In some embodiments, the dimeric SAP-Fc fusion proteins provided herein form stable non-covalent pentamers. In some embodiments, the pentamers are stable for 1, 2, 3, 4, 5, 24 hours or longer. In some embodiments, the pentamers are stable in a size exclusion chromatography column. In some embodiments, the pentamers have a molecular weight of approximately 375 kDa.

[0056] In some embodiments, the dimeric SAP-Fc fusion proteins provided herein form a stable non-covalent decamer. In some embodiments, the decamer is stable for 1, 2, 3, 4, 5, 24 hours or longer. In some embodiments, the decamer is stable in a size exclusion chromatography column. In some embodiments, the decamer has a molecular weight of approximately 750 kDa for the SAP-Fc decamer.

[0057] Further provided herein are pharmaceutical compositions comprising an SAP-Fc fusion protein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be essentially free of any preservatives or other carriers, excipients, or stabilizers. Alternatively, the pharmaceutical composition may optionally contain one or more preservatives, e.g., antimicrobial agents, pharmaceutically acceptable carriers, excipients, or stabilizers as described elsewhere herein, provided that they do not adversely affect the physicochemical stability of the SAP-Fc fusion. Examples of acceptable carriers, excipients, and stabilizers include, but are not limited to, additional buffers, cosolvents, surfactants, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes), and biodegradable polymers such as polyesters. Detailed discussions regarding the formulation and selection of pharmaceutically acceptable carriers, stabilizers, and isomolytes can be found in Remington's Pharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pa., 1990), which is incorporated herein by reference.

[0058] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration using a sterile filtration membrane.

[0059] The pharmaceutical compositions of the present invention are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Liquids or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. pH may be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0060] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the rapid preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for easy syringe handling. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0061] Sterile injectable formulations can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination of the ingredients listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable formulations, the preparation methods are vacuum drying and freeze-drying, which produce powders from their pre-sterile filtered solutions with the active ingredient and any additional desired ingredients added.

[0062] Treatment method Further provided herein are methods for treating amyloid-related disorders, comprising administering the SAP-Fc fusion protein disclosed herein to an individual.

[0063] In some embodiments, the SAP-Fc fusion protein binds to amyloid deposits. In some embodiments, the amyloid deposits may contribute to the pathology of the disease. In other embodiments, the amyloid deposits may suggest amyloidosis or amyloid-related disease in an individual. In some embodiments, the SAP-Fc fusion protein binds to amyloid in an individual having amyloidosis. In some embodiments, the amyloidosis is localized to a specific tissue or organ system, such as the liver, heart, or central nervous system.

[0064] In other embodiments, amyloidosis is systemic amyloidosis. In some embodiments, amyloidosis is familial amyloidosis. In other embodiments, amyloidosis is sporadic amyloidosis. In some embodiments, amyloidosis or amyloid-related disease is AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis in type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch type amyloidosis, cerebral beta-amyloid angiopathy, cavernous encephalopathy, thyroid tumors, Parkinson's disease, Lewy body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndromes, cavernous encephalopathy, reactive chronic inflammation, thyroid tumors, myeloma, or other forms of cancer. In some embodiments, the SAP-Fc fusion protein binds to amyloid associated with normal aging. In other embodiments, SAP-Fc fusion proteins are used for the diagnosis, treatment, or prognosis of amyloidosis or amyloid-related diseases in subjects.

[0065] In some embodiments, the individual or subject being treated is an animal, such as a mammal. In some embodiments, the mammal is a dog, cat, horse, cow, dairy cow, pig, sheep, lamb, goat, primate, mouse, rat, or human. In some embodiments, the individual or subject is a human.

[0066] Host cells, vector, manufacturing method In some embodiments, what is provided herein is a nucleic acid encoding an SAP-Fc fusion protein. In some embodiments, the nucleic acid is contained within a vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is for prokaryotic expression. In some embodiments, the vector is for eukaryotic expression. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector includes a promoter that facilitates the transcription of the SAP-Fc fusion protein. In some embodiments, the promoter is a constitutive or inducible promoter.

[0067] In some embodiments, the SAP-Fc fusion protein is produced by culturing host cells transformed with nucleic acids, preferably expression vectors, containing nucleic acids encoding the polypeptide construct (e.g., Fc manifold, linker, and fusion partner), under suitable conditions that induce or result in the expression of the polypeptide construct. In some embodiments, the conditions suitable for expression vary depending on the selected expression vector and host cell. In some embodiments, a variety of suitable host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines for which applications are found in this disclosure are listed in the ATCC® cell line catalog, available from the American Type Culture Collection. In some embodiments, the SAP-Fc fusion protein of this disclosure is expressed in cells optimized not to glycosylate the protein expressed in the cells by either genetic engineering of the cell line or modification of cell culture conditions, e.g., addition of kifunensin, or use of a naturally non-glycosylating host, e.g., a prokaryote (e.g., E. coli), and in some cases, modification of the glycosylation sequence in Fc is unnecessary.

[0068] In some embodiments, mammalian cells are used as host cells to produce the polypeptides of the Disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells to produce the polypeptides of the Disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537), E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608).

[0069] Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure the correct modification and processing of the expressed polypeptide. In some embodiments, host cells that do not produce the α2,3-linked sialic acid SAP moiety are selected. In some embodiments, host cells that produce the α2,6-linked sialic acid SAP moiety at levels equivalent to wild-type human SAP are selected.

[0070] Once the vector is introduced into host cells for protein production, the host cells are cultured in a conventional nutrient medium that has been appropriately modified for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence.

[0071] In some embodiments, polypeptide constructs, such as polypeptides containing SAP-Fc fusion proteins, are expressed in mammalian expression systems, including systems that introduce expression constructs into mammalian cells using viruses such as retroviruses or adenoviruses. In some embodiments, human, mouse, rat, hamster, or primate cells are utilized. Suitable cells also include, but are not limited to, Jurkat T cells, NIH3T3, CHO, COS, and 293 cells, as well as other known research cells. Alternatively, in some embodiments, proteins are expressed in bacterial cells. Bacterial expression systems are well known in the art, including Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, polypeptide constructs containing Fc variants are produced in insect cells, such as, but not limited to, Sf9 and Sf21 cells, or in yeast cells, such as, but not limited to, organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, a cell-free translation system is used to produce polypeptide constructs containing Fc variants in vitro. In vitro translation systems derived from prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocytes) cells are available and, in some embodiments, are selected based on the expression level and functional characteristics of the protein of interest. For example, as will be recognized to those skilled in the art, in vitro translation is required for some display techniques, such as ribosome display. In addition, in some embodiments, SAP-Fc fusion protein (preotsins) variants are produced by chemical synthesis methods, such as, but not limited to, liquid-phase peptide synthesis and solid-phase peptide synthesis. In the case of in vitro transcription using a non-glycosylating system such as a bacterial extract, Fc is not glycosylated even if a native glycosylation site is present, and therefore, inactivation of Fc is obtained.

[0072] In some embodiments, polypeptide constructs include unnatural amino acids, amino acid analogs, amino acid mimes, or any combination thereof that function similarly to naturally occurring amino acids. Naturally encoded amino acids generally refer to 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolidine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, e.g., a hydrogen-bonded carbon (an a carbon), a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0073] Protein production, recovery, and purification In some embodiments, the host cells used to produce the polypeptides of this disclosure are grown in a medium suitable for culturing selected host cells. Examples of media suitable for mammalian host cells include minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Expi293® expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of media suitable for bacterial host cells include Luria broth (LB) with necessary nutritional supplements, such as selectors, such as ampicillin. In some embodiments, the host cells are cultured at a suitable temperature, e.g., about 20°C to about 39°C, e.g., about 25°C to about 37°C, preferably 37°C, at a CO2 level, e.g., about 5% to 10%. In some embodiments, the pH of the medium is mainly depending on the host organism, e.g., pH about 6.8 to pH 7.4, e.g., pH 7.0. When an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for promoter activation.

[0074] In some embodiments, protein recovery involves disrupting host cells, for example, by osmotic shock, sonication, or lysis. Once the cells are disrupted, cellular debris is removed by centrifugation or filtration. The protein may then be further purified. In some embodiments, the polypeptides of the present disclosure are purified by various methods of protein purification, e.g., chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, solubility difference, or any other standard technique for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and using affinity columns, e.g., Protein A columns (e.g., POROS Protein A chromatography) and chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the polypeptide is conjugated to a marker sequence, e.g., a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His6-tag), which can be conjugated to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein, may be used.

[0075] In some embodiments, polypeptide constructs comprising the polypeptides of the Disclosure, such as SAP-Fc fusion proteins, are produced by cells of a subject (e.g., human) by administering a vector containing a nucleic acid molecule encoding the polypeptides of the Disclosure, such as a viral vector (e.g., retroviral vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector, e.g., modified vaccinia ankara (MVA)), adeno-associated virus vector, and alphavirus vector), for example in the context of gene therapy. Once the vector enters the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it can be used for the expression of the polypeptides disclosed herein. In some cases, the polypeptides are secreted from the cells. In some embodiments, if the treatment of a disease or disorder is the desired outcome, no further action is required. In some embodiments, if protein recovery is desired, blood is taken from the subject and the proteins are purified from the blood by various methods.

[0076] kit Further provided herein are kits comprising SAP-Fc fusion proteins and instructions for use. In some embodiments, the kit includes instructions for use in accordance with any of the methods described herein. In some embodiments, the kit includes instructions for treating amyloid disorders. In some embodiments, it includes instructions for treating systemic amyloid disorders. In some embodiments, the kit includes instructions for treating AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis in type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch type amyloidosis, cerebral beta-amyloid angiopathy, cavernous encephalopathy, thyroid tumors, Parkinson's disease, Lewy body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndromes, cavernous encephalopathy, reactive chronic inflammation, thyroid tumors, myeloma, or other forms of cancer.

[0077] The kit may also include SAP-Fc fusion proteins in containers such as vials, bags, pumps, or syringes. In some embodiments, the SAP-Fc fusion proteins are included in the pharmaceutical composition. Embodiment 1. A fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a human serum amyloid-P (SAP) component protein linked to the N-terminus of a first human Fc domain, the second polypeptide comprises a second human Fc domain but does not contain a human SAP component protein, the first and second Fc domains form a dimer, one of the two Fc domains comprises a knob mutation, and the other Fc domain comprises a hole mutation. 2. The fusion protein according to Embodiment 1, wherein the first polypeptide comprises a knob mutation and the second polypeptide comprises a whole mutation. 3. The fusion protein according to Embodiment 2, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 10, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, or SEQ ID NO: 9. 4. The fusion protein according to Embodiment 1, wherein the fusion protein comprises the first polypeptide and second polypeptide sequences of TNT148, TNT152, TNT157, TNT158, TNT147, TNT159, TNT160, or TNT161 as listed in Table 2. 5. The fusion protein according to Embodiment 1, wherein the Fc domain of the first polypeptide contains a hole mutation, and the Fc domain of the second polypeptide contains a knob mutation. 6. The fusion protein according to Embodiment 5, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12, or SEQ ID NO: 15. 7. A fusion protein containing the structure represented by the following formula, SAP-hinge1-Fc1-L1-hinge2-Fc2 [wherein SAP is the human serum amyloid-P (SAP) component protein, hinge1 is the first hinge sequence, Fc1 is the first Fc domain sequence, L1 is the linker, hinge2 is the second hinge sequence, and Fc2 is the second Fc domain sequence], extending from the N-terminus to the C-terminus. 8. The fusion protein according to Embodiment 7, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23, or SEQ ID NO: 24. 9. The fusion protein according to Embodiment 7, wherein the fusion protein comprises the polypeptide sequence of TNT146, TNT151, TNT155, TNT156, TNT170, or TNT171. 10. The fusion protein according to Embodiment 1 or Embodiment 7, wherein the human serum amyloid-P component protein includes an amino acid substitution at position N32 or N110 based on SEQ ID NO: 17. 11. The fusion protein according to Embodiment 10, wherein the human serum amyloid-P component protein comprises the amino acid sequence shown in SEQ ID NO: 20. 12. The fusion protein according to Embodiment 1 or Embodiment 7, wherein the first and second Fc domains include an amino acid substitution at EU numbering position C226 or C229. 13. The fusion protein according to Embodiment 12, wherein the first and second Fc domains include an amino acid substitution C226S or C229S according to EU numbering. 14. The fusion protein according to Embodiment 1 or Embodiment 7, wherein the first and second Fc domains include an amino acid substitution at amino acid position 11 or 14, numbered based on the amino acid sequence of Sequence ID No. 18. 15. The fusion protein according to Embodiment 14, wherein the first and second Fc domains contain a serine residue at amino acid position 11 or 14, numbered based on the amino acid sequence of Sequence ID No. 18. 16. The fusion protein according to any one of Embodiments 12 to 15, wherein the first or second Fc domain comprises the amino acid sequence shown in SEQ ID NO: 18. 17. The fusion protein according to Embodiment 1 or Embodiment 7, wherein the first and / or second Fc domain contains a mutation that reduces FcRn binding. 18. The fusion protein according to any one of Embodiments 1 to 17, wherein the fusion protein forms a pentamer. 19. A pharmaceutical composition comprising the fusion protein described in any one of Embodiments 1 to 18. 20. A nucleic acid encoding the fusion protein described in any one of Embodiments 1 to 18. 21. A host cell containing the nucleic acid described in Embodiment 20. 22. A method for producing a fusion protein according to any one of Embodiments 1 to 18, comprising culturing a host cell containing a nucleic acid encoding the fusion protein under conditions for expressing the fusion protein. 23. The method according to Embodiment 22, wherein the host cell is a CHO cell or a 293 cell. 24. The method according to Embodiment 22 or Embodiment 23, wherein the host cell does not glycosylate the SAP component protein. 25. A method for treating amyloid disease, comprising administering a fusion protein described in any one of Embodiments 1 to 18 to an individual in need thereof. 26. The method according to Embodiment 25, wherein the amyloid disease includes systemic amyloidosis. 27. A fusion protein having a structure represented by the following formula, from the N-terminus to the C-terminus: hinge1-Fc1-L1-hinge2-Fc2 [wherein hinge1 is the first hinge sequence, Fc1 is the first Fc domain sequence, L1 is the linker, hinge2 is the second hinge sequence, Fc2 is the second Fc domain sequence, the first and second Fc domains contain the amino acid substitution C226S or C229S according to EU numbering, and / or the first and second Fc domains contain a serine residue at amino acid position 11 or 14, numbered based on the amino acid sequence of Sequence ID No. 18]. 28. The fusion protein according to Embodiment 27, wherein the fusion protein further comprises a human serum amyloid-P (SAP) component protein at the N-terminus of the fusion protein. 1. A fusion protein having a structure represented by the following formula, SAP-Fc1-L1-Fc2, from the N-terminus to the C-terminus [wherein Fc1 is a first Fc domain sequence containing a hinge-CH2-CH3, L1 is a linker, Fc2 is a second Fc domain sequence containing a hinge-CH2-CH3, SAP is a human serum amyloid P (SAP) component protein, and Fc1 and Fc2 include amino acid substitutions at EU numbering positions C226 and / or C229]. 2A. The fusion protein according to Embodiment 1A, wherein Fc1 and Fc2 include amino acid substitutions at positions C226 and C229 according to EU numbering. 3A. The fusion protein according to Embodiment 1A or Embodiment 2A, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23, or SEQ ID NO: 24. 4A. The fusion protein according to Embodiment 3A, wherein the fusion protein includes the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23, or SEQ ID NO: 24, which lacks a C-terminal lysine. 5A. The fusion protein according to Embodiment 1A or Embodiment 2A, wherein the fusion protein comprises the polypeptide sequence of TNT146, TNT151, TNT155, TNT156, TNT170, or TNT171. 6A. A fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a human serum amyloid-P (SAP) component protein linked to the N-terminus of a first human Fc domain (Fc1), and the second polypeptide comprises a second human Fc domain (Fc2) but does not contain a human SAP component protein, and the first and second Fc domains form a dimer, with one of the two Fc domains comprising a knob mutation and the other Fc domain comprising a hole mutation. 7A. The fusion protein according to Embodiment 6A, wherein the first polypeptide comprises a knob mutation and the second polypeptide comprises a whole mutation. 8A. The fusion protein according to Embodiment 7A, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 10, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, or SEQ ID NO: 9. 9A. The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 8, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 9; or The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 10, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6. The fusion protein described in Embodiment 8A. 10A. The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, with or without C-terminal lysine; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, with or without C-terminal lysine; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7, which may or may not have a C-terminal lysine; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, which may or may not have a C-terminal lysine; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 8, which may or may not have a C-terminal lysine; and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 9, which may or may not have a C-terminal lysine; or The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 10, which may or may not have a C-terminal lysine, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, which may or may not have a C-terminal lysine. The fusion protein described in Embodiment 8A. 11A. The fusion protein according to Embodiment 6A, wherein the fusion protein comprises the first and second polypeptide sequences of TNT148, TNT152, TNT157, TNT158, TNT147, TNT159, TNT160, or TNT161 as listed in Table 2. 12A. The fusion protein according to Embodiment 6A, wherein the Fc domain of the first polypeptide contains a hole mutation and the Fc domain of the second polypeptide contains a knob mutation. 13A. The fusion protein according to Embodiment 6A, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12, or SEQ ID NO: 15. 14A. The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15; or The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15. The fusion protein described in Embodiment 13A. 15A. The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, with or without C-terminal lysine; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12, with or without C-terminal lysine; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13, which may or may not have a C-terminal lysine; and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12, which may or may not have a C-terminal lysine; The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14, which may or may not have a C-terminal lysine; and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15, which may or may not have a C-terminal lysine; or The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16, which may or may not have a C-terminal lysine, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15, which may or may not have a C-terminal lysine. The fusion protein described in Embodiment 13A. 16A. The fusion protein according to Embodiment 1A or Embodiment 6A, wherein the human SAP is a wild-type SAP that optionally contains the amino acid sequence shown in Sequence ID No. 17. 17A. The fusion protein according to Embodiment 1A or Embodiment 6A, wherein the human SAP comprises an amino acid substitution at position N32 or N110 based on Sequence ID No. 17. 18A. The fusion protein according to Embodiment 17A, wherein the human SAP comprises the amino acid sequence shown in Sequence ID No. 20. 19A. The fusion protein according to Embodiment 6A or any one of Claims 16A to 18A, wherein Fc1 and Fc2 include amino acid substitutions at EU numbering positions C226 and / or C229. 20A. The fusion protein according to Embodiment 19A, wherein Fc1 and Fc2 include amino acid substitutions at positions C226 and C229 according to EU numbering. 21A. A fusion protein according to any one of Embodiments 1A, 2A, 6A, or 16A-20A, wherein Fc1 and Fc2 include the EU-numbered amino acid substitutions C226S and / or C229S. 22A. The fusion protein according to Embodiment 21A, wherein Fc1 and Fc2 include amino acid substitutions C226S and C229S according to EU numbering. 23A. A fusion protein according to any one of Embodiments 1A, 2A, 6A, or 15A-17A, wherein Fc1 and / or Fc2 are numbered based on the amino acid sequence of Sequence ID No. 18 and include amino acid substitutions at amino acid positions 11 and / or 14. 24A. The fusion protein according to Embodiment 23A, wherein Fc1 and / or Fc2 are numbered based on the amino acid sequence of Sequence ID No. 18 and contain serine residues at amino acid positions 11 and / or 14. 25A. The fusion protein according to Embodiment 23A or Embodiment 24A, wherein Fc1 and / or Fc2 comprises the amino acid sequence shown in Sequence ID No. 18. 26A. The fusion protein according to Embodiments 1A, 2A, 6A, or 16A-18A, wherein the first and / or second Fc domain contains a mutation that reduces FcRn binding. 27A. A fusion protein having a structure represented by the following formula, Fc1-L1-Fc2, from the N-terminus to the C-terminus [wherein Fc1 is a first Fc domain sequence containing a hinge-CH2-CH3, L1 is a linker, Fc2 is a second Fc domain sequence containing a hinge-CH2-CH3, Fc1 and Fc2 contain the amino acid substitution C226S and / or C229S according to EU numbering, and / or Fc1 and Fc2 contain a serine residue at amino acid positions 11 and / or 14, numbered based on the amino acid sequence of Sequence ID No. 18]. 28A. The fusion protein according to Embodiment 27A, wherein the fusion protein further comprises a human serum amyloid-P (SAP) component protein at the N-terminus of the fusion protein. 29A. The fusion protein according to Embodiment 28A, wherein the human SAP is a wild-type SAP that optionally contains the amino acid sequence shown in Sequence ID No. 17. 30A. The fusion protein according to Embodiment 28A, wherein the human SAP includes an amino acid substitution at position N32 or N110 based on Sequence ID No. 17. 31A. The fusion protein according to Embodiment 30A, wherein the human SAP comprises the amino acid sequence shown in Sequence ID No. 20. 32A. The fusion protein according to any one of Embodiments 1A to 31A, wherein the fusion protein forms a pentamer or a decamer. 33A. The fusion protein according to any one of Embodiments 1A to 32A, wherein the fusion protein includes a human Fc region. 34A. The fusion protein according to any one of Embodiments 1A to 33A, wherein the fusion protein includes a human IgG1 Fc region. 35A. The fusion protein according to any one of Embodiments 1A to 34A, wherein the fusion protein exhibits reduced aggregation compared to a fusion protein lacking one or more of the aforementioned amino acid substitutions. 36A. A pharmaceutical composition comprising a fusion protein according to any one of Embodiments 1A to 35A, and a pharmaceutically acceptable carrier. 37A. The pharmaceutical composition according to Embodiment 36A, wherein at least 50% of the fusion protein is in the state of pentamers and / or decamers. 38A. A nucleic acid encoding a fusion protein as described in any one of Embodiments 1A to 35A. 39A. A vector containing the nucleic acid described in Embodiment 38A. 40A. A host cell comprising the nucleic acid described in claim 38 or the vector described in embodiment 39A. 41A. The host cell according to Embodiment 40A, wherein the host cell is a CHO cell or a 293 cell. 42A. A method for producing a fusion protein according to any one of Embodiments 1A to 35A, comprising culturing a host cell containing a nucleic acid encoding the fusion protein under conditions for expressing the fusion protein. 43A. The method according to Embodiment 42A, wherein the host cell is a CHO cell or a 293 cell. 44A. The method according to Embodiment 42 or Embodiment 43, wherein the host cell does not glycosylate the SAP component protein. 45A. A method for treating amyloid disease in an individual, comprising administering a fusion protein described in any one of Embodiments 1A to 35A to the individual. 46A. A method for treating amyloid disease in an individual, comprising administering to the individual a fusion protein produced by the method described in any one of Embodiments 42A to 44A. 47A. The method according to Embodiment 45A or claim 46A, wherein the amyloid disease is selected from the group consisting of AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, IAPP amyloidosis of type II diabetes, and Alzheimer's disease. 48A. SAP-Fc fusion protein prepared by the method described in any one of Embodiments 42A to 45A. 49A. The method according to any one of embodiments 45A to 47A, wherein the individual is a human. [Examples]

[0078] Example 1. Manufacturing of SAP-scFc structure SAP-scFc was purified by a protein A affinity (MABSELECT SURE™ LX, CYTIVA®) purification step and a 1700 ml size exclusion chromatography (SEC) purification step. However, these purification steps failed to separate the monomer and dimer from other oligomers (Figure 6A and B). To better separate the monomer and dimer from other oligomers, the purified product from the 1700 ml SEC was further purified by a 496 ml SEC purification step (Figure 7A). SDS-PAGE analysis showed that the fraction obtained in this step contained a mixture of monomers, dimers, and other oligomers (Figure 7B). The fraction "2B1" from the 496 ml SEC was selected for analysis by reducing and non-reducing microfluidic electrophoresis (LABCHIP® GXII, PERKINELMER®). Non-reducing microfluidic electrophoresis of fraction "2B1" showed multiple oligomer peaks, while reducing microfluidic electrophoresis showed a primary monomer peak (Figures 7C-7D). Fraction "2A9" from 496 ml SEC was selected for analysis by size exclusion chromatography-multi-angle light scattering (SEC-MALS). SEC-MALS results indicated that the SAP-scFc protein exists as a high molecular weight complex (Figure 7E). These analyses indicate that the 496 ml SEC purification step did not improve the separation of monomers and dimers from other oligomers. SEC-HPLC analysis of fraction "2A9" after the 496 ml SEC purification step showed a single peak (Figure 7F), while denatured glycosylation mass spectrometry showed monomer, dimer, and trimer signals (Figure 7G). Therefore, it can be hypothesized that SAP-scFc may exist as a complex of monomers, dimers, and trimers combined by non-covalent interactions.

[0079] Furthermore, the purified product from 1700 ml SEC was subjected to further small-scale purification steps using SP SEPHAROSE high-performance strong cation exchange chromatography (HITRAP™ SP HP, CYTIVA®), POROS® XS strong cation exchange chromatography (THERMO FISHER SCIENTIFIC®), PHENYL SEPHAROSE 6 high-speed flow hydrophobic interaction chromatography (HITRAP™ PHENYL FF, CYTIVA®), BUTYL-S SEPHAROSE 6 high-speed flow hydrophobic interaction chromatography (HITRAP™ BUTYL-S FF, CYTIVA®), BIOPROCESS CAPTO PHENYL IMPRES hydrophobic interaction chromatography (HITRAP™ CAPTO PHENYL IMPRES, CYTIVA®), and CHT® ceramic hydroxyapatite calcium affinity cation exchange chromatography (BIO-RAD®). However, none of the small-scale purification methods improved the separation of monomers and dimers from other oligomers.

[0080] Example 2. Modified SAP-Fc fusion protein As described in Example 1, the structure of SAP-Fc presented challenges in the purification process. To improve the expression and purification profile, modifications to the SAP-Fc structure were explored.

[0081] Modification of SAP-Fc to remove cysteine ​​residues within the hinge region Point mutations were introduced to change the cysteine ​​residue at position 226 to serine and to change the cysteine ​​residue at position 229 to serine (C226S / C229S, EU numbering system), and thus, by removing the cysteine ​​residues within the SAP-Fc hinge region, TNT146(293) and TNT146(CHO) were produced (Table 1).

[0082] Modification of SAP-Fc using the "knob-hole" method to generate Fc regions TNT147(293), TNT148(293), and TNT148(CHO) were produced by modifying SAP-Fc using the "knob-hole" method to generate the Fc region (Table 1). Knob mutants were generated by a point mutation from threonine to tryptophan at position 336 (T366W, EU numbering system). Hole mutants were generated by point mutations from threonine to serine at position 366, from leucine to alanine at position 368, and from tyrosine to valine at position 407 (T366S / L368A / Y407V, EU numbering system).

[0083] Modification of SAP-Fc to remove glycosylated sites within SAP TNT151(293) and TNT151(CHO) were produced by modifying SAP-Fc and removing the glycosylation site within SAP (Table 1). Non-glycosylated SAP was generated by point mutations from asparagine at position 32 to serine and from asparagine at position 110 (N32S / N110S, numbered from the first amino acid of SAP).

[0084] Modification of SAP-Fc using the "knob-hole" method to generate the Fc region and to remove glycosylated sites within SAP. TNT152(293) and TNT152(CHO) were produced by modifying SAP-Fc to remove glycosylation sites within SAP and by generating Fc regions using the "knob-hole" method (Table 1). Non-glycosylated SAP and "knob-hole" Fc regions were generated as described above.

[0085] Example 3. Production of SAP-Fc and its modified proteins. Manufacturing of SAP-FC structures The production level of recombinant SAP-Fc protein was evaluated by ELISA. First, MaxiSorp® plates (NUNC) were coated with anti-human IgG primary antibody (1 μg / ml, Beckman Coulter) overnight at 4°C. After washing three times with PBS / 0.05% tween-20, 50 μl of the sample diluted in complete medium was incubated at 37°C for 2 hours. Simultaneously, standard-range human IgG diluted in complete medium was incubated. After three washes, anti-IgG antibody (Beckman Coulter) (1 μg / ml) conjugated to alkaline phosphatase was added and incubated at 37°C for 1 hour and 30 minutes. After a series of final washes, 100 μl of alkaline phosphatase substrate was added, and the reaction was stopped after a few minutes using 3 M NaOH. The plates were read by spectroscopy at 405 nm.

[0086] Next, the obtained SAP-Fc fusion protein is processed by AKTA FPLC Purification was performed using a protein A affinity column (Pierce) by affinity chromatography in a GE Healthcare system, due to the strong affinity of protein A to the Fc segment and the presence of Fc region fragments in the chimeric protein according to the present invention. A UV detector placed downstream of the column allowed for tracking of the progression; when the absorption of the non-retained phase dropped to the threshold, the protein was eluted with 0.1 M glycine at pH 2.6. Various fractions were collected based on the chromatographic peaks. The resulting eluents were then neutralized with Tris at pH 8.8.

[0087] The purified protein was concentrated using an Amicon® Ultra4 30k filter (Millipore), which allows for the removal of molecules smaller than 30 kDa. The solution was placed in the filter and centrifuged at 4000 g for the time required to obtain the desired volume and concentration.

[0088] Purified proteins (or culture supernatants) were detected by Western blotting using denatured gels. The proteins were mixed with a fixed volume of loading buffer (Bio Rad) containing β-mercaptoethanol and boiled for 5 minutes. They were then transferred by electrophoresis on a polyacrylamide gel (SDS-PAGE) consisting of a 7.5% stacking phase and a 10% separation phase. The proteins were then transferred onto a PVDF membrane and saturated with 5% milk. They were then incubated at ambient temperature for 1 hour in the presence of mouse anti-SAP (Abeam) or anti-IgG antibody (1 μg / ml) linked to HRP (BECKMAN COULTER®). Anti-SAP antibodies were identified with goat anti-mouse IgG secondary antibody (0.2 μg / ml) linked to HRP (SANTA CRUZ®). Chemiluminescence was induced by the addition of ECL substrate (PIERCE®) and revealed on an autoradiography membrane (KODAK®).

[0089] Purified proteins (or culture supernatants) were also detected by Western blotting using semi-natural gels. The proteins were mixed with a fixed volume of loading buffer (without β-mercaptoethanol) and then transferred on an SDS-12% PAGE gel. The remaining protocol was the same as for denatured gels.

[0090] Figures 16A and 16B show the SDS-PAGE gels of the purified fusion proteins. In these experiments, TNT146, TNT147, TNT148, TNT155, and TNT160 were purified by a two-step purification process. TNT151 was purified by a single Protein A purification step.

[0091] Example 4. Bonding properties of modified SAP-Fc to fibrils The binding activity of SAP-Fc and SAP-scFc to fibrils and extracts was investigated by ELISA.

[0092] Using the europium-conjugated immunosorbent assay (EuLISA) for humanized IgG, a (sonicated) suspension of 0.83 M rV6Wil fibrils was prepared in phosphate-buffered saline (PBS). The wells of a 96-well microplate were coated with fibrils by adding 50 L to each well. The plate was dried overnight at 37°C. The wells of the microplate were blocked by adding 200 L / well of Tris-buffered saline (TBS) (Thermofisher) (SBT) containing Superblock and 2 mM calcium chloride (Ca), and the plate was left at 37°C for 1 hour. Primary (test) SAP-Fc fusion protein, humanized immunoglobulin, hIgG1, or c11-1F4 were added. The plate was incubated at 37°C for 1 hour. After the washing step (the plate was washed three times with TBS / Ca+ 0.05% tween 20), a 1:3000 dilution of biotinylated goat anti-human IgG (Sigma) by SBTT was added in 100 L / well with secondary antibody. The plate was incubated at 37°C for 1 hour. After another washing step, a 1:1000 dilution of europium / streptavidin (Perkin Elmer) was added in 100 L / well. The plate was incubated at 37°C for 1 hour. After the final washing step, a 100 L / well europium-enhanced solution (Perkin Elmer) was added. Time-resolved fluorescence emission was read using a microplate reader (Wallac).

[0093] The results shown in Figure 8A indicate that the originally provided TNT146(293) and TNT147(293) bind to synthetic rVλ6Wil fibrils with greater affinity (EC50) than the hIgG1 control and c11-1F4 in the ELISA assay. Figure 8B shows that TNT146(CHO), TNT148(CHO), and TNT151(293) bind equally well to rVλ6Wil fibrils, with the exception of TNT152(293). The results shown in Figures 9A-B indicate that the knob-hole and cys mutation forms of SAP-Fc retain amyloid reactivity in vitro.

[0094] The results shown in Figures 10A-10E and Table 4 indicate that all constructs except TNT152(293) and TNT148(CHO) exhibit EC50 values ​​in the range of 1-10 nM. The highest binding affinity to the wtATTR extract was observed for the SAP-scFc reagents TNT146(293), TNT146(CHO), and TNT151(293) (Table 4). In the context of SAP-scFc, non-glycosylation of SAP maintains comparable in vitro amyloid reactivity with similar EC50 values, except for TNT152(293), whose EC50 to the amyloid extract is higher than that of the hIgG1-peptide fusion (Figure 8B and Table 4). [Table 4]

[0095] The binding affinity of SAP-Fc fusions to fibrils and extracts was also tested in a pull-down assay. Synthetic amyloid fibrils from rVλ6Wil were prepared as follows: 1 mL of phosphate-buffered saline (PBS) containing 1 mg / mL of monomer in a solution with 0.01% w / v NaN3 and pH 7.5 was filtered through a 0.2 mm pore size filter and added to a 15 ml conical polypropylene tube (BD BioSciences, Bedford, MA). The reaction mixture was shaken at 225 rpm at a 45° angle for 3–5 days at 37°C until it became opaque.

[0096] Purified human amyloid tissue extracts were prepared using autopsy-derived tissue from patients with light chain (AL) or transthyretin-related (ATTR) amyloidosis, without modification, using the water flotation method described in Pras et al., J Exp Med (1969) 130(4):777-795. The purified amyloid material was isolated in a water wash solution, and amyloid-rich pellets were recovered and stored lyophilized at room temperature until use.

[0097] For the pull-down assay, 25 microliters of 1 mg / mL AL extract or synthetic rVλ6Wil variable domain fibrils (REF) were placed in a 0.5 ml microcentrifuge tube and centrifuged at 21,000 × g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 200 μL of PBS (PBST) containing 0.05% tween-20. 125I-p5+14 (approximately 100,000 counts per minute (CPM); approximately 5 ng of peptide) or 125I-labeled SAP-Fc fusion protein was added to the suspension. The mixture was rotated at room temperature for 1 hour. The sample was then centrifuged twice at 15,000 × g for 10 minutes each. After each step, the supernatant and pellet were separated, and the radioactivity in each was measured using a Cobra II gamma counter (Perkin Elmer) with a 1-minute acquisition. The percentage of 125I-p5+14 or 125I-labeled SAP-Fc fusion proteins bound to the pellet was determined as follows: Pellet CPM / (Pellet CPM + Supernatant CPM) × 100

[0098] In a pull-down assay 125 I-labeled TNT146 and TNT147 showed weak binding affinity to the substrate, with EC50 falling within the nanomolar range (Table 5), suggesting that oxidative radioiodation may adversely affect the product and lead to loss of activity; that amyloid extracts provided as soluble materials may exhibit different binding sites; and that higher concentrations of reagents may promote binding in ELISA format. [Table 5]

[0099] Example 5. In vivo distribution of SAP-Fc in mice AA Amyloidosis Mouse Model AA amyloidosis mice are a mouse strain that stably expresses the human IL6 (huIL6) protein from an inserted transgene. At the National Institutes of Health in the United States, the B6(C)-Tg(H2-Ld-IL-6)Kish(H2 / huIL-6) strain was obtained by backcrossing prototype H2-Ld-IL-6 Tg C57BL / 6 gene-transfected mice with a Balb / c background for more than 20 generations (Kovalchuk et al., PNAS (2002) 99:1509-1514). These mice constitutively express the huIL6 transgene under the control of the mouse major histocompatibility complex class 1 (H2-Ld) promoter. The transgene segregates in a Mendelian autosomal manner (Suematsu et al., PNAS (1992) 89:232-235). Due to the role of IL-6 in maintaining the inflammatory response and lymphocyte proliferation during the immune response, the early generation of these transgenic animals had significant lymphocytosis with high sAA levels and advanced polyclonal plasma cell proliferation in 56% of 18-month-old mice (Kovalchuk et al., PNAS (2002) 99:1509-1514). While the human IL-6 serum level in the early generation of this transgenic line was 0.5-1 ng / mL, backcrossed Balb / c mice bred in our facility for approximately two years had circulating huIL-6 levels approximately 300 times higher (0.3-1 μg / mL) at 8 weeks of age (Solomon et al., Am J Pathol (1999) 154:1267-12724). In response to the pro-inflammatory cytokine huIL6, mice suffer from a chronic inflammatory state characterized by elevated concentrations of circulating sAA-conjugated high-density lipoprotein. Spontaneous onset of AA amyloidosis in these mice typically occurs at 5 months of age and initially manifests as perifollicular deposits only in the spleen, detectable histologically by biopsy (Solomon et al., Am J Pathol (1999) 154:1267-12724). Over the next 3-4 months, amyloid was detected in the periportal vascular structures and sinusoids within the liver, tongue, heart and intestinal villi, as well as in the renal interstitium, glomeruli and papillae.In addition, cylindrical nephropathy was commonly observed, as well as extramedullary hematopoiesis and splenomegaly, due to amyloidosis or lymphoid tissue hyperplasia (or both) resulting from elevated IL-6 levels.

[0100] The properties of amyloid in H2 / huIL-6 mice were immunohistochemically documented using AA-specific monoclonal antibodies (Solomon et al., Am J Pathol (1999) 154:1267-12724). In addition, liquid chromatography-conjugated mass spectrometry showed that isolated tissue-derived AA fibrils consisted of sAA in cleavage forms containing the first 77 N-terminal amino acids (residues 1-77) (Solomon et al., Am J Pathol (1999) 154:1267-12724). No evidence of apolipoproteins AI and AII, or immunoglobulin light chains, was found during immunohistochemical and liquid chromatography-conjugated mass spectrometry of amyloid extracted from these mice.

[0101] The in vivo distribution of SAP-Fc was investigated in wild-type (WT) mice and AA amyloidosis mouse models.

[0102] The in vivo distribution of SAP-Fc in wild-type (WT) mice and AA amyloidosis mouse models was investigated by tissue microautoradiography. The AA amyloidosis mouse model was created by intravenously administering 100 ml of sterile phosphate-buffered saline (PBS) containing 10 mg of isolated amyloid-enhancing factor (AEF, Axelrad et al., Lab Invest (1982) 47:139-146.) to H2-Ld-huIL-6 Tg Balb / c gene transgenic mice that constitutively express the human interleukin-6 transgene. The mice used in these studies were 4-6 weeks old after induction. The AA mouse model is characterized by interstitial cardiac amyloid deposition, marked sinusoidal amyloid deposition in the liver, early nodular perifollicular amyloid deposition in the spleen, and late amyloid deposition in the pancreas, kidneys, adrenal glands, and intestines.

[0103] AA or WT mice were injected with 10 μg of 125I-labeled TNT146(293) and TNT147(293), and then euthanized 48 hours post-injection. From the euthanized WT and AA mice, samples were collected from the spleen, pancreas, left and right kidneys, liver, heart, muscle, stomach, upper and lower intestines, and lung tissue. Each sample was placed in a tare-weighted plastic vial, weighed, and 125I radioactivity was measured using an automated Wizard 3 gamma counter (1480 Wallac Gamma Counter, PERKIN ELMER®). In vivo distribution data were expressed as the percentage of injected dose per gram of tissue (%ID / g). In addition, for histology and autoradiography, each tissue sample was fixed in 10% buffered formalin for 24 hours and embedded in paraffin. For autoradiography, sections 4–6 μm thick were excised from formalin-fixed, paraffin-embedded lumps onto Plus microscope slides (FISHER SCIENTIFIC®), immersed in NTB2 emulsion (EASTMAN KODAK®), stored in the dark, and developed after 96 hours of exposure. Each section was counterstained with hematoxylin. Alternatively, AA or WT mice were injected with 500 μg of unlabeled TNT147(293) and then euthanized 48 hours post-injection. Each tissue sample was fixed in 10% buffered formalin for 24 hours and embedded in paraffin. Sections 4–6 μm thick were excised from formalin-fixed, paraffin-embedded lumps onto Plus microscope slides (FISHER SCIENTIFIC®), and the sections were then stained with anti-human Fc. Tissue amyloid deposits were identified by microscopy in serial tissue sections observed under cross-polarized light after staining with alkaline Congo red. All tissue sections were examined using an optical microscope (DM500, LEICA®) equipped with a cross-polarized filter (for detecting Congo red birefringence). Digital microscopy images were acquired using a cooled charge-coupled device camera (SPOT, DIAGNOSTIC INSTRUMENTS®).

[0104] The results shown in Figures 11A-11C demonstrate that TNT146(293) and TNT147(293) specifically bind to AA amyloid in mice with systemic disease, both with and without the 125I radiolabeling. The specific binding of both TNT146(293) and TNT147(293) to amyloid deposits was confirmed by autoradiography, which showed a lack of AA deposits in the heart.

[0105] The results shown in Figures 12A and 12B indicate that 125I-labeled TNT146(293) and TNT147(293) accumulated in the liver and spleen of AA mice, but not in amyloid-free tissues of WT mice.

[0106] Example 6. Multimerization analysis of SAP-Fc structures High avidity binding of SAP to amyloid is achieved through appropriate polymerization of the protein as pentamers or decamers. On the other hand, large and / or improper aggregates of SAP-Fc can be rapidly removed from the circulation flow, thus adversely affecting their pharmacokinetics and function. Consequently, analysis of higher-order aggregates of SAP-Fc constructs is important for understanding their function in vivo.

[0107] The polymerization and aggregation of SAP-Fc constructs were analyzed by size exclusion chromatography (SEC). A matrix suitable for proteins with molecular weights of 10–1300 kDa was used for this analysis. Commercially available protein preparations were used as molecular weight standards. The estimated molecular weight of the SAP-Fc monomer was approximately 75 kDa, the estimated molecular weight of the SAP-Fc pentamer was approximately 375 kDa, and the estimated molecular weight of the SAP-Fc decamer was approximately 750 kDa.

[0108] The results shown in Figure 13 indicate that while all SAP-Fc constructs had different multimerization structures, TNT146(293) and TNT147(293) were the most uniform. Production in CHO cells compared to HEK-293 cells significantly influenced the multimerization structures of TNT151 and TNT152 constructs (Figure 13).

[0109] The uptake and in vivo distribution of these constructs in mice were also compared. In the spleen, both TNT146(293) and TNT147(293) showed a distribution of approximately 10% (Figure 12 A-B). Other structures showed less distribution in the spleen: TNT151(293) was distributed in approximately 2.5% of the spleen, which was one-quarter lower than TNT146(293) and TNT147(293) (Figure 14A); TNT152(293) was distributed in approximately 2% of the spleen, which was one-fifth lower than TNT146(293) and TNT147(293) (Figure 14B); and TNT148(293) was distributed in approximately 3% of the spleen, which was one-third lower than TNT146(293) and TNT147(293) (Figure 14C). When considered in conjunction with the SEC chromatograms from the multimerization analysis, these results indicate that the 450–669 kDa dimer peak correlates with optimal SAP-Fc uptake in AA mice (Figures 12A–B, 14A–14C, and 13).

[0110] Example 7. Phagocytosis of rVλ6Wil fibrils by THP1 cells Phagocytosis was investigated using the pHrodo red-labeled rVλ6Wil fibril system.

[0111] For solid-phase Wil fibril uptake, 24-well tissue culture plates are coated with type I rat collagen (75 μg / ml in 20 mM acetic acid, 0.4 ml) at room temperature for 2 hours, washed with 0.5 ml of PBS, and coated overnight with 20 μg / well of pHrodo red-labeled rVλ6 fibrils (30% labeling) in 0.5 ml of PBS at 4°C. The wells are washed with 0.5 ml of PBS and 0.5 ml of serum-free phenol red-free RPMI1640 is added. Antibody opsonin or SAP-Fc protein is added, and immediately thereafter, serum-free phenol red-free RPMI1640 (1.2 × 10⁶ cells per 0.5 ml) containing RAW264.7 or uninducible THP-1 cells is added for incubation at 37°C for 4 hours. For uptake measurements, cells from each well are transferred to three replicate wells of a black plastic / clear-bottomed 96-well microplate (Corning) in well-scan mode using a BioTek SynergyHT-1 microplate reader, with excitation at 530 / 25 nm and emission at 645 / 40 nm. Relative fluorescence units are obtained by subtracting background readings from wells incubated in 1 ml of culture medium alone.

[0112] The results shown in Figure 15 indicate that SAP-Fc TNT146(CHO), TNT147(239), TNT148(CHO), and TNT151(CHO) acted as excellent opsonizing agents, while TNT152(CHO) was the least effective reagent, which is consistent with its weaker binding affinity to rVλ6Wil fibrils.

[0113] Example 8. Phase 0 in vivo distribution test AUR03 is an SAP-Fc radiolabeled with 124I using the iodine tube method in this trial. The objectives of the Phase 0 trial are: 1) to determine the in vivo distribution, including off-target binding and target engagement, in patients with systemic amyloidosis; 2) to determine whether AUR03 engages with target amyloid-containing organs, such as the heart; 3) to determine whether AUR03 binds to amyloid-containing organs in both ATTR and AL patients; and 4) to differentiate AUR03 from other drugs by amyloid type or organ for development planning purposes.

[0114] Thirty patients will be recruited into two cohorts. Fifteen ATTR patients with evidence of cardiac involvement will be recruited into cohort 1a, and fifteen AL patients with thoracic and abdominal organ involvement will be recruited into cohort 1b. Patients in both cohorts will be given a single dose of 100 μg of AUR03, and imaging will be performed at one or two clinical sites on days 2 and 5 after injection.

[0115] Blood samples are collected before and after injection for CBC, ClinChem, NT-proBNP, and LDH testing. Blood samples are collected for PK determination, and the radioactivity of the blood samples is measured over 72 hours.

[0116] Example 9: Phagocytosis in vivo A batch of 12 mg of human ALλ(SHI) amyloid containing 10% pHrodo red labeling material was pre-incubated at room temperature (RT) for 30 minutes with 600 μg of TNT146 in Tris-buffered saline containing 2 mM CaCl2. The fluorescence emission of the pHrodo red fluorophore is associated with the acidification of amyloid during phagocytosis by macrophages and potentially neutrophils. Immunosuppressed NU / NU (n=5) mice were administered 2 mg of human ALλ(SHI) as a subcutaneous injection into the left ventral region of the mice. Control NU / NU mice (n=5) were administered a similar subcutaneous dose of 2 mg of human ALλ(SHI) without TNT146 pretreatment. Fluorescence emitted by the pHrodo red fluorophore was continuously tracked by optical imaging using mice anesthetized with 2% isoflurane. Images were collected on days 1, 3, 8, 10, 12, and 14 after amyloid injection. Mice were euthanized on day 15.

[0117] Optical imaging of pHrodo Red revealed an initial increase in emission in TNT146-treated mice, which persisted throughout the entire study (Figure 17A). On day 1 (24 hours after amyloid injection), the fluorescence emission from TNT146-treated amyloid was significantly stronger than that of control animals (Figure 17B). On day 14, TNT146-treated amyloid exhibited noticeably stronger fluorescence emission compared to control animals (Figure 18). These data indicate that the binding of TNT146 to amyloid, when administered subcutaneously to mice, enhances the phagocytic activity of human amyloid.

[0118] Example 10: Complement-enhancing phagocytic effect of amyloid fibrils Step 1: Differentiation of THP-1 cells into M0 macrophages 10⁶ cells / well contained in complete DMEM / F12 (Hyclone, SH 30023.01) supplemented with 10% FBS (Hyclone, SH 30071.03), 1% Pen-Strep (Gibco, 15140-122), and 1% gentamicin (Gibco, 15710-064) were counted and seeded into the center well of a 24-well tissue culture plate (Costar 3526).

[0119] Add 50 ng / mL of PMA (Sigma, P8139) and differentiate the cells in a 5% CO2 incubator at 37°C for 24 hours.

[0120] After 24 hours, the culture medium containing PMA is carefully removed by manual aspiration.

[0121] Replenish each well with 1 ml of complete DMEM-F12 medium and allow the cells to rest for at least 48 hours.

[0122] Step 2: Sample Preparation Rinse the well once with 1 ml of DPBS (Hyclone, SH 30028.02).

[0123] Add 500 μL of serum-free RPMI-1640 (Hyclone, SH30605.01) without phenol red to each well and incubate the plate at 37°C until the start of the assay.

[0124] Prepare the reaction by adding 500 μL of RPMI-1640 to a microcentrifuge tube, followed by the addition of 3 μg of TNT146 or control Fc. Mix thoroughly.

[0125] Add pHrodo Red labeled (pHrodo® Red SE, ThermoFisher, P36600) rVλ6Wil fibril (20 μg) to a microcentrifuge tube.

[0126] Next, add 20 μg of guinea pig complement to half the well. Mix thoroughly and incubate at room temperature for 5 minutes.

[0127] The contents of each microcentrifuge tube are transferred to the corresponding wells of a 24-well plate (at this point, the total volume in each well is 1 mL).

[0128] Gently mix by hand by moving the plate in a vertical motion (rather than a rotational motion).

[0129] To facilitate phagocytosis, the tissue culture plates are incubated in a 5% CO2 incubator at 37°C for 1 hour.

[0130] Step 3: Image acquisition and quantification After incubation for 1 hour, fluorescence associated with pHrodo red is recorded by fluorescence microscopy (Keyence BZ X800 V 1.3.1).

[0131] To ensure that the entire area of ​​each well is included in the document, four images are captured for each well.

[0132] The amount of fluorescence in each image was quantified using image segmentation and Image Pro Premier V 9.0.

[0133] The data was analyzed by calculating the mean and standard deviation of the four observations. Statistical analysis was performed using an independent two-tailed t-test with α = 0.05 (assuming the data is normally distributed) using Prism (v.9.0, GraphPad).

[0134] Activated human THP-1 cells, when opsonized by the addition of TNT146, effectively take up (phagocytose) synthetic rVλ6Wil AL amyloid-like fibrils. In the presence of 20 μg of highly active guinea pig complement, there was a significant increase in the fluorescence intensity of pHrodo red, suggesting enhanced fibril phagocytosis in the presence of TNT146 and complement (Figure 19).

[0135] Example 11: Binding of SAP-Fc fusions to amyloid fibrils in the presence of huSAP. In 96-well microplates, human amyloid extracts (ALκ TAL and ALλ SHI), ATTRwt, and synthetic AL amyloid-like fibrils (rVλ6Wil) were pre-treated by incubation at 37°C for 30 minutes in a human serum amyloid-P component (SAP) solution prepared with 2 mM CaCl2-containing TBS. Stock solutions of rVλ6Wil and amyloid extracts were used at concentrations of 0.83 μM and 0.06 mg / mL, respectively. Samples not incubated in human SAP served as controls. After incubation, the samples were washed, and TNT146 (MW=410 kD), serially diluted from 100 nM in TBS / CaCl2, was added to the microplate wells. After the washing step, bound TNT146 was detected by adding a biotinylated anti-human Fc reagent. Bound TNT146 was quantified by measuring time-resolved fluorescence after adding streptavidin-europium conjugate and developer.

[0136] The mean and standard deviation of the iterative wells were plotted, and the data was approximated using a sigmoid algorithm with a variable gradient (Prism v9.1, Graphpad).

[0137] Pre-binding of human SAP to AL or ATTR amyloid extract or amyloid-like fibrils did not adversely affect the ability of TNT146 to bind to the substrate (Figure 20 A-D).

[0138] Example 12: Binding of SAP-Fc fusions to amyloid fibrils in the presence of huSAP. The wells of a 96-well microplate were coated with synthetic Aβ(1-40) amyloid-like fibrils contained in PBS. The wells were blocked, followed by a washing step, and serially diluted TNT146 (MW=410kD) with TBS / CaCl2 was added to the microplate wells. Binding TNT146 was detected by adding a biotinylated anti-human Fc reagent. Binding TNT146 was quantified by measuring time-resolved fluorescence after adding streptavidin-europium conjugate and developer. Human Fc was used as a control reagent.

[0139] The mean and standard deviation of the iterative wells were plotted, and the data was approximated using a sigmoid algorithm with a variable gradient (Prism v9.1, Graphpad).

[0140] TNT146 binding to Aβ(1-40) amyloid-like fibrils resulted in saturated binding with an estimated EC50 (concentration at 50% half-binding) of 0.4 nM (Figure 21). Fc binding was weak, and although the Ec50 could not be precisely determined, it was estimated to be approximately 0.2 mM.

Claims

1. Starting from the N-terminus and moving towards the C-terminus, the following equation applies: SAP-Fc1-L1-Fc2 [During the ceremony, Fc1 is a first Fc domain sequence containing hinge-CH2-CH3, L1 is a linker, and Fc2 is a second Fc domain sequence containing hinge-CH2-CH3. SAP is a component protein of human serum amyloid-P (SAP). The aforementioned Fc1 and Fc2 each include amino acid substitutions in C226S and C229S according to EU numbering. A fusion protein comprising a structure represented by, The fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, which has or does not have C-terminal lysine, or SEQ ID NO: 23, which has or does not have C-terminal lysine.

2. The fusion protein according to claim 1, wherein the fusion protein comprises an amino acid sequence shown in SEQ ID NO: 1, with or without C-terminal lysine.

3. The fusion protein according to claim 1, wherein the fusion protein comprises an amino acid sequence shown in SEQ ID NO: 23, which may or may not have a C-terminal lysine.

4. Starting from the N-terminus and moving towards the C-terminus, the following equation applies: SAP-Fc1-L1-Fc2 [During the ceremony, Fc1 is a first Fc domain sequence containing hinge-CH2-CH3, L1 is a linker, and Fc2 is a second Fc domain sequence containing hinge-CH2-CH3. SAP is a component protein of human serum amyloid-P (SAP). The Fc1 and Fc2 respectively contain amino acid substitutions in C226S and C229S according to EU numbering, The aforementioned human SAP contains the amino acid sequence shown in Sequence ID No. 20, [The Fc1 and Fc2 are human IgG1Fc domains.] A fusion protein containing the structure represented by .

5. The fusion protein according to claim 4, wherein L1 comprises the amino acid sequence shown in SEQ ID NO:

19.

6. The fusion protein according to claim 4, wherein Fc1 and / or Fc2 comprises the amino acid sequence shown in SEQ ID NO:

18.

7. The fusion protein according to claim 6, wherein the first and / or second Fc domain includes a mutation that reduces FcRn binding ability.

8. The fusion protein according to claim 7, wherein Fc1 and / or Fc2 comprises the amino acid sequence shown in SEQ ID NO:

21.

9. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein forms a pentamer or a decamer.

10. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein exhibits reduced aggregation compared to a fusion protein lacking one or more of the amino acid substitutions.

11. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein at least 50% of the fusion protein is in the state of pentamers and / or decamers.

13. A nucleic acid encoding a fusion protein according to any one of claims 1 to 8.

14. A vector comprising the nucleic acid described in claim 13.

15. A host cell containing the nucleic acid described in claim 13.

16. The host cell according to claim 15, wherein the host cell is a CHO cell or a 293 cell.

17. A host cell comprising the vector according to claim 14.

18. The host cell according to claim 17, wherein the host cell is a CHO cell or a 293 cell.

19. A method for producing a fusion protein according to any one of claims 1 to 8, comprising culturing a host cell containing a nucleic acid encoding the fusion protein under conditions for expressing the fusion protein.

20. The method according to claim 19, wherein the host cell is a CHO cell or a 293 cell.

21. The host cell does not glycosylate the SAP component protein, according to claim 19. method.

22. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 8 for treating amyloid disease in an individual.

23. The pharmaceutical composition according to claim 22, wherein the individual is a human.

24. The pharmaceutical composition according to claim 22 or 23, wherein the amyloid disease is selected from the group consisting of AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, IAPP amyloidosis of type II diabetes, and Alzheimer's disease.

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