Supramolecular filament assembly for protein purification

Abstract

To provide novel immunofiber compositions for protein or peptide purification, and simple and cost-efficient methods and systems using these compositions.SOLUTION: In some embodiments, the immunofibers comprise a customized Z-33 peptide derived from Staphylococcus aureus Protein A, which is used for constructing immuno-amphiphile molecules that assemble into immunofibers in aqueous solution with bioactive epitopes on the surface and have peptide or protein binding ability.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 547,256, filed August 18, 2017. This application is hereby incorporated by reference for all purposes as if fully set forth herein. and is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Filed Properties This application contains a sequence listing which has been submitted via EFS-Web in ASCII format. The ASCII copy is dated 2018 It was created on August 17, 2014, is named P14755-02_ST25.txt, and is 2,433 bytes in size. [Background technology]

[0003] Amphiphilic peptides or peptide conjugates capable of self-assembling into one-dimensional (1D) nanostructures The body has been extensively studied over the past 20 years due to their important biomedical applications. The resulting self-assembled nanostructures have been used as a biological interface. In order to achieve this, various biologically active peptides have been incorporated into the molecular design. While the secondary structure of bioactive peptides presented on supramolecular surfaces allows them to function properly, Controlling it precisely, as needed to achieve this, remains a difficult problem. Generally, the final self-assembled morphology is determined by hydrophobic interactions, hydrogen bonding, electrostatic interactions, and It depends on several interaction factors, including π-π stacking. In the case of the 1D nanostructures, the β-sheet motif provides intermolecular hydrogen bonds, resulting in It is often used to grow α-helical peptides ( Proteins are another important component of the body and are key mediators of many important biomolecular interactions. ether) are also used, less frequently, to create supramolecular nanostructures. For example, Tirrell et al. have reported cylindrical micelles with significant α-helices and Furthermore, micelles of proteins and protein analogs were designed based on their solvent properties, hydrophobic tails, or thermal history. By adjusting the Transitions between various structures in self-assembled peptide nanostructures have been reported occasionally. Although these advances are important, the inherent thermodynamic instability and their supramolecular assembly remain a challenge. Concerns remain regarding the structural uncertainty of α-helical peptides in vivo. do.

[0004] The α-helical secondary structure can be stabilized by the attachment of alkyl chains. However, by adjusting the number of alkyl chains, the In this study, the α-helix to β-sheet transition was rarely observed.

[0005] Bioactive peptides were added to either the C- or N-terminus of the self-assembled peptide motif. Such direct deposition creates bioactive materials for specific biomedical applications. In an effort to modulate the immunogenicity of peptide assemblies, Lilier and coworkers covalently linked the self-assembled peptide Q11 to the antigen OVA peptide. The obtained supramolecular OVA-Q11 nanofibers were found to have enhanced immunogenicity. Until now, biologically active peptides have been synthesized into supramolecular structures while maintaining their biological activity. Many studies have well demonstrated that peptides can be successfully incorporated into nanostructures. However, for epitopes to have biological activity, If an α-helical structure must be maintained, the use of a β-sheet forming sequence is also required. The problem seemed to be the lack of spacing between the α-helical motifs. .

[0006] High affinity antibody-binding particles and materials relate to monoclonal antibodies, which are biological therapeutic agents. Driven by increasing demand, it is rapidly gaining interest in the pharmaceutical industry. Tein A, a well-known antibody-binding ligand, is available from most mammalian species, including humans. Protein A has the ability to specifically bind to the Fc portion of IgG. However, it is large in size. The large size of Protein A limits its industrial use, and therefore many synthetic and Minimized domains have been designed and studied. The Z-domain of Protein A was the first The most well-known synthetic domain is 59 amino acid residues, and binds to IgG1 with a K of approximately 10 nM. d of Further minimization of the Z domain of Protein A significantly alters binding affinity. Without d = 43 nM), a two-helix derivative, Z33, was designed. A high-affinity ligand was identified. On the other hand, methods for displaying ligands on desired substrates can play a key role in the antibody purification process. In the pharmaceutical industry, antibody purification is primarily achieved by using highly selective antibody-binding ligands (e.g. For example, they rely on affinity chromatography based on the immobilization of proteins (e.g., Protein A). However, it suffers from high chromatography media costs and limited capture productivity. It can be efficiently purified using a relatively simple method and batch processing volume bottlenecks can be avoided. Affinity precipitation offers a significant advantage over traditional chromatography by potentially eliminating the block. Only recently has it become an attractive alternative to the fee method.

[0007] A typical example of affinity deposition is the fusion of elastin-like protein (ELP) The Z domain was used to induce the solubility transition of ELP under temperature and salt conditions, resulting in the precipitation of IgG. However, the bacterially expressed ELPs are large, and the ELPs are fused to the respective ligands. Because binding sites are limited and because antibodies may denature at high temperatures, Interest has shifted to finding new substrates for presenting ligands to target proteins.

[0008] Inspired by the elegant molecular design of self-assembled peptide amphiphiles, this study The authors synthesized the protein A mimetic peptide Z33 into a self-assembling immuno-amphiphile. We have previously reported a method for incorporating the target into e-amphiphiles (IA) and The binding ability of the self-assembled immunofibers (IFs) to therapeutic IgG was investigated. The binding affinity between IF and IF was investigated using isothermal differential titration (ITC). These results suggest that the antibody retains a low binding affinity to IgG.

[0009] The inventors have fragmented the peptide and attached it to an alkyl chain to produce flagella. The conformation of the mentholated peptide changes, and the peptide binds to the protein. Yet the properties of self-assembling immuno-amphiphiles can be combined for effective purification. We considered whether the information is being held as such. Summary of the Invention [Problem to be solved by the invention]

[0010] Core building blocks essential for intermolecular hydrogen bonding that allows directional and anisotropic growth of aggregates Self-assembling peptides or peptide conjugates containing short β-sheet sequences as binding motifs By combining these molecules, many one-dimensional (1D) nanostructures can be constructed. Thus, a large number of bioactive filamentous β-sheet aggregates are formed that interface with the cell. Although we have been able to successfully produce the complex, it has toxicity reminiscent of amyloid fibrils. Other supramolecular molecules using α-helical peptides have been investigated due to concerns regarding their potential for The manufacturing strategy was advanced.

[0011] The present inventors have previously identified the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: Q ID NO): 1) Protein A mimetic peptide Z3 with (a motif containing two α-helices) Self-assembling immuno-amphiphiles by directly linking 3 to linear hydrocarbon chains (U.S. Provisional Patent Application No. 62 / 478,795, filed March 30, 2017, (The entire contents of which are incorporated herein by reference as if set forth herein.) The results are The amphipathic peptides shown in Fig. 1 lack the essential β-sheet segment. supramolecular immunoglobulins, preserving their native α-helical structure under physiological conditions. By isothermal titration calorimetry, we have shown that these ions can effectively associate with the IF. Self-assembled immunofibers bind highly specific immunoglobulin G (IgG) antibodies at pH 7.4. It was confirmed that the antibody could bind to the antibody with good affinity, but the binding was not detected in the elution buffer at pH 2.8. It was not possible to do so.

[0012] The present invention provides a method for the preparation of a compound having a structure between an α-helix and a β-sheet by single- or double-strand alkylation. We present a molecular strategy for switching the secondary structure of α-helical peptides. Two peptide sequences isolated from α-helical peptide Z33 derived from protein A The sequence fragments were designed to act as hydrophilic moieties in immuno-amphiphiles (IAs). These self-assembling immunofibers, when combined in solution, exhibit a pH of 7. It is predicted that it can bind to immunoglobulin G (IgG) antibodies with high specificity at .4. . [Means for solving the problem]

[0013] Thus, in some embodiments, the protein-binding peptide is attached to a self-assembling imine. Supramolecular engineering into nanofibers can be effective in protein purification.

[0014] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. Immuno-amphiphiles are provided.

[0015] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. A self-assembling immunofiber comprising an immuno-amphiphile is provided, wherein: The immuno-amphiphiles exhibit an α-helical structure when in aqueous solution at physiological pH. Has.

[0016] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. A self-assembling immunofiber comprising an immuno-amphiphile is provided, wherein: The antibody-binding peptide is Z33 of protein A of Staphylococcus aureus. Hydrophilic amino acid sequence of a peptide, or a functional part, fragment, or derivative thereof It has a conductor.

[0017] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain.

[0003] The present invention provides a self-assembling immunofiber containing immuno-amphiphiles containing a segment. do.

[0018] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain.

[0003] The present invention provides a self-assembling immunofiber containing immuno-amphiphiles containing a segment. wherein the immuno-amphiphile, when in aqueous solution at physiological pH, is an α-heterocyclic compound. It has a ribrick structure.

[0019] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain.

[0003] The present invention provides a self-assembling immunofiber containing immuno-amphiphiles containing a segment. wherein the immuno-amphiphile exhibits a β-syringe structure when in aqueous solution at physiological pH. It has a gate structure.

[0020] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain.

[0003] The present invention provides a self-assembling immunofiber containing immuno-amphiphiles containing a segment. wherein the antibody-binding peptide is a protein of Staphylococcus aureus. It contains a hydrophilic amino acid sequence portion of the Z33 peptide of protein A.

[0021] According to another embodiment, the present invention provides a method for the preparation of a polypeptide comprising an antibody-binding peptide attached to a linear hydrocarbon chain. and an immunofiber composition comprising one or more immuno-amphiphiles, wherein: The antibody-binding peptide is Z33 of protein A of Staphylococcus aureus. Hydrophilic amino acid sequence of a peptide, or a functional part, fragment, or derivative thereof It has a conductor.

[0022] According to one embodiment, the present invention provides a method for protein purification, comprising: The solution containing the above target protein at the first pH level is added to the antibody bound to a linear hydrocarbon chain. an immunofiber composition comprising one or more immuno-amphiphiles containing an antibody-binding peptide; contacting the antibody-binding peptide with Staphylococcus aureus. The hydrophilic amino acid sequence of the Z33 peptide of protein A of Bacillus reus, or a functional portion thereof, or or a fragment or derivative thereof; one or more proteins of interest may be linked to the Fc binding peptide. conjugating to a peptide, or a functional part, fragment, or derivative thereof; The pH level of the solution is adjusted to match the charge characteristics of the antibody-binding peptide and one or more proteins of interest. changing the pH to alter the properties and structure of the protein; and and extracting the protein from said solution.

[0023] According to a further embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: Antibody-binding peptide (Antibody-binding peptide is attached at its N-terminus to a linear hydrocarbon chain) , and the first spacer peptide (the first spacer peptide is P. aeruginosa at its C-terminus) the hydrophilic amino acid sequence of the Z33 peptide of protein A of Staphylococcus aureus, or Binding to an antibody-binding peptide having a functional portion, fragment, or derivative thereof an immunofiber-binding molecule (wherein the first spectrophotometer is a Ser-peptides contain sequences of the general formula XXYYZZ, where XX is a small hydrophobic side chain. and may be the same or different amino acids. YY are two amino acids with positively charged side chains, and may be the same or different amino acids. ZZ are two amino acids with small neutral side chains, and amino acids), which may be the same or different amino acids and Immunofiber spacer molecule (Immunofiber spacer molecule is the N At the end, it has a linear hydrocarbon chain attached to a peptide sequence containing the general sequence XXBB. where XX are two amino acids with small hydrophobic side chains and are the same amino acid or can be different amino acids, where BB is a pair of amino acids with negatively charged side chains. amino acids, which may be the same or different amino acids; Further comprising: Immunofiber compositions are provided.

[0024] According to another embodiment, the present invention provides a method for the preparation of a polypeptide comprising an antibody-binding peptide attached to a linear hydrocarbon chain. Methods for preparing immunofiber compositions containing one or more immuno-amphiphiles wherein the antibody-binding peptide is a Staphylococcus aureus The hydrophilic amino acid sequence of the Z33 peptide of protein A, or a functional part or fragment thereof It has a segment or derivative. [Brief explanation of the drawings]

[0025] [Figure 1] Figures 1A-1C. (1A) Schematic diagram of the Z33 peptide binding to the Fc-portion of IgG. (1B) Sequences of C12-Z33 and 2C8-Z33. The alkyl group and Z33 are shown as yellow and blue shaded regions, respectively. The two α-helices in the Z33 peptide are underlined. (1C) Schematic diagram of the self-assembly of R-Z33 IF and the binding between IF and IgG. [Figure 2] Figures 2A-2F. (2A) Schematic of C12-Z33 self-assembly. (2B) Normalized CD spectra of Z33 peptide and Z33-C12 at pH 7.4 and 2.8. TEM characterization of C12-Z33 at pH 7.4 (2C, D) and 2.8 (2E, F). TEM samples were prepared at a concentration of 100 μM in PBS (pH 7.4) and IgG elution buffer (pH 2.8), respectively. The TEM samples were negatively stained with 2 wt% uranyl acetate. [Figure 3] Figures 3A-3D. ITC profiles of 100 μM C12-Z33 added to a solution of 2 μM IgG1 in (3A) PBS buffer (pH 7.4) and (3B) IgG elution buffer (pH 2.8) at 15 °C. ITC profiles of 100 μM (3C) Z33 and (3D) C12-SZ33 added to a solution of 2 μM IgG1 in PBS at 15 °C, pH 7.4. [Figure 4]Figures 4A-4E. TEM characterization of (4A) 2C8-Z33 with a diameter of 16.8 ± 1.5 nm in PBS at pH 7.4 and (4B) 2C8-Z33 with a diameter of 17.3 ± 1.9 nm in IgG elution buffer at pH 2.8. The TEM sample preparation was similar to that for C12-Z33. (4C) Normalized CD spectrum of 100 μM 2C8-Z33 in PBS at pH 7.4 showed an α-helical secondary structure. (4D) ITC profile of 100 μM 2C8-Z33 dropped into a solution of 2 μM IgG1 in PBS buffer (pH 7.4) and (4E) IgG elution buffer (pH 2.8). [Figure 5] Figures 5A-5D. (5A) Schematic diagram of IF-IgG complex precipitation by 0.6 M Na2SO4 solution. (5B) Photographs of 5 mM C12-Z33 in PBS before (i) and after (ii) the addition of 0.6 M Na2SO4, and of 20 μM IgG1 in PBS containing (iii) 5 mM C12-Z33, (iv) 0.6 M Na2SO4, and (v) 5 mM C12-Z33 and 0.6 M Na2SO4. Precipitation was observed in (ii) and (v). (5C) Absorbance spectra of C12-Z33 and IgG1 + C12-Z33 complex before and after the addition of 0.6 M Na2SO4. Net IgG1 in the supernatant is derived from the IgG1 + C12-Z33 supernatant minus the C12-Z33 supernatant. (5D) Absorbance spectra for 2 mM C12-SZ33 and IgG1+C12-SZ33 complex before and after addition of 0.6 M Na2SO4. [Figure 6] 6A-6B. (6A) Schematic diagram of the design of exemplary embodiments of Helix 1 and Helix 2-based peptide amphiphiles by direct alkylation at C16 and 2C8, respectively. (6B) Schematic diagram of the self-assembly of IA molecules into one-dimensional nanostructures. [Figure 7]Figures 7A-7B. TEM images of various IAs. (7A) Helix1-C16 and (7C) C16-Helix2 show nanofiber morphologies with diameters of 9.5 ± 1.2 nm and 12.4 ± 1.7 nm, respectively. (7B) Helix1-2C8 and (7D) 2C8-Helix2 show nanobelt morphologies with diameters of 10-70 nm and 22.9 ± 1.5 nm, respectively. All samples were prepared in 1 mM water, pH 7.4, and aged overnight before imaging. The TEM samples were negatively stained with 2 wt% uranyl acetate. Scale bar: 200 nm. [Figure 8] Figures 8A-8D. Emission spectra of the reporter dye Nile Red when incubated with (8A) Helix 1-C16, (8B) C16-Helix 2, (8C) Helix 1-2C8, and (8D) 2C8-Helix 2 to determine critical micelle concentration (CMC) values. All spectra shown here are normalized by the emission maximum and exhibit a blue shift when the conjugate concentration exceeds the CMC. The CMC range for each IA is boxed in the legend. Units: μM. [Figure 9] 9A-9B. Normalized CD spectra for 100 μM (9A) Helix 1, Helix 1-C16, Helix 1-2C8, and (9B) Helix 2, C16-Helix 2, 2C8-Helix 2 in water at pH 7.4. [Figure 10] 10A-10B. Normalized CD spectra for various concentrations of (10A) Helix 1-2C8 and (10B) Helix 1-C16 in water at pH 7.4. Concentrations are in μM. [Figure 11]Figures 11A-11F. TEM images of various IAs. (11A) TEM images of Helix 1-C12 and (11B) C12-Helix 2 show nanofiber morphologies with diameters of 12.9 ± 0.9 nm and 13.9 ± 1.5 nm, respectively. Scale bar: 200 nm. Emission spectra for the reporter dye Nile Red upon incubation with (11C) Helix 1-C12 and (11D) C12-Helix 2 to determine critical micelle concentration (CMC) values. Concentrations are in μM. Normalized CD spectra for 100 μM (11E) Helix 1, Helix 1-C8, Helix 1-C12, and (11F) Helix 2, C8-Helix 2, and C16-Helix 2 in water at pH 7.4. [Figure 12] Figure 12. Chemical structures of several exemplary embodiments of fragments of the antibody-binding peptide sequences of Helix1, Helix2, and Z33. [Figure 13] Figure 13. Chemical structures of some exemplary fragments of the antibody-binding peptide sequences of Helix1-C8, Helix1-C12, Helix1-C16, and Helix1-2C8. [Figure 14] Figure 14. Chemical structures of fragments of some exemplary antibody-binding peptide sequences: C8-Helix2, C12-Helix2, C16-Helix2, and 2C8-Helix2. [Figure 15] Figures 15A-15B. Emission spectra for the reporter dye Nile Red upon incubation with (15A) Helix 1-C8 and (15B) C8-Helix 2 to determine critical micelle concentration (CMC) values. All spectra shown here are normalized by the emission maximum. Concentrations are in μM. No detectable peak shift was observed even when the conjugate concentration reached 100 μM. [Figure 16] Figure 16. Normalized CD spectra of Helix1, Helix2, and Z33 at 100 μM in water at pH 7.4. [Figure 17]Figure 17. Analysis of CD spectra of Helix 1- and Helix 2-based IAs. The content of three major secondary structures in (A) Helix 1-based and (B) Helix 2-based molecules. To determine the approximate α-helix, β-sheet, and random coil peptide secondary structures, linear combinations of polylysine-based spectra were used to fit the CD data in the 200-240 nm range. [Figure 18] Figure 18. ThT dye fluorescence with 100 μM Helix 1 and Helix 1-based peptide amphiphiles in deionized water. [Figure 19] Figure 19. (19A) Chemical structure of another embodiment of the binding molecule C12-VVKKGGZ33 and the spacer molecule C12-VVEE. An alkyl tail (orange) was attached to the N-terminus of the peptide sequence. Two valines (VV, red) promote the formation of a one-dimensional structure. Two glutamic acids (EE, blue) are designed as hydrophilic segments in the spacer molecule, and two lysines (KK, blue) are designed for electrostatic interactions between KK and EE, thus allowing alternating packing of the binding molecule and the spacer molecule. Two glycines (GG, green) are designed to further separate Z33 from the alkyl chain. (B) Schematic diagram of the coassembly of C12-VVKKGGZ33 and C12-VVEE. The density of binding molecules on the surface of the coassembled immunofiber can be easily controlled by adjusting the molar ratio of the binding molecule to the spacer molecule. [Figure 20] Figure 20. Representative TEM images of (20A) self-assembled C12-VVEE and (20B) self-assembled C12-VVKKGGZ33. [Figure 21]Figure 21. IgG binding and precipitation yields. (21A) IgG percentage in the supernatant of 20 μM IgG after incubation of C12-VVEE and C12-VVKKGGZ33 at molar ratios of 5:1, 10:1, 25:1, 50:1, and 100:1, followed by addition of 1 M ammonium sulfate. Molar ratio of C12-VVKKGGZ33 to IgG: 10:2. (21B) IgG percentage in the supernatant of 20, 10, and 5 μM IgG after incubation of C12-VVEE and C12-VVKKGGZ33 at molar ratios of 25:1 and 50:1, followed by addition of 1 M ammonium sulfate. The molar ratios of C12-VVKKGGZ33 to IgG are 10:4, 10:2, and 10:1. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of the Invention Staphylococcal protein A (SPA) is a protein found in the cell wall of Staphylococcus aureus. It is a protein first discovered in the United States. It consists of five helical strands folded into a three-helix bundle. Protein A is composed of homologous domains from most mammalian species, including humans. It plays an important role in immunology because it specifically binds to the Fc portion of immunoglobulin G (also known as IgG) Protein A has been the subject of intensive structural and biochemical research. In 1984, the first gene encoding SPA was cloned and sequenced, and Subsequently, numerous, synthetic and minimal IgG binding domains based on Protein A were synthesized. Among them, the Z-58 domain has been used for affinity chromatography and affinity chromatography. It is the first and most well-known synthetic domain widely used in precipitation. A modified binding domain, Z-33, was developed in 1996, but the function of the molecule has changed significantly. not present.

[0027] According to some embodiments, the present invention relates to the amino acid sequence of the antibody binding domain of an IF. Methods for modifying and / or derivatizing immuno-amphiphiles to act as binding units Described herein are methods for producing IgG antibodies or portions or fragments thereof. This is an example of designing and preparing IFs that are useful for binding to IFs in aqueous solutions in the physiological pH range. Once the surface is formed, exposed biologically active epitopes (binding sites) specific to IgG are bound to the surface. They can be heterologously linked.

[0028] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. Self-assembling immunofibers comprising immuno-amphiphiles are provided.

[0029] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. Provided are self-assembling immunofibers comprising one or more immuno-amphiphiles. and wherein the immuno-amphiphile, when in aqueous solution at physiological pH, is an α-heterocyclic compound. It has a ribrick structure.

[0030] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. Provided are self-assembling immunofibers comprising one or more immuno-amphiphiles. wherein the antibody-binding peptide is a protein of Staphylococcus aureus. A hydrophilic amino acid sequence derived from the Z33 peptide of erythropoietin A, or a functional part or fragment thereof. This includes nitrites or derivatives.

[0031] According to one embodiment, the present invention comprises an antibody-binding peptide attached to a linear hydrocarbon chain. Provided are self-assembling immunofibers comprising one or more immuno-amphiphiles. wherein the antibody-binding peptide has the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1), or a functional part, fragment, or derivative thereof Includes.

[0032] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. Self-assembling immunophiles containing one or more immuno-amphiphiles containing a segment Offer a bar.

[0033] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. Self-assembling immunophiles containing one or more immuno-amphiphiles containing a segment and wherein the immuno-amphiphile is in aqueous solution at physiological pH. In some cases, it has an α-helical structure.

[0034] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. Self-assembling immunophiles containing one or more immuno-amphiphiles containing a segment and wherein the immuno-amphiphile is in aqueous solution at physiological pH. In some cases, it has a β-sheet structure.

[0035] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. Self-assembling immunophiles containing one or more immuno-amphiphiles containing a segment wherein the antibody-binding peptide is selected from the group consisting of Staphylococcus aureus, ... It has a hydrophilic amino acid sequence portion of the Z33 peptide of protein A of B. reus.

[0036] As used herein, the term "fragment of an antibody-binding peptide" refers to a fragment of an antibody-binding peptide having the sequence set forth in SEQ ID NO: (S Staphylococcus aureus protein with less than 33 amino acids of EQ ID NO: 1 In some embodiments, the term "Z33" refers to a portion or fragment of the Z33 peptide of phenotype A. A "fragment of an antibody-binding peptide" is a fragment of an antibody-binding peptide having at least one α-helical The fragment of the antibody-binding peptide refers to a portion of the Z33 peptide containing the peptide having the antibody-binding region. The segment may also contain additional amino acids, including the α-helical region. It may or may not contain

[0037] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. and providing an immuno-amphiphile comprising a fragment of said antibody-binding peptide, wherein said fragment of said antibody-binding peptide is a fragment of said antibody-binding peptide. The cleavage fragment contains the amino acid sequence of FNMQQQRRFYEALHD (SEQ ID NO: 2), and is located between Helix 1 and In some embodiments, Helix 1 is referred to as FNMQQQRRFYEALHDK (SEQ ID NO: 3 In another embodiment, Helix 1 comprises the amino acid sequence of FNMQQQRRFYEALHDKK (SEQ ID NO:(S) EQ ID NO): 4).

[0038] According to one embodiment, the present invention provides a fragment of an antibody-binding peptide attached to a linear hydrocarbon chain. and providing an immuno-amphiphile comprising a fragment of said antibody-binding peptide, wherein said fragment of said antibody-binding peptide is a fragment of said antibody-binding peptide. The ment contains the amino acid sequence of PNLNEEQRNAKIKSIRDD (SEQ ID NO: 5), and In some embodiments, Helix 2 is designated as FPNLNEEQRNAKIKSIRDD (SEQ ID NO: 2). NO): 6).

[0039] As used herein, the term "immuno-amphiphile" refers to an "immunofiber." This refers to molecules that can spontaneously assemble into distinct, stable supramolecular nanostructures called IFs. Generally, the IF of the present invention may aggregate in the pH range of about 2.8 to about 7.5. However, their binding properties also depend on pH. More positively charged IFs bind better in higher pH solutions. Conversely, negatively charged IFs associate more readily in lower pH solutions. do.

[0040] In some embodiments, the immuno-amphiphiles of the present invention comprise a carbon atom having between 8 and 22 carbons. The peptides may be linear or branched. There is an upper limit to the number of carbon atoms from the viewpoint of solubility in aqueous solution. Increased water solubility of the construct and, without limitation, favorable secondary structure formation (e.g., , beta-sheet, alpha-helix, poly-proline type II helix, beta-ta Depending on the type of nanoparticle, cylindrical or spherical micelles, hollow nanotubes, toroids, discs, and small particles can be produced. This may promote the formation of well-defined nanostructured structures such as cells.

[0041] As used herein, the term "hydrocarbon chain" is synonymous with the term "aliphatic chain." This is an art-recognized term and includes straight chain, branched chain, and In certain embodiments, the compounds of the present invention include cyclic alkanes, alkenes, or alkynes. The aliphatic group is linear or branched and has 8 to 22 carbon atoms.

[0042] The term "alkyl" is art-recognized and its use herein refers directly to Included are saturated aliphatic groups, including single chain alkyl groups and branched chain alkyl groups.

[0043] As used herein, the term "antibody-binding peptide" refers to a peptide having a length of about 10 -6 M~about 10 -10 Between M K d It binds to a specific portion of an antibody or antibody molecule, such as the Fc portion, with high specificity, e.g., The term "peptide" refers to a peptide that can

[0044] In some embodiments, the antibody-binding peptide is a peptide that binds to Staphylococcus aureus. Hydrophilic amino acids of the Z33 two-helix derivative peptide of the Z-domain of Protein A from Bacillus aureus sequence, or a functional part or fragment or derivative thereof.

[0045] As used herein, the Z33 peptide of Protein A is FNMQQQRRFYEALHDPNLNEEQ It has the amino acid sequence of RNAKIKSIRDD (SEQ ID NO: 1).

[0046] In some embodiments, the antibody-binding peptide comprises a protease linked to a linear hydrocarbon chain. a fragment of the Z33 peptide of tein A, wherein said fragment of the antibody-binding peptide The component contains the amino acid sequence FNMQQQRRFYEALHD (SEQ ID NO: 2) and is designated Helix 1. In some embodiments, Helix 1 is FNMQQQRRFYEALHDK (SEQ ID NO: 3). In another embodiment, Helix 1 comprises the amino acid sequence of FNMQQQRRFYEALHDKK (SEQ ID NO: Q ID NO): 4).

[0047] In some embodiments, the antibody-binding peptide comprises a protease linked to a linear hydrocarbon chain. a fragment of the Z33 peptide of tein A, wherein the antibody-binding peptide is PNLNEE It contains the amino acid sequence of QRNAKIKSIRDD (SEQ ID NO: 5) and is called Helix 2. In some embodiments, Helix 2 is selected from the group consisting of amino acids FPNLNEEQRNAKIKSIRDD (SEQ ID NO: 6). Contains the acid sequence.

[0048] According to another embodiment, the present invention provides a method for the preparation of a polypeptide comprising an antibody-binding peptide attached to a linear hydrocarbon chain. and an immunofiber composition comprising one or more immuno-amphiphiles, wherein: The antibody-binding peptide is Z33 of protein A of Staphylococcus aureus. Hydrophilic amino acid sequence of a peptide, or a functional part, fragment, or derivative thereof In some embodiments, the functional portion, fragment, or derivative is selected from the group consisting of SEQ ID NOS: 1 to 6.

[0049] Other binding peptides may be used in place of the Z33 peptide to bind to other proteins. Those skilled in the art will understand that other suitable antibodies can be used, such as streptavidin. or a functional part or fragment thereof into said immuno-amphiphile. The resulting IF may be used to bind biotinylated compounds.

[0050] The term "amino acid" includes naturally occurring α-amino acids in either the D or L form (e.g., Ala, Arg, Asn, Asp, Cy s, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val ), as well as β-amino acids, synthetic amino acids and unnatural amino acids. amino acid residues are useful in such polypeptides, and the present invention also provides for the natural, genetically encoded amino acid residues. The amino acids that may be utilized in the peptides described herein are not limited to amino acids. Examples of carboxylic acids are given in, e.g., Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, Vol. cular Biology, CRC Press, Inc., and the references cited therein. The RSP Amino Acids LLC website is another source of a wide range of amino acid residues. be.

[0051] References herein to "derivatives" include derivatives of portions of the antibody-binding peptides of the invention, such as flagella. Derivatives also include single or multiple amino acid substitutions, deletions and deletions. Homologues include those from the same species of snake, or from the same genus or genus of snakes. These include functionally, structurally or stereochemically similar peptides derived from the venom of the Streptococcus miliarius. All such homologues are contemplated by the present invention.

[0052] Analogs and mimetics may contain non-naturally occurring amino acids or may be derived from amino acids These include molecules that behave functionally similarly to the peptides, despite not being specifically targeted to the peptide. Screening natural products is one useful method for identifying analogs and mimetics. This is a useful strategy.

[0053] Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to: Although not a steroid, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentaerythritol, Acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omit amine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and and / or D-isomers of amino acids. A partial list of natural amino acids is given in Table 1.

[0054] [Table 1-1] [Table 1-2] [Table 1-3]

[0055] Analogs of peptides of interest contemplated herein include those derived from side chain modifications, peptide synthesis, Incorporation of unnatural amino acids and / or their derivatives into peptide molecules or their analogs, including the use of cross-linking agents and other methods that impose structural constraints. .

[0056] Examples of side chain modifications contemplated by the present invention include reaction with an aldehyde followed by NaBH4 reductive alkylation with methyl acetimidate; amidation with acetic anhydride; Silation; Carbamoylation of amino groups with cyanates; 2,4,6-Trinitrobenzenesulfonyl Trinitrobenzylation of amino groups with succinic anhydride (TNBS); succinic anhydride and tetrahydrobenzoic anhydride. Acylation of amino groups with phthalic acid; and reaction with pyridoxal-5-phosphate. Examples of amino group modification include pyridoxylation of lysine by reduction with NaBH4. can be done.

[0057] The guanidine group of the arginine residue is converted to guanidinium by 2,3-butanedione, phenylglyoxal, and It may be modified by the formation of heterocyclic condensation products with reagents such as dioxal.

[0058] The carboxyl group is activated by carbodiimide activation via O-acylisourea formation, followed by derivatization. It may be modified by derivatization (eg, to the corresponding amide).

[0059] Carboxymethylation of sulfhydryl groups with iodoacetic acid or iodoacetamide; Performic acid oxidation to cysteic acid; mixed disulfide formation with other thiol compounds; maleic acid Reaction with maleic anhydride or other substituted maleimides; 4-chloromercuric benzoic acid, 4-salt Mercuric chloride phenylsulfonic acid, phenylmercuric chloride, 2-mercuric chloride-4-nitrophenol formation of mercury derivatives with mercury, and other mercurials; with cyanate at alkaline pH; It may be modified by methods such as carbamoylation.

[0060] Tryptophan residues can be converted to hydroxy groups by, for example, oxidation with N-bromosuccinimide or 2-hydroxybenzoates. Indole ring cleavage with 5-nitrobenzyl bromide or sulfenyl halides Tyrosine residues may be modified by alkylation. It may also be modified by nitration to form a 3-nitrotyrosine derivative.

[0061] Modification of the imidazole ring of histidine residues was performed by alkylation with iodoacetic acid derivatives or diene. By N-carbethoxylation with ethyl pyrocarbonate good.

[0062] The crosslinking agent may be, for example, (CH2) n Bifunctional imidoesters with spacer groups, Homobifunctional crosslinkers such as glutaraldehyde and N-hydroxysuccinimide esters, and amino-reactive moieties such as N-hydroxysuccinimide and maleimide or dithio The heterocycle usually contains another group-specific reactive moiety such as a moiety (SH) or a carbodiimide (COOH). Bifunctional reagents may be used to stabilize the 3D structure. Chid, for example, C α and N α -methylamino acid incorporation, C of amino acid α Atoms and C β A double bond is introduced between the N and C termini, between two side chains, or between a side chain and N. Alternatively, a cyclic peptide can be formed by introducing a covalent bond such as an amide bond between the C-terminus and the cyclic peptide. The structure may be constrained by forming a bond or analogue.

[0063] As used herein, the term "peptide" refers to a peptide having a length of 4 to 100 amino acid residues, preferably comprises a sequence of about 10 to 80 residues in length, more preferably 15 to 65 residues in length, In a cyclic amino acid, the α-carboxyl group of one amino acid is attached to the main chain of the adjacent amino acid by an amide bond. It is linked to an (α- or β-) amino group.

[0064] In general, in some embodiments, the present invention provides a method for purifying a protein or peptide of interest. The present invention provides a method for immunoprecipitation using the immunofiber of the present invention. The antibody-binding peptide portion of the immunofiber binds to the protein or peptide as follows: can be combined.

[0065] According to one embodiment, the present invention provides an immunoglobulin molecule comprising the Fc portion or a functional portion thereof. The present invention provides a method for purifying a peptide or protein having a fragment thereof, which comprises one or more A solution at a first pH level containing the peptide or protein of interest is added to one or more immuno-amphiphiles. contacting the subject with an immunofiber composition comprising one or more immunogenic substances, - the amphiphile comprises an Fc-binding peptide attached to a hydrocarbon chain, wherein the Fc-binding peptide is a hydrophilic amino acid of the Z33 peptide of protein A from Staphylococcus aureus. one or more having an amino acid sequence, or a functional portion or fragment or derivative thereof; The protein of interest is bound to the Fc-binding peptide, or a functional part or fragment thereof. adjusting the pH level of the solution to change the structure of the Fc-binding peptide; A step of changing the pH to a level that changes the structure so that it no longer binds to one or more proteins of interest. and extracting the dissociated protein or proteins of interest from the solution.

[0066] Generally, an Fc-containing protein is an immunoglobulin or antibody (e.g., IgG type), or an Fc The Fc-containing protein may be a fusion peptide or protein containing a portion thereof. The protein in the sample is mixed with the immunofiber of the present invention in aqueous solution and physiological pH. and binding the immunofiber to the Fc portion of the protein molecule of interest. In one embodiment, the immunofiber is purified by The Z33 portion of the polypeptide A is included, and the Fc portion of an IgG molecule, or a fusion peptide or protein containing the same. It is specific to proteins.

[0067] According to some other embodiments, the immunofiber is then filtered using various filtration methods. and separating the protein from the associated protein using a method (e.g., diafiltration, microfiltration, or ultrafiltration). do.

[0068] In one embodiment, the immunofiber composition of the present invention comprises a linear hydrocarbon-linked In one embodiment, the antibody comprises two or more fragments of the Z33 peptide of Protein A. The immunofibers used in the purification or binding methods contain Helix 1 and Helix 2 peptides. For example, the method may include a mixture of peptides of SEQ ID NOS: 2-4. adding one or more Helix 1 peptides having a nucleotide sequence, and combinations thereof; and one or more Helix 2 peptides having the peptide sequence of SEQ ID NO: 5 or 6. and combinations thereof.

[0069] After the time to bind the immunofibers has elapsed, the immunofibers are released into the solution. The immunofiber-protein complex is then formed in the For example, the salts can be separated by a number of known separation methods, including inducing precipitation with salts and centrifugation. The unbound fiber and protein in the sample can be separated from other components. The separated complex may then be added to another solution at an acidic pH, The muonofibers then lose their binding affinity for the protein. Proteins are removed from the dissociated immunofibers by filtration, such as diafiltration or other means. The dissociated monomers may be removed as well.

[0070] Porous resin (such as beaded agarose) or magnetic beads, or immobilization substrates along with other protein purification methods such as covalent immobilization onto proteins and immunoprecipitation. It is contemplated that the immunofibers of the present invention may be used.

[0071] As used herein, the term "sample" refers to a sample containing the immunofiber of the present invention. It refers to any sample or solution or liquid containing the antibody of interest that can be used to bind. In some embodiments, the sample may be a biological sample.

[0072] In accordance with one or more embodiments of the present invention, the term "biological sample" or "biological fluid" refers to " refers to, but is not limited to, a living or formerly living patient or mammalian source. It is understood that the term "substance" includes any quantity of a substance, such as blood, serum, plasma, Urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovial macro These include, but are not limited to, phages, endothelial cells, and skin.

[0073] The sequences, CMCs, and secondary structures of all IAs in this study are summarized in Table 1. Both and double-stranded alkylation can lead to the formation of one-dimensional filaments, The morphology, CMC, and secondary structure are different. The length and number of alkyl chains affect the identity of the peptide conjugate. For example, Jan van Hest et al. The GANPNAAG (SEQ ID NO: 8) peptide conjugated with a shorter alkyl chain formed aggregates. However, it was found that fibrous aggregates and tubular structures were observed, respectively. , C14 conjugates and C16 or longer conjugates. As reported by researchers, the CMC of single-chain alkylated amphiphiles is higher than that of IAs due to the promotion of their aggregation. The increased hydrophobicity of the alkyl chains decreases with increasing alkyl chain length. The alkyl chain conjugate enhances the stability of the α-helix secondary structure, and It has been previously demonstrated that the amount of α-helix for peptide formation increases. For example, SC4 peptide-amphiphilic An increase in the bactericidal activity of the substance was found by Mayo and Tirell. Our results are in line with those of Forns et al. The degree of helicity of the 16-residue peptide appears to be inconsistent with the system examined by This discrepancy increases with chain length. However, the discrepancy is not significant for the unbound 16-residue peptide. This may be caused by the pre-existing α-helical structure in the protein. showed that longer N-terminally alkylated 2α-helical peptides showed faster α to β transition. We found that the long alkyl chains promoted the formation of β-sheets.

[0074] In conclusion, two families of immuno-amphiphiles have been successfully designed and synthesized. Through molecular design, various alkylation methods, IA molecules can be modified to have different CMC values, morphologies and dimers. Some self-assembly properties, such as the composition of the secondary structure, were found to vary. Our results show that both single-stranded and double-stranded alkylation induce the formation of one-dimensional filaments. This clearly shows that longer single alkyl chains can promote the aggregation of IA molecules. The double-chain alkylation could increase the formation of β-sheets. The formation of α-helices in the ligated filaments may be promoted. However, there is variability in the effect of alkylation among various peptide molecules. This strategy of modulating the number of nucleotides can be further developed and used to produce the desired biological activity. In other self-assembled functional peptide systems, which require specific secondary structures for their synthesis, It can be used.

[0075] According to some embodiments, the peptides and fragments thereof are alkylated using The alkyl chains used for this purpose are 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22 and 24 carbon atoms. The peptides may have carbon lengths from 2 to 24 carbons, including intermediate lengths. The peptide or fragment thereof is alkylated, It may have alkyl chains.

[0076] Another embodiment of the immunofiber composition of the present invention. Previous studies by the present inventors have demonstrated that self-assembled C12-Z33 immunofibers can be efficiently synthesized. The immunofiber showed high IgG binding affinity and IgG deposition ability. Considering the loading, the first immunofiber design mentioned above includes a 10 nanometer The ligand accessibility of the Z33 ligand to an IgG molecule with a diameter of 1 / 4 of a meter is The drawback is that the high density of Z33 ligands may be limited. The surface of the bar is present, but due to steric hindrance, it is difficult to bind IgG and cross-link immunofibers. It was thought that this could prevent the formation of large aggregates that precipitate. Therefore, another embodiment of the immunofiber composition of the present invention is provided.

[0077] According to one or more embodiments, the improved immunofiber binding system comprises a binding molecule (an alkyl group). The antibody-binding peptide (alkyl-XXYYZZ-antibody binding peptide) and the spacer molecule (alkyl-XXBB) were co-assembled. wherein the first spacer pair is provided by combining the first spacer pair in a fiber system. The peptides contain sequences of the general formula XXYYZZ, where XX is a divalent amino acid having a small hydrophobic side chain. YY is a positive integer and is one of two amino acids, which may be the same or different amino acids. Two amino acids with charged side chains, which may be the same or different ZZ are two amino acids with small neutral side chains and are the same amino acid. It may be a cyclic or different amino acid, and may further have a linear hydrocarbon chain at the N-terminus. Immunofiber spectrophotometers bound to peptide sequences containing the general sequence XXBB wherein XX are two amino acids with small hydrophobic side chains; and may be the same or different amino acids, and wherein BB is a negatively charged Two amino acids with side chains, which may be the same or different amino acids. be.

[0078] In an exemplary embodiment, the immunofiber-binding molecule contains 4 to 8 amino acids. and in some embodiments, compared to the C12-Z33 immunofibers provided above. 6 amino acids containing VVKKGG (SEQ ID NO: 9) between the carbon chain and the antibody-binding peptide Z33 Two hydrophobic amino acids, such as valine (VV, red), form a one-dimensional structure. Two positively charged amino acids, such as glutamic acid (EE, blue), are inserted between the spacer molecules. The hydrophilic portion of the molecule is designed to contain two negatively charged amino acids, such as lysine (KK, blue). Designed for electrostatic interactions between positively and negatively charged amino acids, binding and and spacer immunofiber molecule C12-VVKKGGZ33 are packed alternately. Two neutral amino acids, such as glycine (GG, green), were added to the alkyl chain to form an antibody molecule, such as Z33. The binding peptide was designed to further separate the binding molecule and the spacer molecule in aqueous solution. When dissolved, these two molecules form a one-dimensional structure with the binding ligand Z33 protruding above the surface. Uniform co-assembly into immunofibers was predicted (Figure 19).

[0079] According to a further embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: Antibody-binding peptide (Antibody-binding peptide is attached at its N-terminus to a linear hydrocarbon chain) , and the first spacer peptide (the first spacer peptide is P. aeruginosa at its C-terminus) the hydrophilic amino acid sequence of the Z33 peptide of protein A of Staphylococcus aureus, or Binding to an antibody-binding peptide having a functional portion, fragment, or derivative thereof an immunofiber-binding molecule (wherein the first spectrophotometer is a Ser-peptides contain sequences of the general formula XXYYZZ, where XX is a small hydrophobic side chain. and may be the same or different amino acids. YY are two amino acids with positively charged side chains, and may be the same or different amino acids. ZZ are two amino acids with small neutral side chains, and amino acids), which may be the same or different amino acids and Immunofiber spacer molecule (Immunofiber spacer molecule is the N At the end, it has a linear hydrocarbon chain attached to a peptide sequence containing the general sequence XXBB. where XX are two amino acids with small hydrophobic side chains and are the same amino acid or can be different amino acids, and where BB represents two amino acids with negatively charged side chains. amino acids, which may be the same or different amino acids; Further comprising: Immunofiber compositions are provided, for example, those with the amino acid sequence VVEE (SEQ ID NO: : 10) to prepare C12-VVEE as an immunofiber spacer peptide. This may happen.

[0080] The term "amino acid with a hydrophobic side chain" refers to amino acids such as Ala, Val, Ile, and Leu. Those skilled in the art will understand that the term "amino acid having a positively charged side chain" means Arg, His, and Lys. The term "amino acid with a small neutral side chain" means Gly. or Pro. The term "amino acid with a negatively charged side chain" refers to an amino acid such as Asp Or it means an amino acid such as Glu.

[0081] The immunofiber-binding molecule of the present invention (alkyl-XXYYZZ-antibody-binding peptide) and spacer Immunofiber compositions containing the sar molecule (alkyl-XXBB) and other immunofibers The binding system may comprise antibodies and other molecules containing the Fc portion of an antibody, or a portion or fragment thereof. In some embodiments, the immunofibers of the present invention are useful for purifying antibodies against erythrocytes. The composition may be prepared by dissociating any peptide or fusion protein comprising at least a portion of the Fc region of an antibody molecule. It may be used to isolate or purify

[0082] The removal of the last remaining trace impurities from purified proteins is generally performed by polishing. This is called a "licensing" because the preparation is intended for research, diagnostic, or even therapeutic use. In such cases, protein impurities may cause unwanted side effects and should be avoided in subsequent Polishing is generally a separate step from the previous step. This is done by using separation methods, most commonly gel permeation chromatography Despite its polishing effect, this technique has a poor separation capacity. and low productivity, resulting in slow speed. Other approaches, based on hydrophobic chromatography, require optimization for a given process. In some embodiments, the method may be used in a variety of ways, such as: A further polishing step is used to purify the protein of interest.

[0083] Therefore, in view of the above, the present invention provides a method for producing an antibody or Fc-containing peptide or or a method for purifying a protein comprising: a) preparing a soluble immunoglobulin (Ig) comprising: The sample containing the composition was dissolved in an aqueous solution and a physiological pH solution and aged overnight. b) mixing an antibody-containing sample with the IF solution and allowing it to self-assemble into IFs; IF is bound to the Fc portion of an immunoglobulin molecule or an Fc-containing peptide or protein, and Immunofiber-Fc immunoglobulin or immunofiber-Fc containing peptide in liquid or forming a protein complex; c) adding salt and centrifuging The immunofiber-Fc immunoglobulin or immunofiber-Fc-containing peptide d) separating the peptide or protein complex from the solution; Dissociating the IF from IF or Fc-containing peptides or proteins and extracting unbound immunoglobulins. Recovering the immunoglobulin or Fc-containing peptide or protein.

[0084] The following examples provide guidance for practicing representative embodiments of the subject matter disclosed herein. It is included to provide a more complete understanding of the present invention in light of this disclosure and the general level of skill of those skilled in the art. Therefore, it is understood that those skilled in the art will recognize that the following examples are intended to be merely illustrative and not to be outside the scope of the presently disclosed subject matter. It is understood that numerous changes, modifications, and alterations may be made without departing from the spirit and scope of the present invention. The following synthetic descriptions and specific examples are intended for illustrative purposes only. Any restriction on making the compounds of the invention by other methods It should not be construed as such. [Example]

[0085] Example 1 material. All Fmoc amino acids and resins were purchased from Advanced Automated Peptide Protein Technologies (A Fmoc-Lys (Fmoc) was purchased from Novabiochem (San Diego, CA). Therapeutic human IgG1 (IgG1) was obtained from Bristol-Myers Squibb (Boston, MA, USA). SA), and IgG elution buffer was obtained from Thermo Fisher Scientific (Rockford, IL, USA). All other reagents were obtained from VWR (Radnor, PA, USA) and were used without further purification. I used it as received.

[0086] Molecular synthesis. The C12-Z33 and 2C8-Z33 immuno-amphiphiles were synthesized using a similar method. The Z33 peptide was first synthesized using a standard 9-fluorenylmethoxycarbonyl (Fmoc) solid phase Synthesis protocol was used on a Focus XC automated peptide synthesizer (AAPPTEC, Louisville, KY). The C12 (or 2C8) alkyl chain was then attached to the Z33 peptide by shaking overnight at room temperature. Lauric acid (or octanoic acid) / HBTU / DIEA in a ratio of 4 (or 8):4:6 to the Z33 peptide The other alkyl chains were manually coupled at the N-terminus of the amide (after Fmoc removal). Various IAs were generated by manual coupling at the N-terminus or lysine (K) side chain of the peptide. The resulting mixture was shaken overnight at room temperature. Fmoc deprotection was carried out using 20% ​​4-methylpiperidine in DMF. The reaction was carried out for 10 minutes and repeated once. In all cases, the ninhydride The reaction was monitored by phosphorus test (Anaspec Inc, Fremont, CA). TF in a ratio of 92.5:5:2.5 The reacted peptide was cleaved from the solid support using a mixture of A / TIS / H2O for 2.5 hours. The excess TFA was removed by rotary evaporation, and the resulting solution was added to cold distillate. The crude peptide was precipitated by adding diethyl ether. The precipitated peptide and diethyl ether were separated by centrifugation. The ethyl ether was separated at 6000 rpm for 3 min. The peptides were washed twice more with diethyl ether. The solution was then removed by centrifugation.

[0087] Molecular synthesis of IA using fragments of antibody-binding peptides. Peptide amphiphiles were synthesized using a similar method. The synthesis process is shown below. To illustrate this, we take Helix1-C16, Helix1-2C8, C16-Helix2, and 2C8-Helix2 as examples. As a result, FNMQQQRRFYEALHDK (Helix1+Kmtt) (SEQ ID NO: 3) and FNMQQQRRFYEALH The peptide sequence of DKK (Helix1+Kmtt+Kmtt) (SEQ ID NO: 4) was first identified using a standard 9-Fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis protocol was used in the Focus XC automated preparation. K-methylthiotetrazole (Kmtt) was synthesized using a peptide synthesizer (AAPPTEC, Louisville, KY). was added to the C-terminus of the Helix 1 sequence for further reaction. The alkyl tail of octanoic acid (2C8) is in Helix 1+Kmtt and Helix 1+Kmtt+Kmtt, respectively. The side chains of Kmtt were manually coupled to generate Helix 1-C16 and Helix 1-2C8. The mixture was shaken at room temperature overnight. Similarly, for C16-Helix2 and 2C8-Helix2, IRDD (Helix 2) (SEQ ID NO: 5) and FPNLNEEQRNAKIKSIRDD (K-Fmoc-Helix 2) (SEQ ID NO: SEQ ID NO: 6) The peptide sequence was first synthesized on a Focus XC automated peptide synthesizer. -Fmoc was added to the N-terminus of the Helix 2 sequence for further reaction. Palmitic acid (C 16) or the alkyl tail of octanoic acid (2C8) was attached to the N-terminus of Helix 2 or to the K-Fmoc-Helix 2 Both the N-terminus and side chain of K-Fmoc were manually coupled, respectively, and the resulting mixture was then cooled to room temperature. Helix 1-C16 and Helix 1-2C8 were generated by shaking overnight. Fmoc deprotection was performed with 2 in DMF solution. The procedure was repeated once with 0% 4-methylpiperidine for 10 minutes. The reaction was tested using the ninhydrin test for isolated amines (Anaspec Inc, Fremont, CA). The reacted peptide was purified for 2.5 hours using a mixture of TFA / TIS / H2O in a ratio of 92.5:5:2.5. The resulting product was cleaved from the solid support. The excess TFA was removed by rotary evaporation. The crude peptide was precipitated by adding cold diethyl ether. The precipitated peptides and diethyl ether were separated at 6000 rpm for 3 minutes. It was washed twice more with ethyl ether and the solution was removed by centrifugation.

[0088] A Varian ProStar Model 325 preparative HPLC (Agilent Technology) equipped with a fraction collector was used. Varian Polymeric Column (PLRP-S, 100Å, 10 μm) at 25°C. IA was purified by preparative RP-HPLC using a column (150 × 25 mm) containing 0.1% v / v TFA. A water / acetonitrile gradient was used as the eluent at a flow rate of 20 mL / min. The Z33 peptide segment was monitored at 220 nm. The crude material was dissolved in 20 ml of 0.1% TFA in water. The collected fractions were dissolved in 10 ml of the solution, and 10 ml of each solution was injected for each purification step. The fractions were analyzed by MALDI-ToF (Bruker Autoflex III MALDI-ToF instrument, Billerica, MA) to identify the desired product. The fractions containing the product were lyophilized (FreeZone-105°C, 4.5 L lyophilizer, Labconco, Kansas City, CA). City, MO) and stored at -30°C.

[0089] Self-assembly and TEM imaging of immuno-amphiphiles. Immuno-amphiphiles at a concentration of 1 mM were pretreated with HFIP and then dissolved in 1x PBS or deionized water. and incubated overnight at room temperature; 10 μL of the 10-fold diluted sample was applied to a 400-mesh square carbon-copper filter. Spot the cells on a grid (EMS: Electron Microscopy Sciences) and remove the excess with filter paper. The sample was then removed by filtration to form a thin film of the sample on the grid. The sample was then allowed to dry for 5 minutes. Afterwards, 10 μL of 2% uranyl acetate was added to the sample grid, and the excess was removed after 30 seconds. All samples were allowed to dry for at least 3 h before TEM imaging.

[0090] Circular dichroism spectroscopy (CD). CD experiments on self-assembled IA samples were performed using a Jasco J-710 spectropolarimeter (JASCO, Easton, MD, USA). A 1 mm pathlength quartz UV-Vis absorption cell (ThermoFisher Scientific, Pittsburgh, PA) was used. The experiments were carried out using a PBS (Bio-Rad, Inc., USA) at 25°C. The samples were diluted from a 1 mM stock solution in 1x PBS prior to the experiment. The spectra were obtained from three scans in the wavelength range of 190–280 nm. The solvent background spectrum was collected as an average, and the sample spectrum was taken. The collected data were normalized with respect to sample concentration.

[0091] ITC experiment. Isothermal titration calorimetry experiments were performed using a high-precision VP-ITC titration calorimetry system (Microcal Inc.). The IgG1 solution was incubated at 15°C in 1x PBS (pH 7.4 or 2.8) for 1 hour. The IgG1 concentration was titrated using immuno-amphiphiles. The concentration of the immuno-amphiphile was calculated using a mass extinction coefficient of 1.4 at 100 nm. Anal. Biochem. 61.2 (1974): 623-627. The heat generated was determined from the integration of the colorimetric signal. The heat of reaction was obtained by subtracting the heat of dilution. Data were analyzed using MicroCal Origin (trademark). ) package was used.

[0092] CMC measurement. The CMC of Z33 fragment IA containing the fragmented antibody-binding peptide was measured at various concentrations. of these molecules by incubating them with a specific amount of Nile Red. A stock solution of Nile Red was first prepared by dissolving it in acetone at 50 μM. The stock solution was placed in several centrifuge tubes, and the solvent was evaporated at room temperature to obtain dry Nile Red. Peptide solutions of various concentrations were prepared in deionized water and then washed with water containing dried Nile red. The same volume was added to a centrifuge tube containing 100 ml of Nile Red and incubated overnight. The fluorescence was monitored with a Fluorolog fluorometer (Jobin Yvon, Edison, NJ) at a fixed excitation wavelength of 560 nm. The emission spectrum was monitored from 580 to 720 nm. The CMC of IA was characterized by a blue-shift in the emission maximum. This transfer is measured by the incubation of the peptides at their CMC Occurs when the value is exceeded.

[0093] Thioflavin T (ThT) spectroscopic assay. A ThT stock solution was prepared at 50 μM in deionized water. 100 μM Z33 IA containing ThT was vortexed and incubated with an equal volume of ThT stock solution for 1 h. The fluorescence intensity was then measured using a Fluorolog fluorometer (Jobin Yvon, Edison, NJ) with excitation at 440 nm (slit width 5 nm) and emission at 482 nm (slit width 10 nm). It was determined.

[0094] Molecular design of the full-length Z33 immuno-amphiphile. Such as peptide amphiphiles, peptide-polymer conjugates, peptide-drug conjugates, etc. The construction of amphiphilic peptide conjugates has been widely used to fabricate various supramolecular nanostructures. The hydrophilic Z33 peptide sequence (FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD) (SEQ ID NO: SEQ ID NO: 1) and a hydrophobic alkyl chain. The Z33 peptide was designed to act as a building motif for the IF. Derived from Protein A (Figure 1A) that specifically binds to the Fc portion of IgG with high binding affinity (Kd = 43 nM). It is a two-helix derivative of

[0095] Two types of IAs (C12-Z33 and 2C8-Z33 (Figure 1B)) were synthesized by converting the lauric acid moiety (C12) or the two octane The acid moiety (2C8) was synthesized by directly attaching the N-terminus of the Z33 peptide. As shown in Fig. 1, IA self-assembles into IF and is predicted to specifically bind to IgG from the antibody mixture. Pure Z33 peptide was also synthesized to compare the biological activity between the Z33 molecule and Z33-containing IF. Another control molecule, C12-SZ33, was synthesized by attaching C12 to the N-terminus of the scrambled sequence of Z33. All molecules were designed using automated solid-phase peptide synthesis (SPPS) methods and RP-HPLC. The purity and expected molecular weight of the synthesized compounds were confirmed by analytical HPLC. C and mass spectrometry were used to confirm the identity.

[0096] Example 2 Molecular self-assembly and characterization of full-length Z33 immuno-amphiphiles. The self-assembly of the two types of IAs can be easily achieved by a two-step procedure. The IA was separately pretreated in hexafluoroisopropanol (HFIP) to improve its solubility. Any pre-existing nanostructures that may affect the uniformity of the self-assembled morphology were removed. 2, HFIP was removed by evaporation, followed by the addition of deionized water or phosphate buffered saline (PBS). IF binds the alkyl segments by hydrophobic interactions. The formed, biologically active Z33 sequence is soluble in its shell while trapped in the core of the IF. After overnight aging at room temperature, the specimens were then examined under a transmission electron microscope (TEM). and circular dichroism (CD) were used to characterize the morphology of the assembled nanostructures.

[0097] Considering the important role of pH conditions in current IgG purification methods, we investigated the self-assembly behavior of C12-Z33. The activity of the enzyme was evaluated as a function of pH. Neutral pH was usually used as the binding condition, while acidic pH was used. Used to elute antibodies from Protein A affinity columns. Neutral and low pH To investigate the self-assembly behavior at 1000 kJ / cm², PBS (pH 7.4) and IgG elution buffer (pH 2.8) were used. The aqueous environment for the self-assembly of C12-Z33 was used. Morphology of C12-Z33 IF at different pH The C12-Z33 molecule was investigated by TEM (Figures 2C-2F) and CD (Figure 2B). It was found that under these conditions, the polymer was sufficiently soluble and capable of self-assembling into nanofibers. Representative TEM images obtained from a 0 μM solution of C12-Z33 demonstrate the activity of C12-Z33 under physiological and acidic conditions. Under both conditions, C12-Z33 had a diameter of 16.0 ± 1.7 nm (this value corresponds to the diameter of the fully extended peptide molecule). self-assembles into nanofiber structures with lengths less than 100 nm (approximately 22.5 nm for β-sheet structures) The length of the nanofibers is on the scale of micrometers. and could not be adequately controlled.

[0098] To further understand the molecular packing within the self-assembled structures, circular dichroism (CD) was used. The peptide secondary structure was investigated using the NMR spectrum. Strong negative signals were observed at approximately 222 nm (n-π*) and 208 nm (π-π*). The chain length was observed for C12-Z33 and in the self-assembled state, as shown for the pure Z33 peptide. It was suggested that the α-helical secondary structure of the Z33 segment was formed. Based on the measured diameters of the IFs and the IFs, the peptides, when packed into the IFs, It is reasonable to assume that the α-helical secondary structure was maintained. The CD spectrum of C12-Z33 in the IgG elution buffer only partially preserved the α-helical signal. The flatness of the two negative peaks around 222 nm and 208 nm was maintained at 100%. The shift in the CD spectrum was due to the molecular packing of the Z33 segment. This can alter the binding site from its free state, subsequently increasing the specific binding required for the binding site. from the formation of IF, which may affect its binding affinity to IgG due to its unique structure. This may occur.

[0099] Example 3 ITC experiments to measure the binding affinity of IFs. Considering the structural changes in the secondary structure of the Z33 peptide after incorporation into IF, C12-Z33 I To determine whether the formation of F affects the IgG-binding capacity present in the original Z33 peptide. To investigate the binding affinity of the self-assembled C12-Z33 IF, we performed binding to IgG1. The thermodynamic properties of the interaction were investigated by isothermal titration calorimetry (ITC). ITC has been used to analyze the interaction of a large number of proteins with It has been widely used to monitor binding events between ATP and C12-Z33 IF This is an excellent method to investigate whether binding can occur between IgG1 and IgG1. During injection, record the heat associated with the binding reaction and calculate the thermodynamic dissociation constant (K d ), Mol· Directly obtain thermodynamic parameters including enthalpy change (ΔH°) and stoichiometry (N) It is possible.

[0100] [Table 2]

[0101] [Table 3]

[0102] In a typical ITC experiment, a solution of 100 μM C12-Z33 in PBS buffer was incubated overnight, followed by Then, a solution of 2 μM IgG1 in the same buffer at pH 7.4 at 15°C was injected. The binding isotherms are shown in Figure 3A and the thermodynamic parameters recorded per ligand are The results of ITC for C12-Z33 IF binding to IgG1 are summarized in Table 2. The ITC results for C12-Z33 IF binding to IgG1 revealed a K d The enthalpy-driven binding event characterized by the C12-Z33 IF was revealed. To further compare the coupling efficiency, K measured by surface plasmon resonance was d is 43 nM, IgG We synthesized the Z33 peptide, which was shown to bind strongly to IgG1. Binding properties were measured by ITC in PBS, pH 7.4 at 15°C, and typical thermograms and binding The isotherms are shown in Figure 3C. In addition to the 100-fold better affinity, the stoichiometry for Z33 was 2.3. However, the apparent stoichiometry for C12-Z33 was 3.1, and all of the C12-Z33 in the IF was IgG1. It was shown that the IgG1 molecules were not available for binding to the IgG1 molecules. The efficiency of the C12-Z33 molecule was calculated by dividing the stoichiometry of Z33 by the stoichiometry of C12-Z33, which is 74.2%. It can be estimated that:

[0103] Normalization per ligand allows for the determination of the apparent stoichiometry of binding, but not the thermodynamic Comparison of biological parameters is performed after normalization per mole of IgG as shown in Table 2. The binding of Z33 to IgG is contrary to the unfavorable large entropy change. In contrast, the heat of the C12-Z33 bond was characterized by a favorably large enthalpy. The mechanical characteristics were similar, but the magnitude of the enthalpy and entropy changes was smaller. C12-Z33 bonds with a smaller positive (unfavorable) entropy than Z33, but with a negative (f The enthalpy loss for the (avorable) is much larger, resulting in an overall lower binding affinity. The overall loss of favorable bond enthalpy is probably due to the destruction of IF. This may be caused by the unfavorable enthalpy associated with IgG1. The interaction may be limited due to constraints within the IF. Binding affinity may be affected by dissolution from the IF. To demonstrate that the IgG elution buffer (pH 7.5) at 15°C is significantly lower at this low pH, which is suitable for elution, 2.8), we also performed titration of IgG1 with C12-Z33 (Figure 3B).

[0104] To eliminate nonspecific binding between IF and IgG1, a scrambled sequence of the Z33 peptide was used. C12-SZ33 IA was used as a negative control. The self-assembly properties and secondary structure identified using TEM and CD are shown (data not shown). ITC experiments were performed by injecting 100 μM of C12-SZ33 IA into a 2 μM IgG1 solution in PBS, pH 7.4, at 15°C. The binding capacity was measured by the thermograms and binding isotherms shown in Figure 3D. The lines indicate a specific interaction between IgG1 and the Z33 peptide.

[0105] Example 4 To further demonstrate the generality of the IF function, we also performed the double-chain alkylated IA 2C8-Z33. The self-assembly properties and binding affinity to IgG1 were investigated (Figures 4A-E). The nanoscale IF was observed in TEM images, and the α-helical secondary structure was confirmed by CD. The ITC results showed that binding between 2C8-Z33 and IgG1 occurred in PBS (pH 7.4) at 15°C, but No binding was detected in the elution buffer (pH 2.8). The stoichiometry of 2C8-Z33 was 9.1, indicating a lower coupling efficiency. (favorable) bond enthalpy, but the contribution from entropy is smaller unfavorable, which results in slightly better binding affinity (Table 2). From the above results, the present inventors have concluded that self-assembly can be achieved by displaying high density binding sites on the surface. The combined IF has good binding capacity to IgG1, as shown for the original Z33 peptide. Nevertheless, the overall observed value for IF There is a loss in physical binding affinity (which is of enthalpic origin). e) The loss in enthalpy is due to the loss of interaction due to constraints during IF and the destruction of particles. This can be explained by the unfavorable enthalpy contribution associated with I The molecular packing within F can greatly affect their performance in biological activity. The morphological and functional properties of the cells are determined by the

[0106] Example 5 Applicability for purifying immunoglobulins and other molecules with Fc portions. The diversity of constituent amino acids allows for hydrogen bonding, π-π stacking, hydrophobic collapse, and self-assembly. Non-covalent interactions, including electrostatic interactions, between assembled peptide nanofibers The basis for this is broad. For example, the solubility of acidic and basic amino acids varies with ion concentration. The degree of ionization, properties that depend on pH and ionic strength, is therefore The self-assembly process can be accelerated by adjusting the pH or adding salts, and the static The electrical repulsion is reduced, promoting aggregation and even precipitation. Considering the charged amino acid residues in When IgG binds to IF, the IgG-IF complex is It is likely to precipitate by adding salts with high ionic strength (Figure 5A).

[0107] Since C12-Z33 IF has a relatively high binding affinity to IgG1, C12-Z33 IF was used to bind IgG1. As shown in Figure 5B(i-ii), 5 mM C12- Z33 is fully soluble in PBS, but may precipitate in 0.6M Na2SO4 in PBS. The zeta potential of C12-Z33 in PBS solution is -7.61 mV, and the addition of Na2SO4 reduces the charge on the IF surface. For IgG1, 5 mM C12-Z33 and 0.6 M Na2SO4 were used. However, 20 μM IgG1 and 5 mM C12-Z33 were mixed for 5 minutes, followed by 0.6 M Na After the addition of 2SO4, precipitation was observed. To determine the composition of the precipitate, two parallel 5 mM C12-Z33 in 0.6 M Na2SO4 was centrifuged and analyzed by ultraviolet-visible (UV-Vis) spectroscopy. The absorbance change of the supernatant at 280 nm was monitored before and after adding NaSO using the 0.6 The same procedure was performed on a mixture of 5 mM C12-Z33 and 20 μM IgG1 in M ​​Na2SO4. As shown, most of the C12-Z33IF was precipitated by 0.6 M Na2SO4. IgG1-IF complex For the system, its absorbance at 280 nm decreased to a level below the initial absorbance of IgG1. This indicates that IgG1 has been removed from the solution. More clearly, the blue line changes to the green line. The absorbance of the supernatant relative to net IgG1 was plotted by subtracting the value of This suggests that more than 60% of IgG1 was removed from the supernatant. The potential of F to serve as a new affinity precipitant was preliminarily demonstrated.

[0108] Example 6 Molecular design of IA with fragmented antibody-binding peptides. Z33 (FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD) (SEQ ID NO: 1) contains two α-helices. D NO): The peptide sequence of 1) was separated between D and P and alkylated to generate two series of immuno-amphiphiles. The antibodies were: 1) Helix1 (FNMQQRRFYEALHD) (SEQ ID NO: 2)-based immuno-immunoglobulins 2) Helix 2 (PNLNEEQRNAKIKSIRDD) (SEQ ID NO: 5)-based immuno- Amphiphiles. We have demonstrated that the peptide structures after separation, alkylation and self-assembly We were interested in the possibility of structural changes due to the C16 and 2C8 alkyl chains attached to the C-terminus and H-terminus of Helix 1. We attached the peptides to the N-terminus of Helix 1 and Helix 2 (Fig. 6A). The reason for attaching the hexyl tail is to adjust the relative position between the helix and other segments. As shown in the Z33 peptide, IA was able to retain the alkyl selenide under certain conditions. The corresponding fragments are trapped in the hydrophobic core and the peptide sequence is oriented towards the aqueous environment. Using automated solid-phase peptide synthesis (SPPS) and RP-HPLC, All molecules were synthesized and purified using the above method. The purity and expected molecular weight of the synthesized compounds were confirmed by analytical methods. The analysis was confirmed using HPLC and mass spectrometry.

[0109] Example 7 Molecular assembly of IAs bearing fragmented antibody-binding peptides. The self-assembly of IA was carried out in a two-step procedure. IA was first pre-assembled with HFIP. Treatment was performed to remove any pre-existing nanostructures formed during the synthesis and purification process. Then, HFIP was removed by evaporation, followed by the addition of deionized water to a final concentration of 1 mM at pH 7.4. The alkylation affected the morphology of the self-assembled IA. To investigate whether the aggregated morphology of all IAs was achieved, we visualized the aggregated morphology using TEM and measured the aggregated morphology of all IAs at 100 μM. Typical TEM images of the IA solution are shown in Figure 7. Figures 7A and 7C show the Helix 1-C16 and C16-H Both elix2 and elix3 formed long nanofibers with diameters of 9.6±1.3 nm and 12.4±1.7 nm, respectively. This shows that the nanofibers self-assemble into a nanostructure. It was also observed that the fibers joined together to form nanobelts. The scale of the octonic acid (OC) For double-strand alkylation with methyltrimethylsilylpropanol (C8), the results for Helix 1-2C8 and 2C8 were obtained under the same conditions. Filamentous assemblies were found for both Helix 1 and Helix 2 (Figs. 7B and 7D). However, these filaments are relatively short and appear to be more polydisperse in length. The smallest filament was 11.0 for the Helix1-2C8 filament. ±1.4 nm diameter for Helix2-2C8 filaments and 9.3 ±1.3 nm diameter for Helix2-2C8 filaments. The steric effect of the large double chains is thought to play an important role in the morphology of the aggregates. However, the impact of steric effects on different peptide amphiphilic systems varies. The superhelical pitch tends to decrease when the steric effect at the end groups of the alkyl chains is increased. In the Tat peptide-based nanofiber system, Only the quart-C8 conjugates self-assemble into filamentous nanostructures, whereas the di-C8 or mono-C8 conjugates No nanostructures were observed for any of the -C8 conjugates.

[0110] Example 8 Differences between single- and double-chain alkylated IA with fragmented antibody-binding peptides. Further investigations into CMC and secondary structure were carried out to gain a deeper understanding of the various IAs. The critical micelle concentration (CMC) values ​​of all the dyes were determined by using a lipophilic dye that emits fluorescence depending on the polarity of the solvent. The strong fluorescence upon exposure to a hydrophobic environment was measured using Nile Red. In aqueous solutions, the IA and IAs are strongly quenched and red-shifted. When incubated, Nile Red reacts with the hydrophobic molecules formed by self-assembled nanostructures. The CMC of the IAs was measured by the blue-shift of the emission maximum. Figure 8A shows a plot of Nile Red fluorescence intensity versus Helix 1-C16 concentration. The wavelength of the emission maximum is 660 nm up to 0.5 μM, then at a concentration of 1 μM, it decreases from 660 nm to 635 nm. A blue shift to 0.05 occurs first, suggesting that there is a CMC value between them. Similarly, the CMC values ​​for C16-Helix2, Helix1-2C8, and 2C8-Helix2 can be obtained from Figures 8B-8D. Double-chain alkylation shows higher CMC values ​​compared to single-chain alkylation. 2C8 -Helix 2 has a stronger tendency to aggregate than Helix 1-2C8 because the former aggregates at a lower CMC range. Because there is.

[0111] Example 9 Secondary structure studies of alkylated IA using fragmented antibody-binding peptides. Circular dichroism (CD) was used to understand molecular packing within self-assembled structures. As shown in Figure 9, both Helix 1 and Helix 2 are derived from Z33. After the extraction, the α-helical structure was lost and the CD spectrum showed a random coil ( Figure 16). Helix1-C16 and C16-Helix2 assemblies show a strong negative signal around 218 nm (n-π* The formation of a β-sheet secondary structure in both IAs was suggested. have previously been shown to play an important role in promoting the formation of suitable one-dimensional nanostructures. Interestingly, the negative signals around 225 nm (n-π* transition) and 208 nm (π-π* transition) are He1 This was observed in ix1-2C8 and 2C8-Helix2, indicating the presence of an α-helical secondary structure. The D spectrum was fitted by linearly combining the polylysine-based spectrum, and The content of secondary structure in each was evaluated (Figure 17). The β-sheets were Helix 1-C16 and C16-Helix 2 I. A is the major component of A, whereas the α-helix is ​​the dominant secondary structure in double-chain alkylated IA. .

[0112] Example 10 To further identify the components of secondary structure in the self-assembled IA, we bound them to the β-sheet surface. The fluorescent dye thioflavin-T (ThT), which emits increased fluorescence when the Helix 1 base is bound to the nucleus, was used to The presence of β-sheet secondary structure in the IA of the sucrose was investigated. The fluorescence emission of ThT at 482 nm from the doped Helix1 and Helix1-2C8 was similar to that of the pure ThT solution. This is because these IAs have a prominent β-sheet structure. On the contrary, in both pre-incubated Helix 1 and C16, A dramatic increase in fluorescence was observed, which indicates that the β-sheet secondary structure was strongly revealed by CD. This corresponds to the following.

[0113] The CD signal may originate from free monomers rather than self-assembled filaments. To rule out this possibility, the α-helix The results of the dilution of IA (represented by Helix 1-2C8) and β-sheet IA (represented by Helix 1-C16) CD spectra were taken. Figures 10A and 10B show the CD spectra obtained when the IA concentration exceeded the corresponding CMC value. Only in the case of the filamentous structure, the CD signal becomes significant and stable. It was revealed that the packing of the IA is a major contributor to the CD signal.

[0114] As can be seen from the geometry of the α-helix and β-sheet, the axial distance between the helices is approximately 12 Å. However, the β-strands are 5 Å apart, and the formation of β-sheets requires more space than α-helices. This indicates that dense packing is necessary for self-assembled nanostructures. It has been shown that longer alkyl chains tend to pack more tightly and even crystallize. This allows the peptide segments to be closer together. In this case, it is noteworthy that the two C8 chains are asymmetrically bound to Helix 1 and Helix 2. The two C8s bind to two adjacent lysines at the C-terminus of Helix 1, and for Helix 2, One was attached to the N-terminal amine and the other to the side chain of lysine. As the number of chains increases, the steric hindrance increases significantly, the aggregation weakens, and a relatively large CMC is obtained. This results in looser packing and wider diameter filaments.

[0115] Example 11 Effect of single alkyl chain length. Loose packing of double-stranded alkylated IA promotes the formation and stabilization of .ALPHA.-helical assemblies Therefore, we believe that shortening the length of the single alkylated chain may have the same effect. To confirm this assumption, two shorter alkyl chains (lauric acid, C12 octanoic acid, C8) was bound to Helix 1 and Helix 2 separately, and the assemblies were analyzed by TEM and CD. At the highest test concentration of 100 μM, no significant difference was observed for either Helix 1-C8 or C8-Helix 2. No clear nanostructure or emission maximum transition was observed, and these two IAs were This indicates that self-assembly is not possible at a temperature lower than the above. and 11B) are long nanofibers with diameters of 12.2±1.4 nm and 9.1±1.6 nm, respectively. The diameter of the nanofibers depends not only on the length of the chemical structure but also on the packing density. It should be noted that the CMC value is also related to the binding state. A smaller diameter was observed for the C16-linked IA (Figs. 11C and 11D), and It is surprising that a tighter packing than the C12 conjugate is observed due to the strong hydrophobic interactions. In the CD spectra (Figs. 11E and 11F), Helix 1-C8 and C8-He Both lix2 and lix3 produced similar CD results to unbound peptides due to their low aggregation potential. Interestingly, the β-sheet signal appears in the CD spectrum of Helix 1-C12, An α-helical secondary structure was observed in C12-Helix2, confirming our previous assumption. In the case of single-chain alkylation, the self-assembled IA with longer alkyl chains was It can induce tight packing between adjacent IA molecules and promote β-sheet formation On the other hand, the looser packing in the shorter alkylated IA leads to an α-helical structure. This can provide more space for

[0116] [Table 4]

[0117] Example 12 Another method comprising the steps of: Preparation of immunofiber compositions. As described above, the improved immunofiber-based binding system is referred to as the conjugated immunofiber. Immunofiber co-assembled with fiber molecule (C12-VVKKGGZ33) and spacer molecule (C12-VVEE) The binding molecule was designed by combining the carbon chains in a single system (Figure 19A). The peptide Z33 contains six more amino acid residues, VVKKGG, than the originally designed C12-Z33. Two valines (VV, red) promote the formation of a one-dimensional structure. Two glutamic acids (EE , blue) was designed as a hydrophilic segment in the spacer molecule, and two lysines (KK, blue) were By designing for electrostatic interactions between KK and EE, the binding molecule and the spacer The molecules were packed alternately. The alkyl chain was then attached to the antibody-binding peptide (Z33). To further separate the two, two glycines (GG, green) were designed. When the molecules are dissolved in aqueous solution, these two molecules are uniformly reacted with the one-dimensional immunofiber. They can aggregate together, with the binding ligand Z33 protruding from their surface (Figure 19B). The distance between the two Z33 ligands adjusts the molar ratio of the binding molecules and spacer molecules to be co-assembled. The self-assembled C12-VVEE (Fig. 20A) and C12-VVEE (Fig. 20B) A representative TEM image (Figure 20B) shows the cylindrical nanofiber structure in PBS, pH 7.4. Figure 20C shows the structure of nanoparticles co-assembled with C12-VVEE and C12-VVKKGGZ33 at a molar ratio of 10:1. Fiber is shown.

[0118] Example 13 To examine the IgG binding and precipitation capabilities of the new immunofiber binding system, 100 μL of The desired immunofiber solution was mixed with IgG (C12-VVKKGGZ33 and IgG in a molar ratio of 10:1). ), and incubate for 30 minutes, followed by the addition of ammonium sulfate to a final concentration of 1M. Immunofiber and IgG were precipitated. Self-assembled C12-VVKKGGZ33 and IgG were mixed. In this case, no precipitation was observed after the addition of ammonium sulfate. A solution containing IgG and C12-VVEE and C12-VVKKGGZ33 co-assembled at a molar ratio of 10:1 was After adding salt, the solution became cloudy. The precipitate was centrifuged and the IgG concentration in the supernatant was analyzed. Approximately 30% of the IgG was precipitated from the ProA column (TSKgel (登録商標) Protein A-5PW, 20 μm, 3 IgG concentrations were quantified using a 500µL ELISA tube (5 x 4.6mm) and monitored at 280nm. When the amounts of binding molecules and IgG in the solution are the same, the addition of spacer molecules significantly improves the capture of IgG. It has been shown that the binding molecules are spaced apart and then bonded together. By protruding the conjugated ligand Z33 from the surface of the immunofiber, the flexibility of the ligand can be improved. and greater accessibility to IgG.

[0119] Example 14 Increasing IgG binding capacity and optimal precipitation conditions in a co-assembled immunofiber binding system To achieve this, the ratio of binding molecules to spacer molecules, the ratio of binding molecules to IgG, salt concentration, binding and We continue to optimize several factors, including the molar ratio of C12-VVEE and C12-VVKKGGZ33. The molar ratio of C12-VVKKGGZ33 and C12-VVKKGGZ33 was first investigated, and preliminary results are shown in Figure 21. Ratio of C12VVKKGGZ33 to IgG and 5 molar ratios of C12-VVEE to C12-VVKKGG at 1 M ammonium sulfate Z33 (5:1, 10:1, 25:1, 50:1, 100:1) was tested. The percentage of IgG in the supernatant after the treatment is shown. As the content of C12-VVEE increases, more IgG is obtained. precipitated from the solution, reaching a maximum yield of about 75% at 50:1. Note that the precipitation yield decreased at 100:1. It is noted that this may be due to the low solubility of the immunofiber due to its high concentration. Next, we compared the 25:1 and 50:1 immunofluorescence assays, which showed better performance in Figure 3B. Using the Barr system, the molar ratio of C12-VVKKGGZ33 to IgG was adjusted from 10:4, 10:2 to 10:1. As shown in Figure 3B, these two immunofiber systems showed a significant difference between C12-VVKKGGZ33 and IgG. The same trend was followed, with the yield of IgG precipitation increasing as the ratio of Under good conditions, the ratio of C12-VVKKGGZ33 to IgG was 10:1 in a 50:1 immunofiber system. This excellent result allowed us to achieve a 99% IgG precipitation yield. It has been confirmed that Hunofiber has high IgG binding and precipitating ability. We plan to conduct more systematic studies on the optimization of conditions for the production and recovery of nitroxide.

[0120] All references cited herein, including publications, patent applications, and patents, are incorporated by reference. Each document is individually and specifically cited as being incorporated herein by reference, and all of its contents are hereby incorporated by reference. is incorporated by reference herein to the same extent as if set forth herein.

[0121] The terms "a" and "an" and "the" and the context in which the invention is described (particularly in the claims below) The use of similar directives in the context of the range of Unless otherwise clearly contradicted by context, the singular and plural forms are included. The terms "comprising," "having," "including" )" and "containing" are used in open-ended language (i.e., unless otherwise indicated). ", interpreted as "including, but not limited to" The range of values ​​set forth in this specification is not to be construed as limiting the scope of the present invention unless otherwise specified. Insofar as the range is not a single value, it merely serves as a shorthand way of referring individually to each distinct value that falls within the range. and each separate value is intended to be treated as if it were individually described herein. All methods described herein are incorporated herein by reference in their entirety. Unless otherwise indicated or clearly contradicted by context, Any and all examples or exemplary phrases used herein may be Unless otherwise expressly stated in the claims, the term "such as" merely refers to a specific embodiment of the invention. The present disclosure is intended to be illustrative only and is not intended to impose limitations on the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. This document is not to be construed as indicating that

[0122] The preferred embodiments of the present invention are set forth in the best mode known to the inventors for carrying out the invention. These preferred embodiments are described herein, including variations thereof. The present inventors believe that such modifications will be apparent to those skilled in the art upon reading the present invention. The inventors anticipate that the present invention will be applicable to the present invention in the future, and ... Accordingly, the present invention is not limited to the foregoing. The present invention is intended to cover the subject matter of the claims appended hereto, including all modifications and equivalents thereof. Moreover, any combination of the above-described elements in all possible variations thereof is intended to be illustrative and not restrictive. Unless otherwise indicated or clearly contradicted by context, all such terms are encompassed herein.

Claims

1. a) an immunofiber-binding molecule, wherein the immunofiber-binding molecule comprises at its N-terminus a first hydrocarbon chain attached to a spacer peptide, and the spacer peptide is attached at its C-terminus to an antibody-binding peptide, wherein the antibody-binding peptide has a hydrophilic amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; and b) an immunofiber spacer molecule comprising a second hydrocarbon chain attached at its N-terminus to a peptide sequence comprising the amino acid sequence XXBB, where XX are two amino acids with small hydrophobic side chains and can be the same or different amino acids, and BB are two amino acids with negatively charged side chains and can be the same or different amino acids. A self-assembled immunofiber composition comprising:

2. The immunofiber composition of claim 1, wherein the spacer peptide comprises an amino acid sequence of XXYYZZ, where XX are two amino acids with small hydrophobic side chains and may be the same or different amino acids, YY are two amino acids with positively charged side chains and may be the same or different amino acids, and ZZ are two amino acids with small neutral side chains and may be the same or different amino acids.

3. 2. The immunofiber composition of claim 1, wherein the spacer peptide comprises the amino acid sequence VVKKGG (SEQ ID NO: 9).

4. The immunofiber composition of claim 1 , wherein the first hydrocarbon chain is a linear hydrocarbon chain.

5. The immunofiber composition of claim 1 , wherein the first hydrocarbon chain is a C12 alkyl.

6. An immunofiber composition as described in claim 1, wherein the peptide sequence of the immunofiber spacer molecule has the amino acid sequence VVEE (SEQ ID NO: 10).

7. The immunofiber composition of claim 1 , wherein the second hydrocarbon chain is a linear hydrocarbon chain.

8. The immunofiber composition of claim 1 , wherein the second hydrocarbon chain is a C12 alkyl.

9. The immunofiber composition of claim 1 , comprising a plurality of self-assembled immunofiber-binding molecules.

10. 10. The immunofiber composition of claim 1, comprising a plurality of self-assembled immunofiber spacer molecules.

11. 10. The immunofiber composition of claim 1, comprising alternating packing of said immunofiber binding molecules and said immunofiber spacer molecules.

12. The immunofiber composition of claim 11, wherein the spacer peptide of the immunofiber binding molecule contains two lysine amino acids; the peptide sequence of the immunofiber spacer molecule contains two glutamic acid amino acids; and the two lysine amino acids electrostatically interact with the two glutamic acid amino acids, resulting in the alternating packing of the immunofiber binding molecule and the immunofiber spacer molecule.

13. 2. The immunofiber composition of claim 1, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 5:

1.

14. 2. The immunofiber composition of claim 1, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 10:

1.

15. 2. The immunofiber composition of claim 1, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 25:

1.

16. 2. The immunofiber composition of claim 1, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 50:

1.

17. 2. The immunofiber composition of claim 1, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 100:

1.

18. 10. The immunofiber composition of claim 1, wherein said immunofiber binding molecules and said immunofiber spacer molecules homogeneously co-assemble to produce a one-dimensional immunofiber containing protruding binding ligands.

19. The immunofiber composition of claim 1 further comprising an antibody.

20. 20. The immunofiber composition of claim 19, wherein the molar ratio of said immunofiber binding molecule to said antibody is 10:

4.

21. 20. The immunofiber composition of claim 19, wherein the molar ratio of said immunofiber binding molecule to said antibody is 10:

2.

22. 20. The immunofiber composition of claim 19, wherein the molar ratio of said immunofiber binding molecule to said antibody is 10:

1.

23. 20. The immunofiber composition of claim 19, wherein the molar ratio of said immunofiber spacer molecules to said immunofiber binding molecules is 50:1, and the molar ratio of said immunofiber binding molecules to said antibodies is 10:

1.

24. The immunofiber composition of claim 19, wherein the antibody is an IgG.

25. The immunofiber composition of claim 19, wherein the antibody is IgG1.

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