High-affinity supramolecular polymers for binding-induced antibody precipitation and purification

Abstract

The present disclosure relates to systems and methods for purifying proteins that include an Fc region, such as antibodies or Fc fusion proteins. The system includes one or more filler molecules that include a linear hydrocarbon chain conjugated to a peptide amino acid sequence, and one or more ligand molecules that include a linear hydrocarbon chain, a peptide amino acid sequence, a linker, and a Z33 peptide of Staphylococcus aureus protein A. The filler molecules and ligand molecules self-assemble into immunofibers (IFs) under physiological conditions.

Description

Priority claim

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 223,792, filed July 20, 2021, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The text of the computer readable sequence listing submitted herein, entitled "JHU-39610-601_SQL," created on July 18, 2022, and having a file size of 5,022 bytes, is hereby incorporated by reference in its entirety. [Background technology]

[0003] Supramolecular assembly is a bottom-up approach to construct hierarchical and functional nanostructures. Among the recently developed supramolecular polymers, peptide-based materials are particularly attractive due to their biocompatibility, biodegradability, and low toxicity. The presentation of bioactive epitopes on supramolecular substrates allows for efficient molecular / cellular recognition and signaling, as well as active targeting of nanostructures for precise delivery and accumulation of therapeutics at desired sites. Considering the steric hindrance brought about by anchoring these epitopes on the supramolecular polymer surface, the spatial arrangement of epitopes plays a key role in controlling their functionality. Co-assembly of bioactive building units with inert molecules has been shown to effectively modulate epitope density and achieve enhanced bioactivity. Meanwhile, the use of flexible linkers for spacing epitopes in the radial direction of the resulting nanostructures is equally essential to improve epitope accessibility and their interaction with target biomolecules.

[0004] Affinity precipitation has been increasingly explored as a promising alternative to Protein A chromatography for the purification of monoclonal antibodies (mAbs) and other therapeutic proteins, since conventional Protein A-based affinity chromatography methods suffer from limited production capacity and high media costs. During the purification process, it is common to use salts to induce or assist antibody precipitation. However, high salt concentrations are known to destabilize proteins and increase the risk of nonspecific precipitation of impurities present in the solution.

[0005] There remains a need for more efficient systems and methods for capturing and purifying antibodies and other proteins. BRIEF SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides a system comprising: (a) one or more filler molecules comprising a linear hydrocarbon chain conjugated to an amino acid sequence of 2-5 amino acids; (b) one or more ligand molecules comprising a linear hydrocarbon chain, an amino acid sequence of 2-5 amino acids, a linker, and a Z33 peptide of Staphylococcus aureus protein A, or an antibody-binding fragment thereof, conjugated to the linker, wherein the one or more filler molecules and the one or more ligand molecules are capable of self-assembling into immunofibers (IFs) under physiological conditions. Also provided are methods of purifying antibodies and / or fusion Fc proteins using the aforementioned system. [Brief description of the drawings]

[0007] [Figure 1]Figure 1A-1C includes schematic diagrams illustrating the molecular design of immunofiber (IF) building units with OEG (or PEG) linkers and the formation of IFs for monoclonal antibody (mAb) capture. Figure 1A shows the chemical structures of the filler molecule, C12-VVEE, and the ligand molecule, C12-VVKK[linker]Z33. Figure 1B is a schematic diagram illustrating the ligand molecule design with various linkers and the effect of linker length on the presentation of Z33 peptide on co-assembled IFs. Figure 1C illustrates the co-assembly of filler and ligand molecules into supramolecular IFs and the four possible states of mAbs in IF solution: (1) two Fc moieties of a mAb bound to two ligands from two different IFs; (2) two Fc moieties of a mAb bound to two adjacent ligands from the same IF; (3) one Fc moiety of a mAb bound to one ligand on an IF; (4) free mAbs in solution bound to zero, one, or two monomeric ligands that are not assembled into IFs. [Diagram 2] Figures 2A-2H show the characterization of filler and ligand molecules. Figures 2A-2G are representative TEM images of self-assembled filler and ligand molecules (2A: C12-VVEE, 2B: O4, 2C: O8, 2D: O12, 2E: O16, 2F: O36, and 2G: P2000) in PBS with diameters of 7.4 ± 0.6 nm, 11.1 ± 0.9 nm, 12.6 ± 1.2 nm, 13.6 ± 1.2 nm, 14.9 ± 1.3 nm, 18.5 ± 1.8 nm, and 19.3 ± 2.0 nm, respectively. Diameters are given as mean ± SD (n = 30). Scale bar: 100 nm. Figure 2H is a graph illustrating normalized CD spectra of self-assembled ligand molecules with different OEG (or PEG) linkers, suggesting the formation and retention of α-helical secondary structures. [Diagram 3] Figures 3A-3D are graphs illustrating the ITC profiles and binding curves for stepwise injections of 100 μM mAb1 to 40 μM O4 (3A), O8 (3B), O12 (3C), and O16 (3D) in PBS, pH 7.4 at 25°C. [Figure 4]Figures 4A-4E show a comparison of mAb precipitation performance of IFs with different ligand molecules. Figures 4A and 4B are graphs illustrating the mAb precipitation yields for 20 μM and 40 μM mAb1, respectively, incubated with various co-assembled IFs (2.5 mM filler, 100 μM ligand) under 1 M salt or no salt conditions. The data points for G2 with 1 M salt in Figure 4A were replotted from reference. Figures 4C and 4D are graphs illustrating the precipitated mAb mass from 100 μL samples of 20 μM (0.3 mg) (4C) and 40 μM (0.6 mg) (4D) mAb1 incubated with various 100 IFs (2.5 mM filler, 100 μM ligand) under 1 M salt or no salt conditions. Figure 4E includes photographs of co-assembled IFs (2.5 mM filler, 100 μM ligand) after mixing with 40 μM mAb under 1 M salt or no salt conditions for 5 min. All experiments were performed in triplicate and data are presented as mean ± standard deviation. [Diagram 5] Figures 5A-5F show optimization of mAb precipitation yield. Figures 5A and 5B are graphs illustrating the yield and mass of mAb precipitate, respectively, when 100 μL of mAb1 at 40 μM (0.6 mg), 80 μM (1.2 mg), and 133 μM (2 mg) was incubated with IFs at various O16 concentrations. Figure 5C is a graph illustrating a comparison of mAb precipitation yield under 1 M salt and no salt conditions when 40 μM of mAb1 was incubated with optimized IFs (2.5 mM filler, 250 μM O16). Figure 5D is a schematic diagram showing the proposed mechanism of mAb-IF aggregation at increasing ligand concentration at constant mAb concentration. At low, medium, and high ligand concentrations, mAbs in state 3, state 1, and state 2 dominate, respectively. Figure 5E is a graph showing the turbidity of 40 μM mAb with optimized IFs (2.5 mM filler, 250 μM O16) at 2 hours. Figure 5F is a graph showing the effect of binding time on mAb precipitation yield of 40 μM mAb1 incubated with optimized IFs (2.5 mM filler, 250 μM O16). All experiments were performed in triplicate and data are shown as mean ± standard deviation. [Figure 6]6A-6F show the purification of mAbs from clarified bulk cell culture harvest (abbreviated as "mAb CB") and the isolation of mAbs at high titers. FIG. 6A is a schematic of the mAb purification process using IFs in PBS solution. FIG. 6B is a graph showing the mAb precipitation yields of pure mAb1 at 40 μM and 80 μM after two sequential precipitations under salt-free conditions, the yield loss in the washing steps, and the elution yield. FIG. 6C is a graph showing the mAb precipitation yields for mAb1 CB at 40 μM and 80 μM after two sequential precipitations with IF solution under salt-free conditions. FIG. 6D is a schematic of the high titer mAb purification process using solid IFs in lyophilized powder form. FIG. 6E and 6F are graphs showing the precipitation yields for pure mAb1 at 21 mg / mL (6E) and 31 mg / mL (6F) using sequential precipitations with lyophilized IFs under salt-free conditions. All experiments were performed in triplicate and data were presented as the mean ± standard deviation. [Figure 7] FIG. 7 is a graph illustrating mAb precipitation yields for 100 μL of mAb1 at 10 μM and 20 μM after incubation with 100 μL of IFs at various O16 concentrations. IFs with 200 μM O16 gave the best mAb precipitation yields for both mAb concentrations. Definitions

[0008] To facilitate understanding of the present technology, certain terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0009] The terms "nucleic acid," "polynucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably herein and refer to polymers or oligomers of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982)). The terms encompass any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemically modified form thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence may be a nucleic acid that is a member of other types of nucleic acid structures, such as DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (Wang, J. Am. Chem. Soc. (Journal of the American Chemical Society), 122:8595-8602 (2000)), and / or ribozymes, etc.The terms "nucleic acid" and "nucleic acid sequence" can also include strands that contain non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks (e.g., "nucleotide analogs") that can perform the same function as natural nucleotides.

[0010] The term "amino acid," unless otherwise indicated, refers to natural amino acids, unnatural amino acids, and amino acid analogs, in all of their D and L stereoisomers, where their structures permit such stereoisomeric forms.

[0011] Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0012] Unnatural amino acids include, but are not limited to, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allohydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ("4Hyp"), isodesmosine. , alloisoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") including N-methylglycine, N-methylisoleucine, N-alkylpentylglycines ("NAPG") including N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), homoarginine ("hArg"), (S)-N-Fmoc-2-(4'-pentenyl)alanine, and Fmoc-2,2-bis(4-pentenyl)glycine. Other unnatural amino acids that may be employed, examples of which are disclosed in International Patent Application Publication WO 2018 / 106937.

[0013] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0014] As used herein, the term "peptide" includes sequences of from 4 to 100 amino acid residues in length, preferably from about 10 to 80 residues in length, more preferably from 15 to 65 residues in length, and in which the α-carboxyl group of one amino acid is joined to the main-chain (α- or β-) amino group of an adjacent amino acid by an amide bond. Peptides can include natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides can be subsequences of naturally occurring proteins or unnatural (synthetic) sequences.

[0015] As used herein, the term "immuno-amphiphiles" refers to molecules that can spontaneously assemble into discrete, stable supramolecular nanostructures called "immunofibers". In most cases, IFs can assemble in the pH range between about 2.8 and about 7.5. However, the binding properties are also pH-dependent. More positively charged IFs are more likely to assemble in solutions with higher pH, and conversely, negatively charged IFs assemble more readily in solutions with lower pH.

[0016] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. In humans, light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The variable and constant regions are joined in the light and heavy chains by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is made up of three domains: H1 , C H2 and C H3Each light chain is composed of a light chain variable region (herein LCVR or V L The light chain constant region consists of one domain, C L The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are separated by regions that are more conserved called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair is made up of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L ) each form an antibody binding site. The term "antibody" includes antibodies that are part of an antibody multimer (a multimeric form of an antibody), such as a dimer, trimer, or higher order multimer of a monomeric antibody. It also includes antibodies that are linked to or attached to, or otherwise physically or functionally associated with, non-antibody moieties. Furthermore, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and multispecific antibodies, among others.

[0017] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies are typically produced using hybridoma technology as first described by Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976). Monoclonal antibodies can also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), isolated from phage-display antibody libraries (see, e.g., Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol. 1117-25 (2005); and Lonberg, Handb. Exp. Pharmacol. 181:69-97 (2008)). In contrast, "polyclonal" antibodies are antibodies secreted by different B cell lineages within an animal. Polyclonal antibodies are a population of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0018] The terms "Fc region", "Fc fragment", or "fragment crystallizable region" may be used interchangeably herein to refer to the region of a monoclonal antibody that includes the hinge and constant heavy domains (CH2 and CH3). The Fc region mediates downstream effector functions through its interaction with Fc receptors on (innate) immune cells or with C1q, a recognition molecule of the complement system. Interaction with Fc receptors can exert a wide range of immunomodulatory functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in response to infectious pathogens.

[0019] The terms "fragment of an antibody," "antibody fragment," and "functional fragment of an antibody" are used interchangeably herein to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech: 1126-1129 (2005)). Desirably, an antibody fragment contains, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, the following: (i) Fab fragments, which are composed of V L , V H , C L , and C H (ii) the F(a')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; and (iii) the V(a')2 fragment, which is a monovalent fragment consisting of one V domain of a single arm of an antibody. L and V H (iv) Fab' fragments, which result from the disruption of disulfide bridges in the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) domain antibodies (dsAbs), which consist of antibody single variable region domains (V H or V L ) polypeptide.

[0020] As used herein, the term "antibody-binding peptide" refers to a peptide that binds to an antibody or a specific portion of an antibody molecule, e.g., the Fc portion, with high specificity, e.g., about 10 -6 M to about 10 -10 K between M d By "binding" is meant a peptide having the ability to bind to the polypeptide, such as by having

[0021] As used herein, the term "sample" refers to any sample, solution, fluid, or mixture that contains an antibody of interest or an Fc region-containing protein of interest (e.g., an Fc fusion protein) that can be bound using the immunofiber of the present invention. In some embodiments, the sample can be a biological sample. For example, samples include, for example, cell cultures, cell lysates, and / or clarified bulk (e.g., clarified cell culture supernatant). Samples are optionally produced from host cells or organisms that express the antibody or Fc region-containing protein of interest (either naturally or recombinantly). For example, cells in cell culture include host cells that have been transfected with an expression construct that includes a nucleic acid that encodes the antibody or Fc fusion protein of interest. These host cells can be bacterial cells, fungal cells, insect cells, or preferably animal cells grown in culture. As used herein, the terms "biological sample" and "biological fluid" refer to any quantity of a substance from a living or once-living patient or mammal, or from cultured cells. Such substances include, but are not limited to, blood, serum, plasma, urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovial macrophages, endothelial cells, skin, cell cultures, cell lysates, and clarified bulk (e.g., clarified cell culture supernatant).

[0022] The terms "monomer," "monomer subunit," and "monomer unit" are used interchangeably herein and refer to one of the basic structural units of a polymer or oligomer. An "oligomer" is a molecule having from about 1 to about 30 monomers. The architecture of an oligomer can be, for example, linear, branched, or forked. An oligomer is a type of polymer. The term "polymer," as used herein, refers to a substance or material that includes a large molecule, or macromolecule, made up of many monomers (for example, hundreds or thousands). Detailed Description

[0023] The present disclosure is premised, at least in part, on the construction of a supramolecular immunofiber (IF) system by co-assembling rationally designed filler and ligand molecules for affinity precipitation and purification of monoclonal antibodies (mAbs). In some embodiments, the ligand molecule comprises a protein A mimetic peptide that binds to the Fc portion of immunoglobulin G (IgG) from most mammalian species in a pH-specific manner. Additionally, a series of linkers may be incorporated into the ligand design to increase the flexibility and accessibility of the protein A mimetic peptide. Given the dual Fc binding sites on monoclonal antibodies, particularly IgG, and the multivalency of the immunofibers provided herein, the resulting enhancement of multivalent mAb-IF binding may result in IF cross-linking into large complexes and facilitate precipitation of monoclonal antibodies under low-salt or no-salt conditions.

[0024] In this regard, the present disclosure provides a system comprising one or more filler molecules and one or more ligand molecules capable of self-assembling into immunofibers (IFs) under physiological conditions. The term "filler" or "filler molecule" as used herein refers to a molecule, particle, or compound added to a composition that can improve a particular property of the composition. In the context of the present disclosure, one or more filler molecules are designed to regulate the distribution of ligand molecules in the co-assembled immunofibers. The term "ligand" or "ligand molecule" as used herein refers to a substance that forms a complex with a biomolecule to generate a biological effect. In some embodiments, the ligand generates a signal by binding to a site on a target protein (e.g., a receptor). Alternatively, the ligand may be a small molecule, ion, or protein that binds to a nucleic acid molecule, such as a DNA double helix. Other types of ligands include, but are not limited to, steroid hormones, growth factors, neurotransmitters, and other peptides.

[0025] In some embodiments, each of the one or more filler molecules and each of the one or more ligand molecules comprises a linear hydrocarbon chain conjugated to an amino acid sequence. It is recognized that "hydrocarbon" is an organic compound composed entirely of hydrogen and carbon. The linear hydrocarbon chain can be of any suitable length. In some embodiments, the linear hydrocarbon chain comprises 1 to 24 carbon atoms, for example, 1 to 16 carbon atoms, 1 to 14 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, the linear hydrocarbon chain comprises 10-15 carbon atoms (e.g., 10, 11, 12, 13, 14, or 15 carbon atoms). Ideally, the linear hydrocarbon chain comprises 12 carbon atoms. A person of ordinary skill in the art will recognize that there is an upper limit to the number of carbons contained in the linear hydrocarbon chain in order to maintain solubility in aqueous solution.

[0026] Each of the one or more filler molecules and each of the one or more ligand molecules also comprises an amino acid sequence conjugated to the linear hydrocarbon chain. The amino acid sequence present in the one or more filler molecules can be the same or different from the amino acid sequence present in the one or more ligand molecules. Ideally, the amino acid sequences present in the filler molecules and the ligand molecules are designed to promote interactions between the molecules during co-assembly. The amino acid sequence can be any suitable length. In some embodiments, the amino acid sequence present in the filler molecule and / or the ligand molecule comprises 1-20 amino acids, such as 1-5 amino acids, 5-10 amino acids, 1-10 amino acids, 10-15 amino acids, or 15-20 amino acids, etc. For example, each of the one or more filler molecules comprises an amino acid sequence of 2-5 amino acids (e.g., 2, 3, 4, or 5 amino acids), and in some embodiments, 4 amino acids. Similarly, each of the one or more ligand molecules comprises an amino acid sequence of 2-5 amino acids (e.g., 2, 3, 4, or 5 amino acids), and in some embodiments, 4 amino acids. Exemplary amino acid sequences for inclusion in one or more filler molecules and / or one or more ligand molecules include VVXX (SEQ ID NO:2, where "X" represents any amino acid). In some embodiments, each of the one or more filler molecules comprises the amino acid sequence VVEE (SEQ ID NO:3), and each of the one or more ligand molecules comprises the amino acid sequence VVKK (SEQ ID NO:4). However, the disclosure is not limited to these particular amino acid sequences.

[0027] In addition to the linear hydrocarbon chain and the amino acid sequence, each of the one or more ligand molecules further comprises a linker and a Z33 peptide of Staphylococcus aureus protein A or an antibody-binding fragment thereof conjugated to the linker. A "linker" is any chemical moiety capable of stably covalently linking one compound to another compound (e.g., a cell-binding agent, such as a peptide ligand or an antibody, etc.). The linker can be sensitive to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and / or disulfide bond cleavage. In some embodiments, the linker can be any amino acid with a side chain having a free amino group, carboxyl group, or disulfide group. Exemplary amino acids useful as amino acid linkers in the one or more filler molecules and / or one or more ligand molecules of the present invention include lysine (K), glutamic acid (E), arginine (R), and cysteine ​​(C). Other suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Linkers also include charged linkers and their hydrophilic forms, as described herein and known in the art. In some embodiments, the linker can be a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker.

[0028] In some embodiments, the linker is a hydrophilic linker comprising one or more poly(ethylene glycol) (PEG) molecules or one or more oligo(ethylene glycol) (OEG) molecules. Unless otherwise indicated, a "PEG oligomer" or oligoethylene glycol (OEG) is one in which all of the monomeric subunits are ethylene oxide subunits. Typically, substantially all or all of the monomeric subunits are ethylene oxide subunits, although the oligomer may include separate terminal capping moieties or functional groups, for example for conjugation. Typically, PEG oligomers for use in the present disclosure have the following structure: "-(CH2CHO)" depending on whether the terminal oxygen(s) have been replaced, for example, during synthetic transformations. n -" or "-(CH2CH2O) n-1 In some embodiments, the variable (n) ranges from 1 to 30, and the end groups and overall PEG or OEG architecture can vary.

[0029] PEGylation is a process through which polyethylene glycol chains are conjugated to proteins (e.g., therapeutic proteins), peptides, aptamers, enzymes, small molecule drugs, antibodies, and other molecules. Through the PEGylation process, the molecular weight of the conjugated protein increases, resulting in reduced degradation and improved stability in vivo. PEGylation also reduces the immunogenicity of the protein to which it is conjugated. Examples of PEG and OEG linkers and related conjugation methods are described in U.S. Pat. No. 6,716,821; U.S. Pat. No. 9,388,104; U.S. Patent Application Publication No. 2009 / 0285780; and Harris, JM and Chess, RB, Nature Reviews Drug Discovery, 2:214-221 (2003).

[0030] The linker may be composed of any suitable number of PEG or OEG molecules, units, or monomers. In some embodiments, the linker comprises 2-50 (e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 45) PEG or OEG molecules. In other embodiments, the linker comprises 10-20 (e.g., 10, 11, 13, 14, 15, 16, 17, 18, 19, or 20) PEG or OEG molecules, or 30-40 (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) PEG or OEG molecules. For example, the linker may comprise 16 OEG or PEG molecules, or 36 OEG or PEG molecules.

[0031] The ligand molecule disclosed herein further comprises a Z33 peptide of Staphylococcus aureus protein A, or an antibody-binding fragment thereof, conjugated to a linker. Staphylococcal protein A (SPA) is a protein first found in the cell wall of Staphylococcus aureus. SPA is composed of five homologous domains, which are folded into a three-helical bundle. Protein A plays an important role in immunology due to its specific binding to the Fc portion of immunoglobulin G (IgG) from most mammalian species, including humans. Extensive structural and biochemical studies of protein A have been performed. The Z-58 domain of protein A was the first protein A domain to be widely used for affinity chromatography and affinity precipitation. A minimized binding domain, Z-33, was later developed without significantly altering the function of the molecule. The Z33 peptide is a protein A mimic with the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1) and contains two α-helical motifs. The disclosure also encompasses any antigen-binding fragment of a Z33 peptide, or ligand molecule comprising a peptide having an amino acid sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1. The degree of nucleic acid and / or amino acid identity can be determined using any method known in the art, such as, for example, the BLAST sequence database.

[0032] The present disclosure further provides a method for purifying an antibody or Fc fusion protein, which includes (a) dissolving the above system in an aqueous solution at physiological pH and aging overnight, whereby one or more filler molecules and one or more ligand molecules self-assemble into immunofibers (IFs); (b) mixing a sample containing a protein containing an Fc region with IFs under conditions in which the IFs bind to the Fc region to form an immunofiber-protein complex in the solution; (c) separating the immunofiber-protein complex from the solution by adding salt and / or centrifugation; and (d) dissociating the protein containing an Fc region from the IFs. Any suitable protein containing an Fc region can be purified using the methods disclosed herein. For example, the protein containing an Fc region can be an antibody, such as a monoclonal antibody. In other embodiments, the Fc region-containing protein can be an Fc fusion protein. An "Fc fusion protein" is a bioengineered polypeptide that joins the crystallizable fragment (Fc) domain of an antibody with another biologically active protein domain or peptide to generate a molecule with unique structure-function properties and, in some cases, therapeutic potential. Gamma immunoglobulin (IgG) isotypes are often used as a basis for generating Fc fusion proteins due to their advantageous characteristics, such as mobilization of effector functions and increased half-life in plasma. In some embodiments, the system described herein can be used to capture and purify other proteins and Fc regions that do not constitute Fc regions by incorporating customized binding pairs during the design of the filler and ligand molecules.

[0033] "Physiological" or "physiological" pH is the pH that typically occurs in human cells (e.g., human cells in vivo). In this regard, the physiological pH of the human body ranges between 7.35 and 7.45, with an average physiological pH at 7.40. Once the filler molecules and ligand molecules are dissolved in an aqueous solution at a suitable pH, the dissolved system is incubated or "aged" for a period of time sufficient for the one or more filler molecules and one or more ligand molecules to self-assemble into immunofibers (IFs). The incubation or "aging" of the filler molecules and ligand molecules in solution may be for any suitable period of time. In some embodiments, the dissolved system is aged for at least 2 hours, but not more than 48 hours. For example, the dissolved system may be incubated or aged for 2-8 hours, 8-12 hours, 12-16 hours, 16-20 hours, 20-24 hours, 24-36 hours, or 36-48 hours. In some embodiments, the dissolution system is aged overnight (eg, for about 6, 7, 8, 9, 10, 11, or 12 hours).

[0034] After a sufficient maturation period and formation of IFs, the disclosed method includes mixing a sample containing an antibody or Fc fusion protein with the IFs under conditions whereby the IFs bind to the Fc region of the protein (e.g., the antibody or Fc fusion protein) to form an immunofiber-protein complex in solution. Suitable conditions for affinity-based antibody purification methods are known in the art and can be employed in the disclosed methods. For example, suitable reagents and conditions for Protein A-based purification are known in the art and can be used in the context of the present disclosure (see, e.g., Proteus Protein A Antibody Purification Handbook, Bio-Rad (2016); and Fishman, JB, and Berg, EA, Cold Spring Harb Protoc; doi:10.1101 / pdb.prot099143). The complexes formed can then be separated from unbound immunofibers and Fc region-containing proteins (e.g., antibodies or Fc fusion proteins) and other components in the sample by a number of known separation means including, for example, salt-induced precipitation and centrifugation. The separated complexes can then be introduced into another solution at an acidic pH, where the immunofibers lose their binding affinity with the Fc region-containing proteins (e.g., antibodies or Fc fusion proteins).

[0035] The antibody or Fc fusion protein can then be dissociated from the immunofibers by filtration, such as, for example, diafiltration, microfiltration, or other means. In some embodiments, the antibody or Fc fusion protein is dissociated from the IFs by lowering the pH to elution conditions (e.g., pH 2.5-4.0) and filtering or microfiltration. In other embodiments, the antibody or Fc fusion protein can be dissociated from the IFs using sequential precipitation. In this regard, a sample containing an Fc region-containing protein (e.g., an antibody or Fc fusion protein) can be mixed with a first IF solution (prepared as described above), aged or incubated for any suitable amount of time (e.g., 2, 3, 4, 5, or 6 hours), and centrifuged (e.g., at 10,000-20,000 rpm). The resulting supernatant, containing immunofibers complexed with the Fc region-containing protein, may then be mixed with a second IF solution (prepared as described above), aged or incubated for any suitable amount of time (e.g., 2, 3, 4, 5, or 6 hours), and subjected to a second centrifugation (e.g., at 10,000-20,000 rpm). The foregoing process may be repeated any number of times until a desired yield of Fc region-containing protein (e.g., antibody or Fc fusion protein) is reached, and the final precipitate is then washed and resuspended in elution buffer. In some embodiments, the eluted antibody or Fc fusion protein may be fully recovered using membrane separation methods.

[0036] The system and method described herein has several advantages over other protein purification methods known in the art, especially Protein A chromatography, where the capture step is one of the major downstream bottlenecks due to resin capacity limitations and high production costs.Indeed, the IF system and method described herein provides high-throughput purification of monoclonal antibodies, easy handling, and reduced costs.In addition, the disclosed system and method allows for rapid purification of proteins, in that antibody-IF binding and aggregation can be completed within 30 minutes.

[0037] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0038] The following materials and methods were used in the experiments described in the examples.

[0039] Materials. All Fmoc amino acids and resins were obtained from Advanced Automated Peptide Protein Technologies (AAPPTEC, Louisville, KY, USA). Pure mAb1, as well as mAb1 and mAb2 in clarified Chinese hamster ovary (CHO) cell culture harvests, were obtained from Bristol-Myers Squibb (Devens, MA, USA). Cell cultures were clarified using depth filtration. Fmoc-N-amide-O(P)EGn-acid was purchased from PurePEG (San Diego, CA, USA) and BroadPharm (San Diego, CA, USA). Lauric acid (C12) was obtained from MilliporeSigma (St. Louis, MO, USA). Unless otherwise specified, all other reagents were obtained from VWR (Radnor, PA, USA) and used as received without further purification.

[0040] Molecule synthesis. Filler and ligand molecules were synthesized using methods previously described. Briefly, C12-VVEE and C12-VVKKO(P)EGnGGZ33 (n = 4, 8, 12, 16, 36, 45) were synthesized using a standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis protocol on a Liberty Blue automated microwave peptide synthesizer (CEM Corporation, Matthews, NC, USA). The crude products were cleaved from the solid support using a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / HO in a ratio of 92.5:5:2.5 for 2.5 h. Excess TFA was removed by evaporation, and ice-cold diethyl ether was added to precipitate the crude products, followed by centrifugation. The crude product was purified by preparative RP-HPLC using a Varian Polymeric Column (PLRP-S, 100 Å, 10 μm, 150 × 25 mm) at 25 °C on a Varian ProStar Model 325 preparative HPLC (Agilent Technologies, Santa Clara, CA, USA) monitoring the absorbance of the peptide segments at 220 nm. Collected fractions were analyzed by MALDI-TOF (BrukerAutoflex III MALDI-TOF instrument, Billerica, MA, USA), and the fractions containing the product were then lyophilized (LABCONCO). TM FreeZone (LABCONCO TM Free Zone, TM " is a trademark designation in the United States and elsewhere), a 4.5 L freeze dryer, Kansas City, MO, USA), and storage at -30 °C.

[0041] CMC measurements. The CMCs of ligand molecules in PBS were determined using Nile Red, a hydrophobic dye that undergoes a change in fluorescence intensity and emission wavelength (blue shift) when partitioned into hydrophobic domains of supramolecular assemblies. Nile Red was first dissolved in acetone at 20 μM, and 10 μl aliquots were loaded into several centrifuge tubes. After evaporation of acetone at room temperature, 500 μl of fresh ligand solution in PBS was added to the centrifuge tubes containing dried Nile Red at various concentrations and allowed to age overnight for assembly. The fluorescence spectrum of Nile Red was then monitored with a Fluorolog fluorometer (Jobin Yvon, Edison, NJ, USA) with the excitation wavelength fixed at 560 nm; the emission spectrum was monitored from 580 to 720 nm. The ratio of emission intensity at 635 nm (emission maximum of Nile Red in hydrophobic environment) to emission intensity at 660 nm (emission maximum of Nile Red in aqueous environment) was then plotted against the test concentrations to obtain a transition curve, and the CMC value was determined by the intersection of the two fitting lines.

[0042] Self-assembly, co-assembly, and TEM imaging. For self-assembly, filler or ligand molecules were dissolved in PBS (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) to a final concentration of 2.5 mM or 400 μM, respectively, and aged at room temperature for 24 h. To construct co-assembled IFs, filler and ligand molecules were pretreated with hexafluoroisopropanol (HFIP) to remove any pre-existing nanostructures that may have formed during the synthesis and purification process. After HFIP was evaporated, filler and ligand molecules were dissolved in PBS (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) to the desired concentrations, and aged at room temperature for 24 h. Then, 10 μl of each stock sample solution was spotted onto a carbon film-coated copper grid with 400 square mesh (Electron Microscopy Sciences, Hatfield, PA, USA), and the excess was removed with filter paper to leave a thin film of the sample on the grid. After drying the samples for 5 min, 10 μl of 2% uranyl acetate was added to the sample grid and the excess was removed after 30 s. All samples were allowed to dry for at least 3 h before imaging. Bright-field TEM imaging was performed on a FEI Tecnai 12 TWIN Transmission Electron Microscope, and all TEM images were taken with a SIS Megaview III wide-angle CCD camera.

[0043] CD spectroscopy. CD spectra of the self-assembled ligand molecules were collected at 25 °C using a 1 mm path length quartz UV-vis absorption cell (ThermoFisher Scientific, Pittsburgh, PA, USA) on a Jasco J-710 spectrophotometer (JASCO, Easton, MD, USA). Solvent background spectra were acquired and subtracted from the sample spectra. Collected data were averaged from three scans and normalized with respect to ligand concentration.

[0044] ITC experiments. ITC experiments were performed using a high-precision VP-ITC titration calorimeter system (Microcal Inc.). A 40 μM ligand solution was titrated with 100 μM mAb1 in PBS (pH 7.4) at 25 °C. The heat evolved after each injection was determined from the integration of the calorimetric signal. The heat associated with the binding between the ligand molecule and mAb1 was determined by subtracting the heat of dilution. Data analysis was performed using the MicroCal Origin package.

[0045] mAb Precipitation Experiment. One day before the precipitation experiment, IF stock solutions with the desired filler and ligand concentrations were prepared. In the experimental group, pure mAb1 (448 μM, 64.5 g / L) from the concentrated solution was added to 100 μL of IF solution to the desired final concentration and incubated at room temperature for 30 min. The solution was then centrifuged at 15000 rpm for 15 min. For the 1M salt group, ammonium sulfate was added to the mAb-IF mixture to a final concentration of 1M and incubated for an additional 30 min before centrifugation. The supernatant was removed and analyzed by ProA-HPLC (POROS) to determine the protein concentration. TM The analysis was performed using a 20 μm column (Stainless Steel, 2.1 × 30 mm, 0.1 mL). The amount of each precipitate was calculated by subtracting the amount in the supernatant from the amount added. The precipitation yield was calculated by dividing the amount added by the amount of precipitate.

[0046] Sequential precipitation and mAb elution with IF solutions. One day prior to the precipitation experiment, three IF stock solutions were prepared: IF1 (2.5 mM filler, 250 μM O16), IF2 (2.5 mM filler, 750 μM O16), and IF3 (2.5 mM filler, 200 μM O16). Pure mAb1 (448 μM, 64.5 g / L) from the concentrated solution was added to 100 uL of IF1 and 100 uL of IF2 to the desired final concentrations of 40 μM and 80 μM, respectively. After 4 h of incubation, the samples were centrifuged at 15000 rpm for 15 min, and the supernatant was transferred to 100 uL of IF3 for another 4 h of incubation and subsequent centrifugation. Then, 200 μL of PBS and 400 μL of elution buffer (40 mM sodium acetate, pH 3.7) were used to wash and resuspend the precipitates from the two precipitation steps. The supernatants from each precipitation, washing, and elution step were analyzed by ProA-HPLC (POROS) to determine the mAb concentration. TM The analysis was performed using a 20 μm column, stainless steel, 2.1 × 30 mm, 0.1 mL).

[0047] Purification of mAb1 from clarified cell culture harvest. mAb1 in clarified bulk state was incubated with optimized IFs (100 μL) at a concentration of 40 μM or 80 μM for 30 min. The same procedure was performed to precipitate mAb1 and to obtain a pellet by centrifugation. The pellet was then suspended in PBS (400 μL, 40 mM sodium acetate, pH 3.7) and transferred to a dialysis tube (Pur-A-Lyzer Maxi, 50 kDa molecular weight cut-off, Sigma-Aldrich, St. Louis, MO, USA). The resuspended solution was then dialyzed against 40 mM sodium acetate (1 L) at pH 3.7 for 24 h with three changes of dialysis buffer. Protein, filler, and ligand concentrations were determined by ProA-HPLC and RP-UPLC.

[0048] Sequential precipitation of high mAb titers using lyophilized IFs. Six lyophilized IF stock powders (IF4-IF9) were prepared with filler and O16 concentrations of 2.5 mM and 400 μM, respectively, when dissolved in 100 μL. Lyophilized IF powders were prepared one day before the precipitation experiment. IFs were coassembled in water for 24 h before lyophilization. Pure mAb1 from concentrated stock solutions of either (146 μM, 21 g / L) or (215 μM, 31 g / L) was added to lyophilized IF4. After 1 h of incubation, the samples were centrifuged at 15000 rpm for 15 min. After centrifugation, the volume of the supernatant was measured and fresh PBS was added until the final supernatant volume was 100 μL. 100 μL of the supernatant after the first precipitation step was then added to the next lyophilized IF stock powder (IF5) and incubated for another 1 h. This incubation, centrifugation, and supernatant exchange step was repeated until all six lyophilized IFs (IF4-IF9) had been used for mAb precipitation. Supernatants after the first, third, and sixth precipitation steps were analyzed using ProA-HPLC to measure the cumulative amount of mAbs remaining through the precipitation steps. Example 1

[0049] This example illustrates the molecular design and characteristics of the system disclosed herein.

[0050] The immunofiber system was formed by co-assembly of filler and ligand molecules. The filler molecule, C12-VVEE, was designed to regulate the distribution of ligand molecules in the co-assembled IFs (Figure 1A). The Z33 peptide, which has a two-helical motif, was conjugated onto the C-terminus of the ligand molecule, C12-VVKK[Linker]Z33 for specific monoclonal antibody capture (Figure 1A). In the previous ligand design (G2), a double glycine (GG) segment was inserted as a linker between the IF surface and Z33. To further improve the flexibility and accessibility of Z33, we designed a series of ligand molecules, O4, O8, O12, O16, O36, and P2000, with various OEG (or PEG) linkers (n = 4, 8, 12, 16, 36, 45), as summarized in Table 1. [Table 1]

[0051] As shown in Figure 1B, as the length of OEG (or PEG) increases, the Z33 peptide was expected to extend further away from the IF surface. We hypothesized that the incorporation of an OEG (or PEG) linker would further reduce the radial steric hindrance of the IFs and improve the mAb-IF interaction for both Fc binding sites. The filler and ligand molecules were synthesized using an automated solid-phase peptide synthesis (SPPS) method.

[0052] Figure 1C illustrates the process of spontaneous co-assembly of filler and ligand molecules in aqueous solution, leading to the formation of filamentous IFs with Z33 displayed on the IF surface. Upon addition of mAbs, there were four possible states of mAbs in the IF solution. States 1 and 2 represent two Fc moieties of mAb bound to two different IFs or two ligands on the same IF, respectively. In state 3, only one Fc moiety of mAb is bound to one ligand on IFs. Given the equilibrium between ligand monomer and co-assembled structures, in state 4, mAbs could have bound to zero, one, or two monomeric ligands in solution, rather than assembled in IFs. mAb binding and precipitation represent two separate processes. Previous results showed that 1 M salt could not directly precipitate mAb1, but it acts as a strong charge screening agent to precipitate almost all IFs, and thus precipitate mAbs bound to IFs (states 1-3). However, under salt-free conditions, binding to IFs may not be sufficient for mAbs to precipitate. The key to the precipitation of mAbs under salt-free conditions is thought to be the formation of cross-linked mAb-IF complexes, which are primarily induced by mAbs in state 1. Example 2

[0053] This example describes the molecular assemblies and characteristics of immunofibers produced using the systems and methods disclosed herein.

[0054] To evaluate the behavior of the filler and ligand molecules in the coassembled IFs, we first investigated their self-assembly properties in phosphate-buffered saline (PBS), pH 7.4. As shown in Figure 2A, the filler molecules were able to self-assemble into thread-like structures with a diameter of 7.4 ± 0.6 nm. When the OEG (or PEG) linker was used, the critical micelle concentrations (CMCs) of the ligand molecules were relatively higher than the previous G2 ligand, ranging from 5.1 to 10.5 μM (Table 1). Representative transmission electron microscopy (TEM) images revealed that each ligand molecule could self-assemble into thread-like structures with diameters of 11.1 ± 0.9 nm (O4), 12.6 ± 1.2 nm (O8), 13.6 ± 1.2 nm (O12), 14.9 ± 1.3 nm (O16), 18.5 ± 1.8 nm (O36), and 19.3 ± 2.0 nm (P2000), respectively (Figure 2B-2G). As expected, the diameter of the IFs increased with increasing linker length. To gain insight into the molecular packing within the self-assembled ligand molecules, circular dichroism (CD) spectra were collected for each of the newly designed ligand molecules. The negative peaks around 208 nm and 222 nm suggest the conservation of α-helical secondary structure in all the self-assembled ligand molecules, as shown for the original Z33 peptide (Figure 2H). Isothermal titration calorimetry (ITC) was used to determine the binding affinity between monoclonal human IgG1 (144 kDa, abbreviated as "mAb1") and the ligand molecules O4-O16. The dissociation constants Kd ranged between 50 nM and 70 nM, similar to the reported value for the free Z33 peptide (26 nM), indicating that the conjugation of OEG linkers in the ligand molecules did not significantly impair their mAb binding affinity (Figures 3A-3D). Example 3

[0055] This example demonstrates the effect of linker length and ligand selection on antibody yield.

[0056] To compare the performance of the ligands, 100 μM of the ligand molecules were individually coassembled with 2.5 mM of the filler molecules in 100 μL of PBS (pH 7.4) using a similar method as described above. Filler molecules at 2.5 mM (solubility limit) were found to give the best mAb precipitation results and were used consistently in the experiments described here. Pure mAb1 from the stock solution was then added to the different IF solutions to reach a final concentration of 20 μM and incubated for 30 min at room temperature. To investigate the effect of salt on the mAb precipitation yield, two parallel experiments were performed for each coassembled IF system: one with 1 M salt and one without salt (Figure 4A). After centrifugation, the supernatants were analyzed by ProA-HPLC to determine the remaining mAb concentration and to calculate the mAb precipitation yield. When using 1M salt and replacing the GG linker with the O(P)EG linker in the ligand design, the mAb precipitation yield showed a significant improvement from 65% to more than 88%. Notably, mAb precipitation yields of over 95% were obtained for O4-O36. Since there is no bias for IF precipitation efficiency under 1M salt conditions, this increase in mAb precipitation yield from G2 to the new ligand molecules with OEG (or PEG) linkers leads to a significant improvement in mAb binding efficiency, especially as a result of the increased length and flexibility of the linker. A slight decrease in mAb precipitation yield was observed for P2000, possibly due to entanglement of the long PEG chains that impair the accessibility of the Z33 peptide for mAb capture.

[0057] Under salt-free conditions, the mAb precipitation yields were generally lower than those in the salt group, while an upward trend was observed with increasing OEG linker length. Unlike the salt group, the mAb precipitation yields under salt-free conditions were determined by a combination of the mAb binding efficiency and the precipitation efficiency of the mAb-IF complex. As indicated by the salt group data set, there was no obvious difference in the mAb binding efficiency from O4 to O36. The increase in the mAb precipitation yield under salt-free conditions represents an improvement in the mAb precipitation efficiency, possibly caused by the improved cross-linking between IFs. More importantly, the difference in yields between the 1M salt and salt-free groups gradually decreased with increasing linker length, leading to a yield difference of less than 10% for O36. This was considered very promising for mAb precipitation in that the improved mAb-IF interactions achieved by using OEG or (PEG) linkers could potentially replace the salt contribution to mAb precipitation.

[0058] To confirm the above observations and to get a preliminary understanding of the mAb binding capacity for this IF system, the experiment was repeated with a higher mAb1 concentration (40 μM). As shown in Figure 4B, a similar yield trend was observed, indicating an improvement in mAb binding efficiency with increasing OEG linker length, except for a slight upward trend in the 1M salt group for O4-O36. Despite a consistent decrease in mAb precipitation yield from 20 to 40 μM mAbs, the precipitated mAb mass for 100 μL samples (20 μM: 0.3 mg, 40 μM: 0.6 mg) was actually comparable or higher than 40 μM mAbs (Figures 4C-4D), indicating that the Z33 peptide displayed on the IF surface was not fully saturated by 20 μM mAb1 and could potentially be further utilized if more mAbs were added.

[0059] The difference in mAb precipitation yields can also be revealed by the turbidity of the IF solutions. The IF solutions of O12, O16, O36, and P2000 became turbid within 5 min after mAb addition, which was not observed in the first three IF systems. Figure 4E shows sample vials of IFs from O4 to O16 after mixing with 40 μM mAb1 for 5 min under 1 M salt or no-salt conditions. In most cases, the turbidity of the salt group was relatively higher than that of the no-salt group, suggesting a higher mAb precipitation yield for the salt groups. Meanwhile, the turbidity increased from O4 to O16, following the yield trend shown in Figure 4B.

[0060] These results demonstrate that increasing the linker length can simultaneously improve the mAb binding efficiency of the ligand molecule as well as the precipitation efficiency of the mAb-IF complex. Although O36 showed the best mAb precipitation yield in all conditions, O16 was more desirable in terms of synthesis yield and material cost and was selected for subsequent experiments. Example 4

[0061] This example describes the optimization of monoclonal antibody precipitation under salt-free conditions using the methods disclosed herein.

[0062] Previous studies have shown that the ligand:mAb molar ratio plays the most important role in the precipitation yield of mAb. To optimize the mAb precipitation yield under salt-free conditions and to investigate whether the O16 IF system can be efficiently applied to high concentrations of mAb, mAb1 at three concentrations, 40 μM (6 mg / ml), 80 μM (12 mg / ml), and 133 μM (20 mg / ml), was incubated with IFs formed by 2.5 mM filler molecules and O16 ligand molecules at various concentrations in 100 μL of PBS. Figures 5A and 5B show the plots of mAb precipitation yield and mass vs. O16 concentration, respectively, and the optimum was observed between 6:1 and 9:1 ligand:mAb molar ratios for all three mAb concentrations. At the optimal O16 concentration, the mAb precipitation yields for 40 and 80 μM mAb were about 85%, while at 133 μM mAb only about 65% of mAb was precipitated, indicating a decline in IF performance when scaling up to higher mAb concentrations. To better understand why the mAb precipitation yield did not reach 100% at the optimal O16 concentration, parallel experiments were performed with 40 μM mAb with optimal IFs (2.5 mM filler, 250 μM O16) under both 1 M salt and no-salt conditions (Figure 5C). More than 99% of mAb1 was precipitated when 1 M salt was present, indicating complete capture of mAb1 on IFs. However, only 88% of mAb1 was precipitated in the no-salt group, which was attributed to insufficient IF precipitation that may result from limited IF cross-linking.

[0063] To understand the trends for mAb precipitation yields observed in Figures 5A and 5B, state 3, state 1, and state 2 would likely predominate at low, medium, and high ligand concentrations, respectively (Figure 5D). At low ligand concentrations, all ligands are saturated by excess mAbs, leaving little room for binding of the second Fc site (state 3). The initial yield increase could be attributed to an increase in dual-site mAb binding for IF crosslinking as the ligand concentration increases (state 1). After the optimal operating point, a further increase in ligand concentration results in a decrease in yield, which could be caused by a decrease in crosslinking efficiency, since the opportunity for mAbs to bind to two Z33s from the same IF (state 2) becomes much higher with the reduced spacing of the O16 ligands on the IFs surface.

[0064] To gain a better understanding of the kinetics of aggregation, the turbidity of a mixture of 40 μM mAb1 and optimized IFs (2.5 mM filler, 250 μM O16) was monitored by absorbance at 350 nm for 24 h. As shown in Figure 5E, the turbidity increased with time and reached a plateau within 30 min, suggesting rapid initial aggregation. To correlate the turbidity of the solution with the mAb precipitation yield, the supernatant was analyzed for 30 min, 2 h, and 24 h (Figure 5F). Consistent with the turbidity studies, the binding and aggregation of mAb-IF was nearly complete within 30 min, allowing a precipitation yield as high as 83% to be reached. A slight increase in yield was observed at 2 h (88%) and 24 h (92%), but this was not revealed by the less sensitive turbidity studies, which came at the expense of the time efficiency of the mAb precipitation process. Example 5

[0065] This example describes the sequential precipitation and elution of a monoclonal antibody.

[0066] To further improve the mAb precipitation yield, two sequential precipitation steps were performed to precipitate the remaining mAb. As depicted in Figure 6A, the supernatant from the first precipitation step was added into fresh IF solution. Then, a washing step was performed with PBS to remove non-specifically bound impurities in the precipitate from the two precipitation steps. To resuspend the precipitated mAbs, elution buffer (40 mM sodium acetate, pH 3.7) was added to dissociate the mAb-IF complex.

[0067] As a proof of concept, sequential precipitations were performed with pure mAb1 at 40 μM and 80 μM. For the first precipitation, mAb1 at 40 μM (6 mg / mL) and 80 μM (12 mg / mL) were incubated with IFs containing 250 μM and 750 μM O16, respectively, and the optimized conditions were indicated by Figure 5A. More than 82% of mAb1 was precipitated for both groups, while 8–20 μM mAb1 remained in the supernatant (Figure 6B). Based on another O16 concentration optimization set shown in Figure 7, IFs containing 200 μM O16 were used for the second precipitation step. Finally, a final precipitation yield of more than 97% and an elution yield of ~88% were achieved for both mAb concentrations, with little yield loss during the washing steps (Figure 6B). To ensure sufficient recovery of eluted mAbs, a membrane separation step using a 50 kDa cutoff membrane was performed to separate mAb1 (~144 kDa) and dissociated IFs (monomer size <6 kDa) (Figure 6A). This sequential precipitation with dissolved IFs was then applied to the separation of mAb1 from clarified cell culture harvest at 40 μM and 80 μM, where mAb1 precipitation yields of over 86% and 90% were achieved for the respective mAb titers (Figure 6C).

[0068] To achieve high mAb precipitation yields at mAb titers above 20 mg / mL, sequential precipitations were performed with fresh lyophilized IFs containing 2.5 mM filler and 400 μM O16 mixed with 100 μL of either 21 mg / mL or 31 mg / mL pure mAb. Prior to centrifugation, the mAbs were incubated with the lyophilized IFs for 1 h. After the centrifugation step, the supernatant still containing the mAbs was measured and fresh PBS was added to reach a final volume of 100 μL. The precipitation steps were repeated with freshly prepared lyophilized IFs at the same concentration until a total of six precipitation steps were completed (Figure 6D). The supernatants after the first, third, and sixth precipitation steps were analyzed using ProA-HPLC to measure the cumulative amount of mAbs remaining throughout the precipitation process. As shown in Figures 6E and 6F, nearly all mAbs were precipitated after six incubation and centrifugation steps, with yields reaching 96% and 98% for mAb titers of 21 mg / mL and 31 mg / mL, respectively. Furthermore, after only three precipitation steps, the precipitation yield of the mAb increased significantly from around 20% to 80%, indicating that the optimal ligand:protein ratio is achieved early in the process, as more protein is captured and separated with each successive precipitation step.

[0069] The above examples illustrate the design and construction of a series of supramolecular IF systems containing OEG (or PEG) linkers, and demonstrate the influence of epitope topography in the radial direction of IFs on the biological activity of IFs. The described results revealed that increasing the length of the linker to OEG16 can simultaneously improve monoclonal binding and precipitation efficiency under salt-free conditions. However, too long linkers show a negative effect on the function of the resulting supramolecular polymer. Importantly, the mAb precipitation yield under salt-free conditions can be efficiently optimized by adjusting the ligand concentration to reach the desired monoclonal antibody binding state. The strategy in engineering the linker for even better epitope presentation sheds important light on the design of supramolecular polymers for specific molecular recognition and targeted drug delivery. The supramolecular IF system described here serves as an efficient alternative for monoclonal antibody purification, and can be applied to capture and purification of other molecules of interest by incorporating customized binding pairs into the system design. References

[0070] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. 1.Adelizzi, B.;Van Zee, NJ;de Windt, LNJ;Palmans, ARA;Meijer, EW Future of Supramolecular Copolymers Unveiled by Reflecting on Covalent Copolymerization. Journal of the American Chemical Society 2019, 141, 6110-6121. 2. Aida, T.; Meijer, E. W. Supramolecular Polymers - We've Come Full Circle. Israel Journal of Chemistry, 2020, 60, 33-47. 3.Stupp, SI;Clemons, TD;Carrow, JK;Sai, H.;Palmer, Supramolecular and Hybrid Bonding Polymers. Israel Journal of Chemistry 2020, 60, 124-131. 4.Webber, MJ;Appel, EA;Meijer, EW;Langer, R. Supramolecular Biomaterials. Nature Materials 2016, 15, 13-26. 5.Shy, AN; Kim, BJ; Xu, B. Enzymatic Noncovalent Synthesis of Supramolecular Soft Matter for Biomedical Applications. Matter 2019, 1, 1127-1147. 6. Aida, T.; Meijer, EW; Stupp, S. Functional Supramolecular Polymers. Science 2012, 335, 813-817. 7. Sato, K.; Hendricks, MP; Palmer, LC; Stupp, SI Peptide Supramolecular Materials for Therapeutics. Chemical Society Reviews 2018, 47, 7539-7551. 8.Cui, HG;Webber, MJ;Stupp, SI Self-Assembly of Peptide Amphiphiles: From Molecules to Nanostructures to Biomaterials. Biopolymers 2010, 94, 1-18. 9.Acar, H.;Srivastava, S.;Chung, EJ;Schnorenberg, MR;Barrett, JC;LaBelle, JL;Tirrell, M. Self-Assembling Peptide-Based Building Blocks in Medical Applications. Advanced Drug Delivery Reviews 2017, 110, 65-79. 10.Su, H.;Wang, FH;Ran, W.;Zhang, WJ;Dai, WB;Wang, H.;Anderson, CF;Wang, ZY;Zheng, C.;Zhang, PC;Li, YP;Cui, HG The Role of Critical Micellization Concentration in Efficacy and Toxicity of Supramolecular Polymers. Proceedings of the National Academy of Sciences of the United States of America 2020, 117, 4518-4526. 11.Wang, HM;Feng, ZQQ;Xu, B. Supramolecular Assemblies of Peptides or Nucleopeptides for Gene Delivery. Theranostics 2019, 9, 3213-3222. 12.Moore, AN;Hartgerink, JD Self-Assembling Multidomain Peptide Nanofibers for Delivery of Bioactive Molecules and Tissue Regeneration. Accounts of Chemical Research 2017, 50, 714-722. 13. VandenBerg, MA; Sahoo, JK; Zou, L.; McCarthy, W.; Webber, MJ Divergent Self-Assembly Pathways to Hierarchically Organized Networks of Isopeptide-Modified Discotics under Kinetic Control. ACS Nano 2020. 14.Wang, FH;Xu, DQ;Su, H.;Zhang, WJ;Sun, XR;Monroe, MK;Chakroun, RW;Wang, ZY;Dai, WB;Oh, R.;Wang, H.;Fan, Q.;Wan, FY;Cui, HG Supramolecular Prodrug Hydrogelator as an Immune Booster for Checkpoint Blocker-Based Immunotherapy. Science Advances 2020, 6. 15.Chen, CH;Palmer, LC;Stupp, SI Self-Repair of Structure and Bioactivity in a Supramolecular Nanostructure. Nano Letters 2018, 18, 6832-6841. 16.Storrie, H.;Guler, MO;Abu-Amara, SN;Volberg, T.;Rao, M.;Geiger, B.;Stupp, SI Supramolecular Crafting of Cell Adhesion. Biomaterials 2007, 28, 4608-4618. 17.Du, X.W.;Zhou, J.;Xu, B. Supramolecular Hydrogels Made of Basic Biological Building Blocks.Chemistry-an Asian Journal, 2014, 9, 1446-1472. 18.Sur, S.;Tantakitti, F.;Matson, JB;Stupp, SI Epitope Topography Controls Bioactivity in Supramolecular Nanofibers. Biomaterials Science 2015, 3, 520-532. 19.Pashuck, ET;Duchet, BJR;Hansel, CS;Maynard, SA;Chow, LW;Stevens, MM Controlled Sub-Nanometer Epitope Spacing in a Three-Dimensional Self-Assembled Peptide Hydrogel.ACS Nano 2016, 10, 11096-11104. 20.Guler, MO;Hsu, L.;Soukasene, S.;Harrington, DA;Hulvat, JF;Stupp, SI Presentation of Rgds Epitopes on Self-Assembled Nanofibers of Branched Peptide Amphiphiles. Biomacromolecules 2006, 7, 1855-1863. 21.Hudalla, GA;Sun, T.;Gasiorowski, JZ;Han, HF;Tian, ​​YF;Chong, AS;Collier, JH Gradated Assembly of Multiple Proteins into Supramolecular Nanomaterials. Nat. Mater. 2014, 13, 829-836. 22.Cheetham, AG;Zhang, PC;Lin, YA;Lock, LL;Cui, HG Supramolecular Nanostructures Formed by Anticancer Drug Assembly. Journal of the American Chemical Society 2013, 135, 2907-2910. 23.Trent, A.;Marullo, R.;Lin, B.;Black, M.;Tirrell, M. Structural Properties of Soluble Peptide Amphiphile Micelles. Soft Matter 2011, 7, 9572-9582. 24.Bolton, GR;Mehta, KK The Role of More Than 40 Years of Improvement in Protein a Chromatography in the Growth of the Therapeutic Antibody Industry. Biotechnol. Prog. 2016, 32, 1193-1202. 25.Gronemeyer, P.;Ditz, R.;Strube, J. Trends in Upstream and Downstream Process Development for Antibody Manufacturing. Bioengineering 2014, 1, 188-212. 26.Mullerpatan, A.; Chandra, D.; Kane, E.; Karande, P.; Cramer, S. Purification of Proteins Using Peptide-Elp Based Affinity Precipitation. Journal of Biotechnology 2020, 309, 59-67. 27.Venkiteshwaran, A.;Heider, P.;Teyssevre, L.;Belfort, G. Selective Precipitation-Assisted Recovery of Immunoglobulins from Bovine Serum Using Controlled-Fouling Crossflow Membrane Microfiltration.Biotechnology and Bioengineering 2008, 101, 957-966. 28.Fausnaugh, J. L.;Pfannkoch, E.;Gupta, S.;Regnier, F. E. High-Performance Hydrophobic Interaction Chromatography of Proteins. Anal Biochem 1984, 137, 464-72. 29. Baldwin, R. L. How Hofmeister Ion Interactions Affect Protein Stability. Biophysical Journal 1996, 71, 2056-2063. 30.Majumdar, R.;Manikwar, P.;Hickey, JM;Samra, HS;Sathish, HA;Bishop, SM;Middaugh, CR;Volkin, DB;Weis, DD Effects of Salts from the Hofmeister Series on the Conformational Stability, Aggregation Propensity, and Local Flexibility of an Igg1 Monoclonal Antibody.Biochemistry 2013, 52, 3376-3389. 31. Swartz, AR; Xu, XK; Traylor, SJ; Li, ZJ; Chen, W. One-Step Affinity Capture and Precipitation for Improved Purification of an Industrial Monoclonal Antibody Using Z-Elp Functionalized Nanocages. Biotechnology and Bioengineering 2018, 115, 423-432. 32.Li, Y.;Lock, LL;Wang, YZ;Ou, SH;Stern, D.;Schon, A.;Freire, E.;Xu, XK;Ghose, S.;Li, ZJ;Cui, HG Bioinspired Supramolecular Engineering of Self-Assembling Immunofibers for High Affinity Binding of Immunoglobulin G. Biomaterials 2018, 178, 448-45. 33. Braisted, AC; Wells, JA Minimizing a Binding Domain from Protein A. Proc. Natl. Acad. Sci. USA 1996, 93, 5688-5692.

[0071] In the context of describing the invention (particularly in the context of the following claims), use of the terms "a," "an," "the," and "at least one" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are merely intended to facilitate a better understanding of the invention, and are not intended to impose limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0072] Preferred embodiments of the invention will now be described, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those of ordinary skill in the art as appropriate, and intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. (a)One or more filler molecules comprising a linear hydrocarbon chain conjugated to an amino acid sequence of 2 to 5 amino acids; (b)One or more ligand molecules comprising a linear hydrocarbon chain, an amino acid sequence of 2 to 5 amino acids, a linker, and the Z33 peptide of Staphylococcus aureus protein A or an antibody-binding fragment thereof conjugated to the linker comprising a system, wherein the one or more filler molecules and the one or more ligand molecules have the ability to self-assemble into immunofibers (IFs) under physiological conditions.

2. The system according to claim 1, wherein the Z33 peptide comprises the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1).

3. The system according to claim 1, wherein each of the one or more filler molecules and the one or more ligand molecules comprises a linear hydrocarbon chain 12 carbons in length.

4. The system according to claim 1, wherein each of the one or more filler molecules and the one or more ligand molecules comprises the amino acid sequence VVXX (SEQ ID NO: 2).

5. The system according to claim 4, wherein the filler molecule comprises the amino acid sequence VVEE (SEQ ID NO: 3).

6. The system according to claim 4, wherein the ligand molecule comprises the amino acid sequence VVKK (SEQ ID NO: 4).

7. The system according to claim 1, wherein the linker comprises one or more oligo(ethylene glycol) (OEG) molecules or one or more polyethylene glycol (PEG) molecules.

8. The system according to claim 7, wherein the linker comprises 2 to 50 OEG molecules or 2 to 50 PEG molecules.

9. The system according to claim 8, wherein the linker comprises 36 OEG molecules or 36 PEG molecules.

10. The system according to claim 8, wherein the linker comprises 16 OEG molecules or 16 PEG molecules.

11. A method for purifying a protein comprising the Fc region of an antibody, comprising dissolving the system according to any one of claims 1 to 10 in an aqueous solution at physiological pH and aging overnight, whereby the one or more filler molecules and the one or more ligand molecules self-assemble into immunofibers (IFs); (b) Mixing a sample containing a protein comprising an Fc region with the IFs under conditions where the IFs bind to the Fc region to form an immunopher - protein complex in solution; (c) Separating the immunopher - protein complex from the solution by addition of salt and / or centrifugation; and (d) Dissociating the protein comprising the Fc region from the IFs A method comprising the above steps.

12. The method according to claim 11, wherein the protein comprising the Fc region is an antibody.

13. The method according to claim 11, wherein the protein comprising the Fc region is an Fc fusion protein.

14. The method according to claim 11, wherein the protein comprising the Fc region is dissociated from the IFs by lowering the pH to elution conditions and by filtration or microfiltration.

15. The method according to claim 11, which is completed within 30 minutes.

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