Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
  • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28023—Fibres or filaments
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
  • C07K1/303—Extraction; Separation; Purification by precipitation by salting out
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
  • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• bioactive epitopes such as cell adhesion motif RGD and neurite-promoting sequence IKVAV (SEQ ID NO: 2)
• RGD cell adhesion motif
• IKVAV neurite-promoting sequence IKVAV
• Webber et al. investigated bioactive PA nanofibers displaying the RGDS (SEQ ID NO: 3) epitope on the surface therapeutic delivery of bone-marrow mononuclear cells (BMNCs), implying an enhanced biological adhesion.
• BMNCs bone-marrow mononuclear cells
• Collier and coworkers covalently linked the self-assembling peptide Ql l to an antigen OVA peptide and found that the resultant supramolecular OVA-Ql 1 nanofibers possess enhanced immunogenicity.
• a typical example of affinity precipitation is the use of elastin-like-protein (ELP) fused Z-domain to precipitate IgG through the temperature and salt triggered solubility transition of ELP. 39"40
• ELP elastin-like-protein
• ID nanostructures are constructed by self-assembly of peptides or peptide conjugates containing a short ⁇ -sheet sequence as the core building motif essential for the intermolecular hydrogen bonding that promotes directional, anisotropic growth of the resultant assemblies. While this molecular design strategy has led to the successful production of a plethora of bioactive filamentous ⁇ -sheet assemblies for interfacing with cells, concerns associated with potential toxicity reminiscent of amyloid fibrils have promoted other supramolecular crafting strategies with a-helical peptides.
• IFs supramolecular immunofibers
• IgG monoclonal antibody immunoglobulin G
• the present invention comprises the direct conjugation of the Protein A of Staphylococcus aureus mimicking peptide Z33, having the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1), a motif containing two a-helices, to linear hydrocarbons to create self-assembling immuno- amphiphiles.
• the results show that the resulting amphiphilic peptides can, despite lacking the essential ⁇ -sheet segment, effectively associate under physiological conditions, into supramolecular immunofibers (IFs) while preserving their native ⁇ -helical conformation. Isothermal titration calorimetry measurements confirmed that these self-assembling immunofibers can bind to the immunoglobulin G (IgG) antibody with high specificity at pH 7.4, but no detectable binding occurred in elution buffer, pH 2.8.
• IgG immunoglobulin G
• the IFs of the present invention have pH dependent specific binding which enables the precipitation and purification of the target IgG antibodies.
• the supramolecular engineering of protein binding peptides into filamentous assemblies are useful for effective protein purification.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain and wherein the peptide takes an a-helical conformation when in an aqueous solution at physiological pH.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain, wherein the antibody binding peptide has a hydrophilic amino acid sequence of the Z33 peptide of Protein A of Staphylococcus aureus, or a functional portion or fragment or derivative thereof.
• the present invention provides an immuno-amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain, wherein the antibody binding peptide has the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1), or a functional portion or fragment or derivative thereof.
• the present invention provides a method for purification of an antibody or an Fc fusion protein, comprising the steps of: a) dissolving the immuno-amphiphile in an aqueous solution at physiological pH and aging overnight to make it self-assemble into immuofibers (IFs); b) mixing a sample containing the antibody or Fc fusion protein with the IFs, and allowing the IFs to bind the Fc portion of the antibody or Fc fusion protein and form an immunofiber-antibody complex or an immunofiber-Fc fusion protein complex in solution; c) separating the immunofiber-antibody complex or
• the immunofiber-Fc fusion protein complex from the solution by adding salt and centrifugation; and d) dissociating the IFs from the antibody or Fc fusion protein and collecting the unbound antibody or Fc fusion protein.
• the IFs may be separated from the antibody or Fc fusion protein by lowering the pH to elution condition and filtration or microfiltration.
• FIG. 1A Schematic illustration of the Z33 peptide binding to Fc- portion of IgG
• IB The sequences of C12-Z33 and 2C8-Z33. Alkyl groups and Z33 are indicated with the yellow and blue shaded areas, respectively. The two a-helices in Z33 peptide are underlined.
• FIG. 2A-2F (A) Schematic illustration of self-assembly of C12-Z33.
• B Normalized CD Spectra of Z33 peptide and Z33-C12 at pH 7.4 and 2.8, respectively.
• TEM characterization of C12-Z33 at pH 7.4 (C, D) and 2.8 (E, F).
• the TEM samples were prepared at concentration of 100 ⁇ in PBS (pH 7.4) and IgG elution buffer (pH 2.8) separately.
• the TEM samples were negatively stained with 2 wt% uranyl acetate.
• FIGS 3 A-3D ITC profiles for the titration of 100 ⁇ C 12-Z33 into a solution of 2 ⁇ IgGl at 15 °C in (A) PBS buffer, pH 7.4, and (B) IgG elution buffer, pH 2.8. ITC profiles for the titration of 100 ⁇ (C) Z33 and (D) C12-SZ33 into 2 ⁇ IgGl in PBS at 15 °C, pH 7.4.
• FIGS 4A-4E TEM characterization of 2C8-Z33 in (A) PBS at pH 7.4 with a diameter of 16.8 ⁇ 1.5 nm and (B) IgG elution buffer at pH 2.8 with a diameter of 17.3 ⁇ 1.9 nm.
• the preparation of TEM sample was similar with that of C12-Z33.
• C Normalized CD spectra of 100 ⁇ 2C8-Z33 in PBS at pH 7.4 showed a-helix secondary structures.
• FIGS 5A-5D Schematic illustration of the precipitation of IFs-IgG complexes triggered by 0.6 M Na2S04 solution.
• B Photographs of 5 mM PBS solution of C12-Z33 (i) before and (ii) after addition of 0.6 M Na2S04 and 20 ⁇ PBS solutions of IgGl with (iii) 5 mM C12-Z33, (iv) 0.6 M Na 2 S0 4 , and (v) 5 mM C12-Z33 and 0.6 M Na 2 S0 4 . Precipitation were observed in (ii) and (v).
• FIG. 7 CMC Measurement of C12-Z33. Emission spectra of the reporter dye Nile Red monitored by a Fluorolog fluorometer (Jobin Yvon, Edison, NJ) after incubated with a series of concentrations of C12-Z33. Excitation wavelength was fixed at 560 nm; emission spectra were monitored 580-720 nm. The CMC of C12-Z33 is determined by a blue-shift of the emission maximum, where the transition indicates the dye partitioning into the hydrophobic compartment of assembled nanostructures. All spectra shown here are normalized by the emission maximum. The CMC range for C12-Z33: 2-5 ⁇ .
• FIGS 8A-B Figures 8A-B. RP-HPLC (8A) and MALDI-TOF MS (8B) characterization of RB-C12-Z33.
• the RP-HPLC spectrum confirms the purity of the product (>99%).
• the expected mass is 4838.5.
• the peak at 4840.2 corresponds to [M+H] + .
• Figures 9A-D TEM images of 100 ⁇ (9A) C12-SZ33 and (9C) RB-C12-Z33 in PBS, pH 7.4. Both molecules self-assembled into nanofibers with diameters of 11.5 ⁇ 1.5 nm and 13.8 ⁇ 1.8 nm respectively.
• Normalized CD spectra of 100 ⁇ (9B) C12-SZ33 and (9D) RB-C12-Z33 nanofibers in PBS at pH 7.4 showed ⁇ -sheet and a-helix conformation, respectively.
• FIG. 1 Figures lOA-C. Confocal fluorescence images of 100 ⁇ RB-C12-Z33 incubated with 2 ⁇ FITC-IgG in PBS (pH 7.4) show co-localization of the fluorescence signal of Rhodamine B with that of the FITC.
• A Image of Rhodamine B fluorescence.
• B Image of FITC fluorescence and
• C merged image of (A) and (B). Scale bar: 20 ⁇ .
• Staphylococcal protein A is a protein originally found in the cell wall of Staphylococcus aureus. It is composed of five homologous domains that fold into a three- helix bundle. Protein A plays an important role in immunology due to its specific binding to the Fc-portion of immunoglobulin G (aka IgG) from most mammalian species, including human. Extensive structural and biochemical studies of protein A have been conducted. The first gene encoding SPA was cloned, sequenced and expressed in 1984 followed by numerous synthetic and minimized IgG-binding domain based on protein A. Among them Z-58 domain is the first and most famous synthetic domain to be widely used in affinity chromatography and affinity precipitation. Another minimized binding domain Z-33 was obtained in 1996 without significantly changing the function of the molecule.
• the present invention provides methods for the modification and/derivatization of the amino acid sequence of the antibody binding domain of SPA into immuno-amphiphiles which serve as the building unit for IFs. Described herein are examples of the design and creation of IFs useful in binding IgG antibodies or portions or fragments thereof. Once immunofibers are formed in aqueous solution at physiological pH ranges, the exposed bioactive epitopes (binding sites) displayed on the surface are able to specifically bind IgG.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain and wherein the immuno-amphiphile has an a-helical conformation when in an aqueous solution at physiological pH.
• the present invention provides an immuno- amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain, wherein the antibody binding peptide has a hydrophilic amino acid sequence of the Z33 peptide of Protein A of Staphylococcus aureus, or a functional portion or fragment or derivative thereof.
• the present invention provides an immuno-amphiphile comprising an antibody binding peptide conjugated to a linear hydrocarbon chain, wherein the antibody binding peptide has the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1), or a functional portion or fragment or derivative thereof.
• immuno-amphiphiles means a molecule that can spontaneously associate into discrete, stable supramolecular nanostructures termed
• the IFs can assemble in a pH range between about 2.8 to about 7.5.
• the binding properties are also pH dependent. Those IFs which are more positively charged are easier to associate with higher pH solutions, and conversely, negatively charged IFs will associate easier in lower pH solutions.
• a hydrophilic peptide is conjugated to a hydrocarbon tail having between 8 and 22 carbons, and is linear or can be branched. There is an upper limit to the number of carbons in view of solubility in an aqueous solution.
• the hydrophilic peptide increases the aqueous solubility of the nanostructure and can promote the formation of well- defined nanostructure architectures including, but not limited to, cylindrical or spherical micelles, hollow nanotubes, toroids, discs and vesicles, through preferred secondary structure formation, e.g. beta sheet, alpha helix, poly proline type-II helix, beta turn.
• antibody binding peptide means a peptide that has the ability to bind an antibody, or a specific portion of an antibody molecule, for example, the Fc portion, with high specificity, such as having a Kd of between about 10 "6 M to about 10 "10 M.
• the antibody binding peptide is the hydrophilic amino acid sequence of the Z33 two-helix derivative peptide of the Z-domain of Protein A of
• Staphylococcus aureus or a functional portion or fragment or derivative thereof.
• the Z33 peptide of Protein A has the amino acid sequence of FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1).
• amino acid includes the residues of the natural a-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, lie, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as ⁇ -amino acids, synthetic and non-natural amino acids.
• a-amino acids e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, lie, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val
• Many types of amino acid residues are useful in the polypeptides and the invention is not limited to natural, genetically-encoded amino acids.
• amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the reference cited therein. Another source of a wide array of amino acid residues is provided by the website of RSP Amino Acids LLC.
• references herein to "derivatives” includes parts, fragments and portions of the inventive antibody binding peptides of the present invention.
• a derivative also includes a single or multiple amino acid substitution, deletion and/or addition.
• Homologues include functionally, structurally or sterochemically similar peptides from venom from the same species of snake or from within the same genus or family of snake. All such homologues are contemplated by the present invention.
• Analogs and mimetics include molecules which include molecules which contain non-naturally occurring amino acids or which do not contain amino acids but nevertheless behave functionally the same as the peptide. Natural product screening is one useful strategy for identifying analogs and mimetics.
• Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
• Table 1 A partial list of known non-natural amino acid contemplated herein is shown in Table 1.
• Analogs of the subject peptides contemplated herein include modifications to side chains, incorporation of non-natural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptide molecule or their analogs.
• side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBlrU; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
• modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBlrU; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);
• the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
• the carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
• Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
• Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
• Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
• Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
• peptides can be conformationally constrained by, for example, incorporation of Ca and Na-methylamino acids, introduction of double bonds between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
• peptide includes a sequence of from four to 100 amino acid residues in length, preferably about 10 to 80 residues in length, more preferably, 15 to 65 residues in length, and in which the a-carboxyl group of one amino acid is joined by an amide bond to the main chain (a- or ⁇ -) amino group of the adj acent amino acid.
• the immunofibers can be separated from bound antibodies using several filtration methods, such as, for example diafiltration.
• the present invention provides methods for purification of an antibody or an Fc fusion protein by mixing the antibody or Fc fusion protein in a sample with the immunofibers of the present invention in an aqueous solution at physiological pH, and allowing the immunofibers to bind the Fc portion of the antibody or Fc fusion protein.
• the immunofibers comprise the Z33 portion of Protein A and are specific for the Fc portion of antibodies. After a period of time to allow the immunofibers to bind, the immunofibers form immunofiber-antibody or immunofiber-Fc fusion protein complexes in solution.
• the complexes formed can then be separated from the unbound immunofibers and antibodies or Fc fusion proteins and other components in the sample by many 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 for the antibodies or Fc fusion proteins.
• the antibody or Fc fusion protein can then be separated from the dissociated immunofibers by filtration, such as diafiltration or other means, and the dissociated monomers can be removed as well.
• sample means any sample or solution or fluid or mixture containing an antibody of interest or an Fc fusion protein of interest which can be bound using the immunofibers of the present invention.
• the sample can be a biological sample.
• the sample includes, for example, cell cultures, cell lysates, and clarified bulk (e.g., clarified cell culture supernatant).
• the sample is produced from a host cell or organism that expresses the antibody or Fc fusion protein of interest (either naturally or recombinantly).
• the cells in a cell culture include host cells transfected with an expression construct containing a nucleic acid that encodes an antibody or Fc fusion protein of interest.
• host cells can be bacterial cells, fungal cells, insect cells or, preferably, animal cells grown in culture.
• Bacterial host cells include, but are not limited to E. coli cells. Examples of suitable E. coli strains include: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleave foreign DNA.
• Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells.
• Insect cells that can be used include, but are not limited to, Bombyx mori, Mamestra drassicae, Spodoptera frugiperda, Trichoplusia ni, Drosophilia melanogaster .
• a number of mammalian cell lines are suitable host cells, including for example, CHO, COS, PER.C6, TM4, VERO076, DXB11, MDCK, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1, C3H10T1/2, Colo205, 293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP- 1, PC12, Mix, murine myelomas (e.g., SP2/0 and NSO) and C2C12 cells, as well as transformed primate cell lines, hybridomas, normal diploid cells, and cell
• biological sample or “biological fluid” includes, but is not limited to, any quantity of a substance from a living or formerly living patient or mammal or from cultured cells.
• 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).
• the protein purified using the present invention is an antibody.
• antibody is used in the broadest sense to cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, immunoadhesins and antibody-immunoadhesin chimerias.
• an “antibody fragment” includes at least the Fc portion of an antibody and typically an antigen binding or variable region thereof.
• the term "monoclonal antibody” is used in the conventional sense to refer to an antibody obtained from a population of substantially homogeneous antibodies such that the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. This is in contrast with polyclonal antibody preparations which typically include varied antibodies directed against different determinants (epitopes) of an antigen, whereas monoclonal antibodies are directed against a single determinant on the antigen.
• monoclonal in describing antibodies, indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• monoclonal antibodies used in the present invention can be produced using conventional hybridoma technology first described by Kohler et al, Nature 256:495 (1975), or they can be made using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
• Monoclonal antibodies can also be isolated from phage antibody libraries, e.g., using the techniques described in Clackson et al, Nature 352:624-628 (1991); Marks et al, J. Mol.
• the monoclonal antibodies described herein include “chimeric” and “humanized” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
• Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• the monoclonal antibodies described herein also include "human" antibodies, which can be isolated from various sources, including, e.g., from the blood of a human patient or recombinantly prepared using transgenic animals.
• transgenic animals include KM-MOUSE® (Medarex, Inc., Princeton, NJ) which has a human heavy chain transgene and a human light chain transchromosome (see WO 02/43478),
• XENOMOUSE® (Abgenix, Inc., Fremont CA; described in, e.g., U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al), and HUMAB- MOUSE® (Medarex, Inc.; described in, e.g., Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al.
• Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
• Fmoc amino acids and resins were purchased from Advanced Automated Peptide Protein Technologies (AAPPTEC, Louisville, KY, UXSA), and Fmoc- Lys(Fmoc) were obtained from Novabiochem (San Diego, CA, USA).
• the therapeutic human IgGl (IgGl) was obtained from Bristol-Myers Squibb (Boston, MA, USA), and IgG elution buffer was sourced from Thermo Fisher Scientific (Rockford, IL, USA). All other reagents were obtained from VWR (Radnor, PA, USA) and used as received without further purification.
• C12-Z33 and 2C8-Z33 immuno-amphiphiles were synthesized using similar methods.
• Z33 peptide were first synthesized on the Focus XC automatic peptide synthesizer (AAPPTEC, Louisville, KY) using standard 9- fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis protocols.
• the C12 (or 2C8) alkyl chain was then manually coupled at the N-terminus (after Fmoc removal) of Z33 peptide with lauric acid (or octanoic acid)/HBTU/DIEA at a ratio of 4 (or 8): 4: 6 relative to the Z33 peptide, shaking overnight at room temperature.
• Fmoc deprotections were performed using a 20% 4-methylpiperidine in DMF solution for 10 minutes, repeating once. In all cases, reactions were monitored by the ninhydrin test (Anaspec Inc., Fremont, CA) for free amines. Completed peptides were cleaved from the solid support using a mixture of TFA/TIS/H2O in a ratio of 92.5:5:2.5 for 2.5 hours. Excess TFA was removed by rotary evaporation and cold diethyl ether was added to precipitate the crude peptide. By centrifugation method, precipitated peptide and diethyl ether were separated at 6000 rpm for 3 minutes. Peptides were washed another 2 times with diethyl ether and solution was removed by centrifugation.
• the IAs were purified by preparative RP-HPLC using a Varian Polymeric Column (PLRP-S, 100 A, 10 ⁇ , 150 ⁇ 25 mm) at 25 °C on a Varian ProStar Model 325 preparative HPLC (Agilent Technologies, Santa Clara, CA) equipped with a fraction collector. A water/acetonitrile gradient containing 0.1% v/v TFA was used as eluent at a flow rate of 20 ml/min. The absorbance peak was monitored at 220 nm for Z33 peptide segments. The crude materials were dissolved in 20 ml of 0.1% aqueous TFA, and each purification run was carried out with a 10 ml injection.
• MALDI-ToF BrukerAutoflex III MALDI-ToF instrument, Billerica, MA
• those containing the desired product were lyophilized (FreeZone -105 °C 4.5 L freeze dryer, Labconco, Kansas City, MO) and stored at -30 °C.
• CD Circular Dichroism Spectroscopy
• the samples were instantly diluted from the 1 mM stock solution to 100 ⁇ in 1 xPBS prior to the experiment.
• the spectra were collected in the wavelength range of 190-280 nm as the average of three scans.
• a background spectrum of the solvent was acquired and subtracted from the sample spectrum. Collected data was normalized with respect to sample concentration.
• ITC Experiment Isothermal titration calorimetry experiments were performed using a high precision VP-ITC titration calorimetric system (Microcal Inc.). The IgGl solution was titrated with immuno-amphiphiles in 1 *PBS (pH 7.4 or 2.8) at 15 °C. The IgGl concentration was calculated using the mass extinction coefficient of 1.4 at 280 nm for a 0.1% (1 mg/ml) IgG solution. The concentration of immuno-amphiphles was determined by total nitrogen assay (Anal. Biochem., 61 ,2 (1974): 623-627). The heat evolved after each injection was obtained from the integral of the calorimetric signal. The heat associated with the binding of immuno-amphiphiles to IgGl was obtained by subtracting the heat of dilution. Analysis of the data was performed using MicroCal OriginTM package.
• HFIP hexafluoroisopropanol
• Representative TEM images from a solution of 100 ⁇ C12-Z33 revealed that C12-Z33 self-assembled into nanofibrous structure under both physiological condition and acidic condition with a diameter of 16.0 ⁇ 1.7 nm, a value that is less than the length of the fully extended peptide molecule (about 22.5 nm in ⁇ -sheet conformation).
• the length of the nanofibers was shown in micro-meter scale and could not be well-controlled.
• circular dichroism CD was used to study the peptide secondary structure.
• CD spectra for C12-Z33 in PBS solution or IgG elution buffer only maintained partial a-helix signals
• the ellipticity of the two negative peaks at around 222 nm and 208 nm changed compared with Z33 peptide in the same buffer.
• the shift of the CD spectra may result from the formation of the IFs that can change the molecular packing of Z33 segment from its free state and may subsequently influence its binding affinity to IgG due to the specific conformation required for the binding sites.
• ITC Experiment for Measuring Binding Affinity of IFs. Given the conformation change in the secondary structure of Z33 peptide after incorporation into IFs, it is of great interest to know if the formation of C12-Z33 IFs would influence the IgG binding ability existing in original Z33 peptide.
• thermodynamic properties of the binding to IgGl were investigated by isothermal titration calorimetry (ITC). ITC has been widely employed to monitor the binding events between great numbers of proteins and ligands, 43"45 which is an excellent method to explore if the binding could occur between C12-Z33 IFs and IgGl.
• thermodynamic dissociation constant Kd
• ⁇ ° molar enthalpy change
• N stoichiometry
• thermodynamic parameters should be done after normalization per mole of IgG as shown in Table 2.
• the binding of Z33 to IgG was characterized by a large favorable enthalpy opposed by a large unfavorable entropy change.
• the thermodynamic signature for the binding of C12-Z33 was similar although the magnitudes of the enthalpy and entropy changes were smaller.
• C12-Z33 binds with a less unfavorable entropy than Z33, the loss in favorable enthalpy is even larger which results in an overall lower binding affinity.
• C12-SZ33 with scrambled Z33 peptide sequence was used as negative control.
• This C12-SZ33 IAs shows similar self-assembly properties and secondary structures characterized with TEM and CD (data not shown).
• ITC experiment was carried out by injecting 100 ⁇ C12-SZ33 IAs into 2 ⁇ IgGl solution at 15 °C in PBS at pH 7.4 to measure their binding ability. The thermograms and binding isotherms in Figure 3D suggests specific interactions between IgGl and the Z33 peptide.
• the diversity of constituent amino acids provides a broad basis for non-covalent interactions including hydrogen bonding, ⁇ - ⁇ stacking, hydrophobic collapse, and electrostatic interactions between self-assembling peptide nanofibers.
• solubility of acidic and basic amino acids is determined by the degree of ionization, a property that is pH and ionic strength dependent.
• the self-assembly process of charged peptides can thus be facilitated by tuning the pH or adding salts to reduce the electrostatic repulsions, promote aggregation and even precipitation.
• a spectacular advantage of the inventive immunofiber system of the present invention relies in the easily-tunable solubility.
• C12-Z33 IFs were chosen to study the possibility to precipitate IgGl because of its relatively high binding affinity to IgGl.
• 5 mM C12-Z33 could be well dissolved in PBS solution but precipitated in a PBS solution of 0.6 M Na2S04.
• the zeta potential of C12-Z33 in PBS solution is -7.61 mV and the addition of Na2S04 could screen the charges on the surface of IFs and thus induce precipitation.
• IgGl it is well dissolved in 5 mM C12-Z33 as well as 0.6 M Na2S04.
• the absorbance at 280 nm was reduced to a level below the initial absorbance of IgGl, indicating the removal of IgGl from the solution. More clearly, the absorbance of supernatants of net IgGl was plotted via subtracting the value of green line from the blue line, suggesting more than 60% IgGl was removed from the supernatant. So far, the possibility of our IFs to serve as a new affinity precipitation agent was preliminary proved.
• the difficulty to identify the IgG structure in TEM limited the visualization of binding between immunofibers and IgG directly.
• the preformed C12-Z33 and C12-SZ33 immunofibers were incubated with 10 nm IgG-labelled Au nanoparticles for 2 h separately. After placing a drop of each solution onto a TEM grid, the grid was blotted with a filter paper and left to dry naturally. Then, we carefully washed the grid 3 times with PBS buffer, in an effort to remove the unbounded Au nanoparticles before staining the sample with uranyl acetate.
• IgG- coated Au nanoparticles could be bound to the C12-Z33 in the monomer state and then taken away in the washing steps, because the IgG concentration (0.33-0.66 ⁇ ) in the IgG-coated Au nanoparticles was below the critical micelle concentration value for C12-Z33, which is 2- 5 ⁇ (Fig. 7).
• labelling the C12-Z33 with the fluorescent dye allows for direct imaging of fluorescent immunofibers and FITC-IgG under a confocal laser scanning microscope.
• Rhodamine B has been extensively used for staining biomaterials.
• Rhodamine B labelled C12-Z33 (RB-C12-Z33) was synthesized by adding a lysine at the N-terminal of Z33 (Fig. 8)
• Rhodamine B and C12 were conjugated to the amine group on the side chain and backbone of the newly added lysine separately. It was assumed that the fluorescent labelling will not interfere with the binding ability of C12-Z33 because the dye will stay in the hydrophobic core after self-assembling into the immunofibers together with the C12 and it's located remotely from the binding sequence Z33.
• the self-assembled fibrous morphology in PBS was confirmed using TEM (Fig. 9C).
• FITC-IgG 100 ⁇ RB-C12-Z33 diluted from a 1 mM stock solution and 2 ⁇ FITC-IgG were premixed in PBS. 30 ⁇ solution was spotted on a clean microscope slide right before imaging and covered by a coverslip to obtain a thin layer of liquid. Fluorescence images (Figs. lOA-C) were then taken by a confocal laser scanning microscope and showed co-localization of fluorescence signals from both RB-C12-Z33 (red) and FITC-IgG (green). It was found that FITC-IgG formed large bright assemblies that were never observed in the pure FITC-IgG solution and the solution incubated with C12-SZ33 at same conditions (data not shown).
• FITC-IgG Compared to the bright fluorescence of FITC-IgG when incubated with RB-C12-Z33, low fluorescent signals were detected in the PBS solution or after incubated with C12-SZ33. This showed that FITC-IgG was dispersed well in the PBS buffer or in the presence of C12-SZ33, while the binding between RB-C12-Z33 and FITC- IgG induced the aggregation of FITC-IgG and the strong fluorescence.

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