WO2006110292A2 - Temperature-triggered immobilization and purification of antibodies - Google Patents

Abstract

This invention provides compositions and methods for capture and purification of antibodies. Protein sequences with inverted solubility temperature profiles are fused to antibody-binding proteins. Fusions of elastin-like peptides (ELPs) and protein G and/or protein L can be readily purified and employed to capture and purify immunoglobulins. To avoid the drawbacks of conventional affinity chromatography, ELP fusion proteins consisting of an elastin-like polypeptide made up of the pentapeptide VPGVG repeating units and Ig-binding ligands, protein A, protein G, protein L, and hybrid ligands such as protein LG are used for immobilization and purification of monoclonal or polyclonal antibodies from a material containing these antibodies by simple temperature transition cycling.

Description

TEMPERATURE-TRIGGERED IMMOBILIZATION AND PURIFICATION

OF ANTIBODIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of a prior U.S. Provisional

Application number 60/665,479, Temperature-Triggered Immobilization and Purification of Antibodies, by Jae- Young Kim, et al., filed March 25, 2005. The full disclosure of the prior application is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] Part of the funding for research into the subject matter of this application was provided by the National Science Foundation. The Federal government may have certain rights and interests to claims granted from the present application.

FIELD OF THE INVENTION

[0003] The invention provides fusion proteins for immobilization, separation and/or purification of antibodies from a biological material. The fusion proteins are capable of reversible phase-separation, affording selective recovery of antibodies by simple environmental triggers, in particular, temperature triggers. The invention also provides methods for immobilization and purification of antibodies. Fusion proteins of an elastin- like peptide (ELP) with antibody binding protein sequences can self assemble on hydrophobic surfaces or precipitate from solution under conditions of high temperature. Purification schemes based on manipulation of antibody binding and fusion protein precipitation can provide efficient capture of antibodies in solution, separation from impurities and elution of purified antibodies. BACKGROUND OF THE INVENTION

[0004] Antibodies, because of their highly specific binding nature, are valuable tools for environmental monitoring and in vitro and in vivo diagnostic applications. The advantages of antibodies, such as sensitivity, reliability, and accuracy, render them suitable for use in an array format; offering the potential to extend molecular analysis beyond the limitations of DNA microarrays (Templin, M. F., Stoll, D., Schwenk, J. M., Potz, O., Kramer, S., and Joos, T. O. (2003) Protein microarrys; Promising tools for proteomic research. Proteomics 3, 2155-2166). Antibody therapy is another application that has been used for the prevention and treatment of infectious diseases, including respiratory infections, diphtheria, hepatitis, and measles (Keller, M. A. and Stiehm, E. R. (2000) Passive immunity in prevention and treatment of infectious diseases. Clin. Microbiol. Rev. 13, 602-614). Antibodies can be useful in applications protecting against biological terrorism and against bacteria resistant to antibiotics. A breakthrough in antibody applications has been achieved with the application of monoclonal antibodies (mAb) which represent a virtually unlimited source of uniform, pure, and highly specific binding molecules (Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells can secrete antibody of predefined specificity. Nature 256, 495-497. In addition, recent advances in recombinant technology have broadened the use of mAbs as therapeutic agents for the treatment of diseases like cancer. Efficacy of antibody-based cancer therapies have been improving significantly (Nat. Rev. Cancer 1, 118-129); and: since 1995, five antibodies have been approved for the treatment of cancer (Carter, P., 2001).

[0005] Large-scale production of antibodies can be provided by use of transgenic animals or hybridoma technology (Vandekerckhove, B. A. E., Jones, D., Punnonen, J., Schols, D., Lin, H.-C, Duncan, B., Bacchetta, R., Vries, J. E., and Roncarolo, M.-G. (1993) Human Ig production and isotype switching in severe combined immunodeficient-human mice. J. Immunol. 151, 128-137; Mckinney, K. L., Dilwith, R., and Belfort, G. (1995) Optimizing antibody production in batch hybridoma cell culture. J. Biotechnol. 40, 31-48). In the case of multifeed strategies, final antibody titers of 1-2 g/L have been attained (Bibila, T. A. and Robinson, D. K. (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol. Prog. 11, 1-13). The purification of antibodies, however, presents an additional challenge due to the broad range of sources such as blood, milk, cell culture supernatant, and extracts from different cells, low antibody concentration, excessive amounts of contamination proteins, and the requirement of high purity.

[0006] Several Ig-binding bacterial proteins have been isolated, characterized, and used as affinity ligands in research, analysis and industry. The most widely used affinity ligand is the Staphylococcal protein A (SpA), which is a cell wall component of Staphylococcus aureus, that binds to immunoglobulin G (IgG) from several mammalian species (Moks, T., Abrahmsen, L., Nilsson, B., Hellman, U., Sjoquist, J., and Uhlen, M. (1986)). Staphylococcal protein A consists of five IgG-binding domains. (EMr. J. Biochem. 156, 637-643). SpA has been extensively applied in immunoassays, owing to its high specific avidity for the Fc portion of IgG without interrupting its antigen-binding ability (Langone, J. J. (1982)). Protein A of Staphylococcus aureus and related immunoglobulin receptors are produced by streptococci and pneumonococci. {Adv. Immunol. 32, 157-252; Lilsson, B., Abrahmsen, L., and Uhlen, M. (1985), Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4, 1075-1080). However, the binding affinity of SpA has been reported to be strongly dependent on the source of IgG and the pH of binding buffer (Lindmark, R., Thren-Tolling, K., and Sjoquist, J. (1983) Binding of Immunoglobulins to Protein A and Immunoglobin Levels in Mammalian Sera. J. Immunol. Methods 62, 1-13).

[0007] Another affinity ligand is protein G from the group G streptococci, which offers binding to broader IgG subclasses, different affinity constants, and less pH dependence (Bjorck, L. and Kronvall, G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent, J. Immunol. 133, 969-974; Akerstrom, B. and Bjorcks, L. (1986) A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. /. Biol. Chem. 261, 10240-10247). Because of these benefits, protein G can be used for the immobilization and purification of IgG with improved binding properties when compared with SpA.

[0008] Still another type of ligand is protein L, an immunoglobulin light chain- binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, (Bjorck, L. (1988) Protein L: A novel bacterial cell wall protein with affinity for Ig L chains. J. Immunol. 140, 1194-1197). Whereas SpA and protein G bind IgG with high specificity, protein L has the strong binding affinity specific to other immunoglobulins, such as IgM, IgA, IgE, and IgD. This makes protein L useful for immobilization and purification of these types of antibodies (Kastern, W., Sjobring, U., and Bjorck, L. (1992) Structure of Peptostreptococcal Protein L and identification of repeat Immunoglobulin light chain-binding domain. /. Biol. Chem. 267, 12820-12825).

[0009] Probably the most widely used purification techniques for antibodies are affinity chromatography schemes based on contact with protein A, G, or L immobilized on appropriate support (Fassina, G., Ruvo, M., Palombo, G., Verdoliva, A., and Marino, M. (2001) Novel ligands for the affinity-chromatographic purification of antibodies. /. Bioch. Bioph. Meth. 49, 481-490). However, conventional affinity chromatography requires coupling procedures and commercially available affinity sorbents that can be prohibitively expensive. Isolation of the antibody affinity ligands can be complex and expensive, even where expression has been enhanced by genetic modification of the bacteria (Huse, K., Bohme, H.-J., and Scholz, G. H. (2002) Purification of antibodies by affinity chromatography. /. Biol. Chem. 51, 217-231). Moreover, immobilization of the ligands on solid supports can slow binding kinetics and decrease the affinity of the ligands for the antibodies. Protein A immobilized on rigid ceramic composites have been found to lose about 75% of its original IgG-biding capacity (Guerrier, L., Flayeux, L, Schwarz, A., Fassina, G., and Boschetti, E. (1998) IRIS 97: an innovative protein A-peptidomimetic solid phase medium for antibody purification. /. MoI. Recognit. 11, 107-109). Ligand leakage from columns is a significant problem that can interfere with analysis of the purified antibody and validation of mAbs for human therapeutic use. Leakage and conformational effects on binding affinities can depend on the nature of the matrix and coupling chemistry (Godfrey, M. A. J., Kwasowski, P., Clift, R., and Marks, V. (1992). A sensitive enzyme- linked immunosorbent assay (ELISA) for the detection of staphylococcal protein A (SpA) present as a trace contaminant of murine immunoglobulins purified on immobilized protein A. /. Immunol. Methods 149, 21-27). Such leakage can require additional purification steps that can negate the advantages of affinity purifications.

[0010] Poly-N-isopropylacrylamide (PNIPAM), is a thermally reversible polymer that can be used in the solution-phase affinity separation analogous to affinity chromatography. These solution-phase techniques have been applied in combination with various conjugation partner ligands, such as streptavidin, antibodies, and antibody fragments (Ding, Z., Long, C. J., Hayashi, Y., Bulmus, E. V., Hoffman, A. S., and Stayton, P. S. (1999) Temperature control of biotin binding and release with a streptavidin-poly(N- isopropylacrylamide) site-specific conjugate. Bioconjugate Chem. 10, 395-400; Kumar, A., Kamihira, M., Galaev, I. Y., Mattiasson, B., and Iijima, S. (2001) Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature- sensitive polymers, Biotechnol. Bioeng. 75, 571-580; Fong, R. B., Ding, Z., Hoffman, A. S., Stayton, P. S. (2002) Affinity separation using an Fv antibody fragment-"smart" polymer conjugate. Biotechnol. Bioeng. 79, 271-276). Although this polymer has been shown adaptable to some antibody affinity ligands, it requires laborious polymer synthesis and chemical conjugation steps to practice (Chen, J. P. and Hoffman, A. S. (1990) Polymer- protein conjugates; π. Affinity precipitation separation of human immunogammaglubulin by a poly(N-isopropylacrylamide)-protein A conjugate, Biomaterials 11, 631-634). And yet, this time consuming coupling can still present the problems of affinity chromatography, such as the leakage of affinity ligands, and functionality decrease due to steric hindrance and/or reduced mass transport limitation.

[0011] In view of the above, a need exists for methods of antibody affinity purification that avoid the problems associated with covalent linkage of the ligands to solid supports. It would be desirable to have antibody binding in solution to ligands that are efficiently and simply removable from process streams. The present invention provides these and other features that will be apparent upon review of the following.

SUMMARY OF THE INVENTION

[0012] The inventors of the present invention have surprisingly discovered that fusion proteins consisting of elastin-like polypeptide (ELP) and antibody-affinity ligands enable a temperature directed purification and isolation of antibodies from biological samples. Thus, according to the present invention, there are provided ELP fusions with antibody affinity ligands (e.g., immunoglobulin-binding proteins) that can be utilized for the immobilization and purification of antibodies. The inventive ELP fusions with different types of affinity ligands can be specifically applied to the immobilization and purification of antibodies of various types and from a variety of different sources. [0013] The inventive methods for immobilization, purification and isolation of antibodies exhibit significant advantages over currently used affinity purification procedures in purification of antibodies. First of all, they obviate the need for chromatographic techniques. Second, the costs for conducting the inventive methods are relatively low since they do not require sophisticated technical equipment, but only standard equipment, such as centrifuges and incubators. Further, the conditions used for immobilization and separation of the antibodies are very gentle since the methods rely on relatively low stress manipulations, such as centrifugation, buffer modifications and temperature adjustments. Immunoglobulin purification using the inventive fusion proteins can be performed to provide excellent purity and recovery. The efficiencies of antibody recovery using the inventive fusion proteins is much higher than those reported for chromatographic separations. Another advantage is that the inventive fusion proteins can be reused many times without losing binding affinity.

[0014] The present invention provides fusion proteins for purification of immunoglobulins or antibodies. The fusion proteins can comprise, e.g., one or more elastin-like polypeptides fused to one or more antibody-affinity ligand. In a preferred embodiment, the elastin-like polypeptide consists of two or more pentapeptide VPGVG (SEQ ID NO: 1) repeating units. In preferred embodiments, the antibody-affinity ligand of the fusion protein includes an immunoglobulin-binding protein, such as, e.g., a wild-type protein or mutated protein. In many embodiments, the fusion protein is able to bind to at least a chain of IgG, IgM, IgA, IgE or IgD and/or fragments thereof. Typical Ig-binding proteins of the fusion protein include, e.g., protein A from Staphylococcus aureus or a fragment thereof, protein G from the group G streptococci or a fragment thereof, protein L from Peptostreptococcus magnus or a fragment thereof, and/or a hybrid molecule comprising at least two of these proteins or fragments thereof.

[0015] The fusion proteins can be constructed with a spacer peptide sequence separating the elastin-like polypeptide from the antibody-affinity ligand. In some cases, the spacer sequences can be engineered to separate two or more different antibody-affinity ligands from each other.

[0016] In an aspect of the invention, a nucleic acid molecule is provided with a nucleotide sequence encoding a fusion protein for purifying immunoglobulins or antibodies. The nucleic acid can include, e.g., a first nucleotide sequence encoding a elastin-like polypeptide which consists of the two or more pentapeptide VPGVG repeating units and at least one second nucleotide sequence functionally linked to the first nucleotide sequence and encoding one antibody-affinity ligand. In preferred embodiments, the second nucleotide sequence encodes a wild-type or mutated immunoglobulin-binding protein, such as, e.g., a protein A nucleotide sequence from Staphylococcus aureus or a fragment thereof, a protein G nucleotide sequence from the group G streptococci or a fragment thereof, a protein L nucleotide sequence from Peptostreptococcus magnus or a fragment thereof, or a sequence of a hybrid protein comprising at least two of the these proteins or fragments thereof.

[0017] The nucleic acid molecule can functionally incorporate a third nucleotide sequence encoding a spacer wherein the third nucleotide sequence is located between the first (elastin-like peptide) and second (immunoglobulin-binding protein) nucleotide sequences.

[0018] The present invention includes vector nucleic acids having a sequence encoding a fusion protein for purifying immunoglobulins or antibodies. Such a vector can encode a fusion protein sequence operably linked to one or more regulatory units, which are able to drive the expression of the fusion protein from the nucleic acid in a suitable environment. The vector can include one or more sequences for an elastin-like peptide and one or more sequences of immunoglobulin-binding proteins. The regulatory units can include, e.g., a promoter, an enhancer, a transcriptional terminator and/or a ribosome- binding site.

[0019] In one aspect of the invention, host cells carry at least one of the vectors.

The host cells can be eukaryotic or prokaryotic cells. In a preferred embodiment, the host cell is a mammalian cell.

[0020] A kit for immobilization and/or purification of antibodies can be provided including at least one fusion protein of the invention, at least one nucleic acid encoding a fusion protein of the invention, at least one vector of the invention, and/or at least one host cell of the invention. [0021] Methods for isolating and/or purifying antibodies from an antibody containing sample are useful aspects of the invention. For example, the methods can include the steps of: 1) mixing a fusion protein of the invention (e.g., a fusion of ELP and an immunoglobulin-binding protein) with a liquid medium sample containing an antibody of interest; 2) incubating the mixture for a sufficient time under conditions conducive to binding in order to enable the antibody to bind with the fusion protein, thus forming a fusion/antibody complex; 3) recovering the complex thus formed from the sample at a precipitation temperature; 4) incubating the recovered complex at a solubilizing temperature in an aqueous solution; 5) providing conditions that cause the fusion protein to be unbound from the antibody; and, 6) precipitating the fusion protein from the solution and recovering the antibody in the solution. In the methods, the mixture can be incubated at a binding temperature ranging, e.g., more than about 0⁰C, or from about 4°C to about 4O⁰C, from about 15⁰C to about 37°C, from about 20⁰C to about 30⁰C, or at about 25°C. In typical embodiments, the complex of antibody and fusion protein is recovered by precipitation at a relatively high temperature ranging from about 50⁰C to about 2O⁰C, from about 45°C to about 25⁰C, from about 35°C to about 4O⁰C, or about 37⁰C. In preferred embodiments, the precipitated complex of antibody and fusion protein can be recovered by solubilization at a relatively low temperature ranging from about O⁰C to about 20⁰C, from about 2°C to about 15°C, from about 3°C to about 8°C, or about 4°C.

[0022] The methods of the invention can selectively or generally immobilize and/or purify various antibodies. The antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies and/or fragments thereof. For example, the methods can be used to efficiently purify one, or any combination of: members of the greater immunoglobulin family of proteins, IgG, IgM, IgA, IgE, IgD, and/or fragments thereof.

DEFINITIONS

[0023] Unless otherwise defined herein or below in the remainder of the specification, all technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0024] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a component" can include a combination of two or more components; reference to "antibodies" can include mixtures of feed, and the like.

[0025] Although many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0026] As used herein, the term "fusion protein" generally refers to a fusion protein comprising one or more ELP sequence and one or more immunoglobulin-binding affinity proteins (e.g., antibody-affinity ligands).

[0027] The term "immunoglobulin chain", as used herein, refers to regions of an immunoglobulin, such as all or part of a heavy chain or light chain.

[0028] The term "inverse phase transition", as used herein, refers to a transition from substantial solubility to substantial insolubility of a fusion protein with an increase in temperature (e.g., at a relatively high temperature), or a transition from substantial insolubility to substantial solubility of a fusion protein with an a decrease in temperature (at a relatively low temperature). For example, many fusion proteins of the invention (complexed with target antibody or not) can be substantially insoluble in aqueous environment at relatively low temperatures (e.g., O⁰C to 1O⁰C), yet they can be substantially soluble in the aqueous environment at relatively high temperatures (e.g., 30°C to 45⁰C). This phenomenon is the inverse of the solubility profile on most molecules and complexes in solution.

[0029] As used herein, the term "complex" generally refers to immunoglobulins bound to an ELP-affinity ligand fusion protein. Such a complex may be single bound pairs in solution or can include complexes with more than one binding pair, e.g., even including binding arrangements resulting in a three-dimensional lattice. In preferred embodiments, complexes are in solution or suspensions smaller than colloidal suspensions at relatively low solution temperatures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure IA shows purification of fusion proteins by a process of inverse phase transition. The purity of the fusion proteins is analyzed by 10% SDS-PAGE gel. Lane G contains ELP-ProG; Lane L contains ELP-ProL; and Lane LG contains ELP-ProLG.

[0031] Figure IB shows the purification of fusion proteins by inverse phase transition with the purity of the fusion proteins analyzed by Western-blot analysis with detection by goat IgG-alkaline phosphatase conjugate.

[0032] Figure 1C shows the purification of fusion proteins by inverse phase transition with the purity of the fusion proteins analyzed by Western-blot analysis with detection by human IgM-horseradish peroxidase conjugate.

[0033] Figure 2 shows turbidity profiles of ELP fusions with antibody-affinity ligands.

[0034] Figure 3 shows the immobilization of antibodies on a hydrophobic surface.

[0035] Figure 4 shows the recovery of IgG from buffer containing purified mouse-

IgG (A) and rabbit-IgG (B). The IgG lane contains purified mouse-IgG or rabbit-IgG; the Sup lane contains the supernatant fraction; the Elute lane contains the elution fraction; and the P lane contains the pellet fraction.

[0036] Figure 5 shows IgG purification from the supernatant of hybridoma cell culture. The C1B7 lane contains supernatant of hybridoma cell culture; the G lane contains ELP-ProG; the Sup lane contains the supernatant fraction; the Elute lane contains the elution fraction; and the P lane contains the pellet fraction.

[0037] Figure 6 shows IgG purification from mouse-serum (A) and rabbit-serum

(B). The Serum lane contains mouse-serum or rabbit-serum; the Sup lane contains the supernatant fraction; the Elute lane contains the elution fraction; and the Plane contains the pellet fraction. [0038] Figure 7 shows a repeated IgG purification from rabbit serum. 1, 2, and 3, are the number of repeat purifications involved. The S lanes contain supernatant fractions. The E lanes contain elution fractions.

DETAILED DESCRIPTION

[0039] The present invention provides fusion proteins for immobilizing and purifying immunoglobulins or antibodies. The fusion proteins can comprise an elastin-like polypeptide fused (typically by recombinant technologies) to at least one antibody-affinity ligand. Temperature-triggered immobilization and purification of antibodies is provided. ELP fusions consisting of elastin-like polypeptides (ELPs) made up of, e.g., pentapeptide VPGVG repeating units and Ig-binding ligands (e.g., SpA, protein G, protein L, and protein LG). The ELP fusion proteins can be synthesized biologically and purified by simple temperature transition cycling between precipitating warm temperatures and solubilizing cool temperatures. The fusion proteins can capture antibodies for immobilization on hydrophobic surfaces. Antibodies can be purified with high recovery by fusion protein capture and immobilization or precipitation out of a sample.

[0040] Problems associated with affinity chromatography can be avoided by utilizing tunable biopolymers capable of reversible phase-separation, affording selective recovery of antibodies by simple environmental triggers. Elastin-like polypeptide (ELP), consisting of the repeating pentapeptide VPGVG, can undergo a reversible phase transition from water-soluble forms into aggregates as the temperature increases (Urry, D. W., (1992) Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog. Biophys. MoI. Bio. 57, 23-57). In addition, the thermally triggered hydrophobic nature of the ELP moiety can enable the self-assembly of antibodies onto hydrophobic surfaces, providing a simple and reversible method to functionally immobilize antibodies (Shimazu, M., Mulchandani, A., and Chen, W. (2002) Thermally triggered purification and immobilization of elastin-OPH fusions. Biotechnol. Bioeng. 81, 74-79).

[0041] Elastin-like polypeptides (ELPs), consisting of the repeating pentapeptide

VPGVG, are capable of reversible inverse phase-separations, affording selective recovery of antibodies by simple environmental triggers. ELP can undergo a reversible phase transition from water-soluble forms into aggregated forms as the temperature increases. The ELPs have an unusual inverse temperature transition property that can be conveniently manipulated to readily separate ELP-containing fusions and complexes, e.g., from typical biological solutions or suspensions. Whereas most molecules are more soluble in warm solutions and less soluble in cold solutions, ELP can have the reverse behavior in aqueous environments. At relatively warm temperatures, e.g., above ambient temperatures (e.g., above 25°C, or between about 15°C and about 60⁰C), the ELPs, and associated fusions or complexes, can be reversibly insoluble. However, at relatively cold temperatures, e.g., below ambient temperatures (e.g., below 25°C, or between about -5⁰C and about 20⁰C), the ELPs, and associated fusions or complexes, can be soluble in aqueous environments.

[0042] The thermally triggered hydrophobic nature of the ELP moiety enables the generation of a fusion protein consisting of a thermally responsive ELP and an antibody- affinity ligand, such as an immunoglobulin-binding protein, wherein the environmentally sensitive solubility is imparted to the antibody-affinity ligand. If the antibody-affinity ligand of the fusion protein has a bound antibody, then the environmentally sensitive solubility can also be imparted to the antibody. Using the thermally triggered soluble- insoluble phase transition of the ELP portion of such a fusion protein, it is thus possible to immobilize an antibody and afterwards to elute and separate the antibody from the fusion protein. The inventive fusion proteins thus allow the purification of antibodies in a very simple and efficient way.

[0043] The elastin-like polypeptide comprises pentapeptide VPGVG (SEQ ID No.

1) repeating units and can undergo a reversible, soluble to insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature. The inventive fusion proteins can retain the inverse transition behavior of ELPs, thus undergoing a inverse temperature dependent soluble-insoluble phase transition. This temperature- sensitive behavior of the inventive fusion proteins enables the binding and separation of fusion/antibody complexes from samples. After removal from the sample, the fusion/antibody can be resolubilized at a relatively low temperature. The antibody can be unbound from the fusion using conditions (e.g., pH, cation chelation or ionic strength) typically used to elute antibodies from the particular immunoglobulin-binding protein employed in the fusion. Finally, purified antibody can remain in solution while the fusion is precipitated from the solution at a relatively warm temperature.

[0044] In an exemplary embodiment, the inventive fusion proteins can be soluble at a relatively cold temperature of about 4°C (and typically at temperatures ranging from about 0⁰C to about 10⁰C) in an aqueous sample. Antibodies can be bound to the fusion protein in solution at these temperatures. The complexes formed between the fusion proteins and the antibodies can remain in solution or suspension at relatively cold temperatures. In contrast, at a temperature of about 37°C (and typically at temperatures ranging from about 25⁰C to about 40⁰C) the inventive fusion proteins can form aggregates removable from the sample, e.g., by centrifugation or by hydrophobic interaction with a hydrophobic substrate. The remaining aqueous sample material, including contaminants, can be removed, e.g., by decanting. The aggregates can be resolubilized in an elution buffer at a relatively cold temperature. The elution buffer can be formulated to have conditions, e.g., of pH and/or salt concentration, that result in the antibody becoming unbound from the fusion protein. Thus the fusion protein and antibody can each be freely soluble in the aqueous elution buffer. The fusion protein can be substantially removed from the solution by again increasing the temperature above the inverse transition temperature wherein the fusion is no longer soluble in the aqueous buffer. The fusion protein can be removed by precipitation and/or hydrophobic interaction, leaving behind highly purified antibody in the solution.

[0045] According to the invention, the elastin-like polypeptide can comprise, e.g., pentapeptide VPGXG repeating units wherein X is any amino acid except proline (preferably a conservative variant, such as a hydrophobic amino acid). Preferably the pentapeptide has the sequence VPGVG. In a preferred embodiment of the invention the elastin-like polypeptide consists of more than two repeating units, more than five VPGVG repeating units, preferably more than 10 VPGVG repeating units, more preferably more than 50 VPGVG repeating units and most preferred more than 100 VPGVG repeating units. Of course it is possible that the Elastin-like protein contains 200, 300 or 500 repeating pentameric units. Any two or more of the oligomeric repeats may be separated by one or more amino acid residues (e.g., spacer peptides) that do not eliminate the phase transition behavior of the inventive fusion proteins. [0046] The antibody-affinity ligand contained in the inventive fusion protein is preferably an immunoglobulin-binding protein. According to the invention the antibody- affinity ligand, in particular the Ig-binding protein can be a wild-type protein or naturally or laboratory mutated protein. In the context of the present invention "mutated protein" means that the amino acid sequence of the protein shows in comparison to the wild-type protein one or more sequence differences. The mutated amino acid sequence can result in a different binding of antibodies to the inventive fusion proteins in comparison to the wild- type protein. In a preferred embodiment of the invention, the mutated antibody-affinity ligand results in an improved binding of the antibodies to be isolated to the inventive fusion proteins. Examples for mutated antibody-affinity ligands can include, without being restricted to, substitution of one or more amino acid residues by different amino acid residues which have chemical characteristics which are similar to that of the replaced amino acid residues, deletion of one or more amino acid residues and addition of one or more amino acid residues. The mutated amino acid sequence of the antibody-affinity ligand can be due to a spontaneous mutation of the nucleic acid encoding the antibody-affinity ligand, for example genetic recombination, but can also be brought about by an artificially induced mutagenesis, e.g. by the use of a chemical or physical mutagens or by a vitro mutagenesis and site-directed mutagenesis, e.g. by a recombinant technology. Methods for artificial introduction of mutations in a amino acid sequences are known to the person skilled in the art and are described for example in Sambrook, J. and Russell, D. W. (2001), Molecular cloning - a laboratory manual, 3rd ed., Cold Spring Harbor, New York.

[0047] In a particularly preferred embodiment of the invention, the Ig-binding protein is selected from the group consisting of protein A from Staphylococcus aureus or a fragment thereof, protein G from the group G streptococci or a fragment thereof, protein L from Peptostreptococcus magnus or a fragment thereof, or a hybrid molecule comprising at least two of these proteins or fragments thereof.

[0048] Staphylococcal protein A (SpA), a cell wall component of Staphylococcus aureus, can specifically bind immunoglobulin G (IgG) from several mammalian species. It has high specific avidity for the Fc portion of IgG without interrupting its antigen-binding ability. However, the binding affinity of SpA is strongly dependent on the source of IgG and the pH of binding buffer. A fusion protein comprising protein A therefore can be used for the immobilization, isolation and purification of IgG chains.

[0049] Protein G from the group G streptococci offers binding to broader IgG subclasses and exhibits different affinity constants, and less pH dependence in comparison to protein A. Because of these benefits, fusion proteins containing protein G as antibody- affinity ligand protein G can be used for the immobilization, isolation and purification of IgG with improved binding properties when compared with SpA.

[0050] Protein L is an Ig light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. Different from SpA and protein G, protein L has a strong binding affinity to IgM, IgA, IgE, and IgD. Thus, fusion proteins comprising protein L as antibody-affinity ligand can be used for the immobilization, purification and isolation of IgM, IgA, IgE, and IgD.

[0051] "Hybrid molecules" are antibody-affinity ligands comprising fragments of at least to different Ig-binding proteins. A preferred example of a hybrid molecule is a hybrid between protein L and protein G. Thus, a preferred example of the inventive fusion protein relates to a protein which contains a fusion between the Elastin-like polypeptide and a hybrid GL protein. This inventive fusion protein combines advantageously the binding properties of proteins G and L and can be used for the immobilization, purification and isolation of IgG, IgM, IgA, IgE, and IgD.

[0052] In one embodiment of the invention, the fusion "immunoglobulin-binding protein" includes a peptide hapten, epitope or entire antigen specifically bound by an antibody of interest. Such a fusion construct can allow capture, immobilization and purification of an antibody of desired specificity.

[0053] According to the invention a "fragment" of the Ig-binding protein is a domain or sequence portion of these proteins that can specifically recognize an sequence of the immunoglobulin chain and bind thereto.

[0054] In another preferred embodiment of the invention the fusion protein comprises in addition to the elastin-like polypeptide two, three or more different antibody- affinity ligands. If the inventive fusion proteins contains more than one antibody-affinity ligand, for example two, three or more different Ig-binding proteins or fragments thereof, also these different antibody-affinity ligands can be separated from each other by spacer sequences.

[0055] The inventive fusion proteins can specifically used for binding, immobilization, purification and/or isolation of an antibody from any material containing this antibody. Examples of antibodies to be immobilized and/or isolated according to the invention include, without being restricted to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies or fragments thereof such as Fab fragments, Fv fragments, scFV fragments, scFv homodimers and the like. Fab fragments consist of assembled complete light and truncated heavy chains, whereas Fv fragments consist of VH and VL chains non- covalently associated. A review of such fragments is given in Conrad et al., Plant MoI. Biol., (1998), 38, 101-109.

[0056] Another aspect of the present invention relates to a preferably purified and isolated, nucleic acid molecule that has a nucleotide sequence which encodes a fusion protein for purifying immunoglobulins or antibodies. The inventive nucleic acid molecule can be a DNA or RNA molecule. The nucleic acid molecule comprises a first nucleotide sequence encoding a elastin-like polypeptide which consists of the pentapeptide VPGXG repeating units, in particular VPGVG repeating units, and at least one second nucleotide sequence functionally linked to the first nucleotide sequence and encoding one antibody- affinity ligand. The second nucleotide sequence can encode a wild-type or mutated antibody-affinity ligand, in particular a wild-type or mutated immunoglobulin-binding protein. In a preferred embodiment, the second nucleotide sequence contained in the inventive nucleic acid molecule encodes protein A from Staphylococcus aureus or a fragment thereof, protein G from the group G streptococci or a fragment thereof, protein L from Peptostreptococcus magnus or a fragment thereof, or a hybrid protein comprising at least two of the these proteins or fragments thereof. In another embodiment of the invention the nucleic acid molecule can comprise at least a third nucleotide sequence that encodes a spacer sequence. The third nucleotide sequence is located between the first and second nucleotide sequences. The expression of the inventive nucleic acid molecule results in a suitable environment, for example a host cell, in particular a mammalian host cell, in the generation of the inventive fusion proteins. [0057] Another aspect of the invention relates to a vector comprising a nucleic acid with a nucleotide sequence encoding a fusion protein for purifying immunoglobulins or antibodies. Upon introduction of the vector in a suitable host cell the inventive vector can be used for generating and producing the fusion protein of the invention in large amounts. In the vector the nucleic acid encoding the fusion protein is operably linked to one or more regulatory units which are able to control the transcription of the nucleotide sequence encoding the fusion protein and translation of the fusion protein in a given host cell. The regulatory units controlling the expression of the fusion protein to be included in the vector have to be selected according to the type of host cells into which the vector is to be used for expressing the fusion protein. For example, a vector to be introduced into an eukaryotic host cell should at least contain upstream of the nucleotide sequence encoding the fusion protein a promoter which is active in the host cell selected and, downstream of this nucleotide sequence, a transcriptional terminator sequence active in the host cell selected. Further regulatory units include, without being restricted to, enhancer elements and ribosome-binding sites.

[0058] Still another aspect of the invention relates to a host cell carrying at least one inventive nucleic acid molecule or at least one inventive vector. The host cell can be a eukaryotic or prokaryotic cell. Preferred examples of prokaryotic host cells include bacterial cells, such as cells of Escherichia coli or Bacillus subtilis. Preferred examples of eukaryotic host cells include, without being restricted to, insect cells, fungal cells and mammalian cells.

[0059] Another aspect of the present invention relates to a kit for immobilization, separation and/or purification of antibodies. The kit comprises at least one means for isolating an antibody, which is contained in a container. The kit can comprise one or more inventive fusion proteins, one or more inventive nucleic acids encoding a fusion protein, one or more inventive vectors, and/or one or more inventive host cell, each of which can be contained in a separate container. For example, if the kit comprises four different inventive fusion proteins to be used for the immobilization and/or isolation of four different antibodies, the four fusion proteins can be contained in separate containers.

[0060] The present invention also relates to methods for isolating and/or purifying antibodies from an antibody-containing sample comprising the steps of: 1 ) mixing a fusion protein of the invention (e.g., a sequence of repeated ELPs fused with an immunoglobulin-binding protein) with an antibody containing sample in a liquid medium;

2) incubating the mixture under conditions conducive to binding of the antibody to the fusion protein, thus forming a complex between the antibody and fusion protein;

3) substantially separating the complex from the remainder of the sample by precipitation or hydrophobic interaction at a temperature ranging from about 25°C to about 40⁰C;

4) resolubilizing the complex at a temperature ranging from about 0⁰C to about 1O⁰C;

5) incubating the resolubilized complex under conditions that cause the antibody to become unbound (elute) from the fusion protein; and,

6) separating the fusion protein from the antibody by precipitation or hydrophobic interaction at a temperature ranging from about 25°C to about 40⁰C.

It is often possible to elute the antibody from the fusion protein without previously resolubilizing the complex; particularly where the complex has been separated by hydrophobic interaction onto a hydrophobic surface. Conditions for elution vary, e.g., with the particular immunoglobulin-binding protein of the fusion, but can include adjustment of the solution to a specific pH or ionic strength. Preferably, complex and fusion precipitations take place at about 37°C. Soluble conditions for the fusion and complex are typically at about 4⁰C.

[0061] The inventive method can be used for the isolation and purification of antibodies from substantially any aqueous sample containing antibodies, thereby obtaining an antibody preparation with high purity and high biological activity. The sample material containing antibodies can be derived from a broad range of sources such as blood, milk, cell culture supernatant, and extracts from any of various cells. The inventive method can be employed for the large scale isolation of antibodies, for example from fermenters with antibody producing mammalian cells, but also for the isolation of minimal quantities of antibodies from a human sample such as blood.

[0062] In an exemplary embodiment, an ELP fusion with SpA can be useful for phase-separation schemes, allowing simple separation of ELP-SpA-IgG by thermal precipitation. A solution of pure IgG can then be prepared, e.g., by dissolution at cool temperatures, elution of the IgG from the SpA, precipitation of the ELP-SpA at warm temperatures, and centrifugation leaving only IgG in the supernatant. The final ELP-SpA precipitate can be reused in additional purification processes. The present invention also includes construction of ELP fusions with protein G and/or protein L, or other proteins having affinity interactions with antibodies of interest.. These fusions are found to be useful in the selective immobilization and purification of antibodies. Fusion proteins like these have been found useful for the rapid, efficient, and economical immobilization and purification of a wide range of antibodies.

[0063] I n another aspect of methods, ELP-ligand fusions can be used as components in affinity based analytical techniques. For example, at warm temperatures, the fusion proteins can self assemble on a hydrophobic substrate. Antibodies can be bound and used to capture antigens of interest. The presence of the antigen can be detected using a second antibody against the antigen, typically having reporter-label. Based on the present specification, one skilled in the art can appreciate the utility of the ELP-ligand-Ig moieties in sandwich assays such as Western Blot and ELISA assays.

FUSIONS OF ELPs WITH ANTIBOD Y-BIKDING PROTEINS

[0064] Fusion protein compositions of the invention generally include one or more amino acid sequences with thermally controllable inverse phase solubility covalently attached to one or more protein sequences having an affinity for one or more type of immunoglobulin. For example, useful and functional fusion protein structures can be prepared by expression of constructs including repeat elastin-like peptide (ELP) sequences (e.g., VPGVG) with known antibody-binding sequences (e.g., antibody-specific ligands from bacteria).

[0065] One elegant aspect of such ELP-ligand constructs is the ease of purification.

Highly purified solutions of the fusions can be prepared without sophisticated processing equipment, but for a centrifuge and a temperature controlled incubator. To prepare fusions of the invention, nucleic acids with the desired ELP and ligand sequences can be prepared by DNA synthesis, cloning from natural sources, and/or obtained from generally available recombinant plasmids containing the sequences. The ELP and ligand sequences can be combined using well known techniques of genetic recombination, e.g., using restriction endonucleases and ligases. The fusion sequence can be inserted into a suitable expression vector and expressed as a fusion protein, e.g., in a bacterial host cell. Instead of complicated purification schemes (typically lysis, centrifugation, filtration and several orthogonal chromatography steps - with a 10% recovery, or less), the fusion can be simply and efficiently recovered by a series of temperature cycling and centrifugation steps. For example, a large part of a bacterially expressed ELP-ligand fusion can be harvested in high purity by lysing the bacteria, centrifuging the debris at low temperatures (e.g., at 2-8⁰C), separating and warming the supernatant (e.g., to about 37⁰C) so that the fusion protein becomes insoluble, centrifuging the fusion from solution, and preparing a pure solution of the fusion protein (at any desired concentration) by dissolving the pellet in a cool aqueous buffer.

[0066] In some embodiments, the fusion proteins include SpA, protein L and/or protein G fused to ELP repeats. Since proteins L and G have been shown to possess different binding affinity and specificity from SpA for antibodies, it can be of interest to designing ELP fusions with antibody-binding domains of these proteins so that a wide variety of immunoglobulins can be targeted from various possible antibody sources. For example, amplified fragments coding for protein L and G can be fused to the 3 prime end of a gene coding for the ELP domain. For a fusion containing both protein L and protein G, a fragment coding for protein LG was amplified and fused with ELP domain.

[0067] The fusion proteins are readily produced in E. coli BL21(DE3) using well known techniques and purified by two cycles of inverse temperature transition. The purity of the fusion proteins can be determined by SDS-PAGE followed by silver staining (see Figure IA) and bands corresponding to the expected sizes of the fusions are observed. In the case of protein L and protein LG fusions, partially degraded products can be detected as observed previously (Kihlberg, B-M., Sjobring, U., Kastern, W., and Bjorck, L. (1992) Protein LG: A Hybrid Molecule with Unique Immunoglobulin Binding Properties. J. Biol. Chem. 267, 25583-25588). Approximately 10 mg of ELP-ProG and 2 mg of ELP-ProL and ELP-ProLG can be obtained in a 25 mL culture.

[0068] The presence of antibody-binding domains on the fusions can be confirmed by Western blot analysis using goat IgG-AP (Figure IB) and human IgM-HRP conjugates (Figure 1C). Consistent with the binding preference, a strong interaction is observed between protein G and the goat IgG-AP conjugate, while protein L has high affinity for human IgM but not for goat IgG. Neither conjugate interacted with ELP, indicating the antibody binding functionality of the fusion proteins lies in the protein L and G domains.

[0069] The temperature transition properties of the ELP fusions can be studied by turbidity measurements. As shown Figure 2, similar profiles from ELP fusions are observed as for independent ELP. This shows that the transition property of the ELP is not affected by antibody affinity ligands in fusions.

ANTIBODY IMMOBILIZATION BY ELP FUSION PROTEINS

[0070] The thermally tunable hydrophobic property of the ELP fusions provides a simple and non-covalent means to immobilize antibodies onto a support surface using the specific adhesion based on hydrophobic interaction. At higher temperatures, ELPs interact with substantially more hydrophobic character. This phenomenon can be manipulated to physically capture ELP fusions, e.g., on hydrophobic solid supports, such as, many plastic surfaces, hydrophobic plates, hydrophobic resin beads, and the like. The solid support bound ELP fusions can capture and immobilize antibodies from the surrounding environment. Alternately, the ELP fusions can bind antibodies in solution before capture onto hydrophobic substrates at elevated temperatures.

[0071] In one embodiment, antibody-immobilization capability can be demonstrated by first immobilizing ELP fusions onto hydrophobic polystyrene microplates, then capturing antibodies with detectable markers from a solution. For example, ELP-ligand fusion proteins can be aggregated at 37⁰C for 30 min in the presence of a hydrophobic substrate, where they self assemble on the surface. In one controlled experiment, ELP and ELP fusions were captured on the hydrophobic surfaces of different microtiter plate wells. Labeled antibodies of different types were found to be immobilized with high specificity by particular ELP fusions. The amount of immobilized antibodies was measured by the HRP (horse radish peroxidase) activity. Extensive immobilization of either goat IgG or human IgM was observed using the ELP fusions. The binding preferences of the affinity ligand proteins were consistent with the binding affinity of protein G or protein L not fused with ELPs (see Table 1, below). ELP without an antibody binding domain or HRP were used as controls and virtually no binding was observed. These results also demonstrate that the protein G and protein L domains are solely responsible for the antibody binding and even aggregated ELP fusions are presented in an accessible orientation to interact with the target antibodies.

Table 1. Binding of antibody by ELP fusions as indicated by HRP activity (ΔA490)

[0072] The amounts of antibody captured can be controlled by simply adjusting the level of immobilized ELP fusions. For example, a serial dilution of ELP-ligand can be immobilized onto microplate wells and the quantity in each well detected using an antibody- reporter conjugate. In one embodiment, ELP-SpA fusions were serially diluted into wells of a polystyrene microplate. Donkey IgG-HRP conjugate was subsequently added to each well, the wells rinsed and a reporter HRP substrate added to signal the amount of antibody binding. The results show that antibody immobilization was dependent on the concentration of ELP-SpA initially added to each well, as shown in Figure 3.

PURIFICATION OF ANTIBODIES USING ELP-LIGAND FUSION PROTEINS

[0073] Antibodies can be purified easily and specifically, without sophisticated equipment or techniques, using the ELP-ligand fusions of the invention. The present techniques and compositions offer diverse capabilities suitable for purification of various antibodies from many different sources. Antibodies can be purified and/or concentrated from relatively pure solutions or from complex solutions or suspensions having any number of contaminants. Different types of antibodies can be selectively purified away from other types of antibodies. ELP fusions can capture the antibodies in solution or while bound to a solid support. Antibodies can be eluted from the ELP fusions in pure form while the fusions remain bound to a solid support. In some embodiments, antibodies are be eluted from the ELP fusions in solution and the fusions removed by centrifugation at cold temperatures, leaving pure antibodies in solution. Alternately, antibodies bound to precipitated ELP fusions can be eluted in pure form at warm temperatures, leaving behind precipitated ELP fusions.

[0074] In an exemplary embodiment, IgG purification capability of the fusions can be demonstrated using ELP-ProG. Initial demonstrations of IgG purification were conducted using purified mouse and rabbit IgGs. After 30 min incubation at room temperature with the ELP-ProG fusion, bound IgG was recovered by thermal precipitation. In both cases, substantially complete recovery of the ELP-ProG was achieved. After solubilization at cool temperatures, the bound IgG was eluted (elution fraction) from the ELP-ProG-IgG complex with an ice-cold elution buffer (pH 2.6) and the ELP-ProG fusion was subsequently separated by thermal precipitation and centrifugation into the pellet fraction. Each fraction was analyzed by silver stained SDS-PAGE and then quantified using a Bio-Rad Gel Doc 2000 Gel Documentation System and Quantity One software. As shown in Figure 4, around 92% of rabbit IgG and 68% of mouse IgG were recovered in this one step process.

[0075] Complex sample matrices typically associated with IgG purification do not significantly reduce recovery efficiency for processes with ELP fusions. For example, recovery of antibodies from a supernatant of a hybridoma cell culture (C1B7), which produces mouse IgG against human acetylcholinesterase, can be similar to recovery from pure antibody solutions. By employing a similar procedure as described above, each fraction can be recovered and analyzed. Again, substantially complete recovery of the ELP fusions can be obtained. Because of the low concentration of IgG in the supernatant of a typical hybridoma cell culture, enhanced chemiluminescence (ECL) can be applied to signalize and quantify each fraction (Figures 5). A recovery efficiency of 64% has been calculated for antibodies from such a hybridoma culture; thus demonstrating that the presence of cell culture supernatant has no significant effect on the binding efficiency or the precipitation efficiency of the ELP-ProG fusion.

[0076] For purification of IgG from serums, mouse and rabbit serums were examined. The serum samples were prepared by centrifugation to remove non-soluble proteins. Purification was performed as before and the efficiency was determined by silver staining (Figures 6A and 6B). Essentially, a single band representing the recovered IgG was detected in the elution fractions, while the other serum proteins remained in the supernatant fractions. Parallel to the results with the purified antibodies, around 60% of IgG in the mouse serum was recovered, while almost 90% of IgG in the rabbit serum was recovered. These results confirmed that no significant interference occurred during purification due to the presence of other non-specific proteins in the serums.

[0077] In addition to the ease of purification and the high efficiency, another significant advantage of this strategy is the possibility of repeated usages of ELP-ProG. The regeneration and re-binding efficiency was evaluated with rabbit serum. The same ELP- ProG fusion was used for three times for IgG purification (Figure 7). Each elution fraction showed the same recovery efficiency and purity, demonstrating that the ELP-ProG fusion can be reused for IgG purification several times without losing binding affinity and the inverse transition property. This result opens possible applications for ELP fusions to be useful with harsh conditions such as low pH without losing functionality.

EXAMPLES

[0082] The following examples are offered to illustrate, but not to limit the claimed invention.

[0083] ELP fusions with antibody affinity ligands can be utilized for the immobilization and purification of antibodies, in which the ELP domain offers inverse transition in homogeneous condition and affinity ligands operate to combine with antibodies. It can be demonstrated that the immobilization of antibodies is specific, e.g., between ELP-SpA and IgG. IgG purification by ELP-ProG was performed with excellent purity and recovery. The lower recovery found for mouse IgG compared to rabbit IgG may be due to the low binding affinity between protein G and one of mouse IgG subtype (IgGl). The efficiencies of recovery are higher than those reported for chromatographic separation (Dancette, O. P., Taboureau, J.-L., Tournier, E., Charcosset, C, and Blond, P. (1999), Purification of immunoglobulins G by protein A/G affinity membrane chromatography, /. Chromatogr. B 723, 61-68; Thomas, T. M., Shave, E. E., Bate, I. M., Gee, S. C, Franklin, S., and Rylatt D. B.(2002), Preparative electrophoresis: a general method for the purification of polyclonal antibodies, J. Chromatogr. A 944, 161-168). ELP fusions with different types of affinity ligands can apply for the immobilization and purification of different sources and types of antibodies. Moreover, these ELP fusions can be reusable without losing binding affinity.

[0084] Example 1 - Construction of Expression Vectors

[0085] DNA manipulations were performed according to standard procedures unless specified otherwise (Sambrook, J. and Russell, D. W. (2001) Molecular cloning - a laboratory manual, 3^rd ed., Cold Spring Harbor, New York). PCR was performed using the Taq DNA polymerase (Promega, Madison, WI) according to the manufacturer instruction. E. coli JM109 (recAl supEM endAl hsdRll gyrA96 relAl thi A(lac-proAB) F' [traD36 proAB⁺ lacl^q lacL ΔM15]) and BL21(DE3) (hsdS gal (λclts857 indl Sam7 nin5 lac\JV5- T7 gene I)) were grown on LB agar for solid culture and in terrific broth for liquid culture. All media contained 0.1 mg/mL of ampicillin for selection. Plasmid pET-Ela78h6 (Kostal, J., Mulchandani, A. and Chen, W. (2001) Tunable biopolymers for heavy metal removal. Macromolecules 34, 2257-2261) was used as the source of the ELP gene and plasmid pLG (Kihlberg et ah, 1992) as the source of the protein G and L gene. Plasmid pELP-SpA was used for ELP-SpA fusion.

[0086] A DNA fragment coding for the protein G and protein L from pLG were amplified as 407-bp and 905-bp PCR fragment using primer sets of Upper-G (SEQ ID No. 2: 5 '-tec ccc ggg agg agg agg agg aac tta caa att-3'), Lower-G (SEQ ID No. 3: 5 '-tat ggt gac ctt cag gta ccg taa agg tc-3') and Upper-L (SEQ ID No. 4: 5'-tcc ccc ggg agg agg agg agg aaa aga aga aac -3'), Lower-L (SEQ ID No. 5: 5'-tat ggt gac ctg caa ate taa tat taa tag-3'). The protein LG fragment was amplified as 1319-bp PCR fragment using Upper-L and Lower-G primer set. The PCR products were digested with Xmal and BstEII and inserted into a similarly digested ρET-Ela78h6, resulting in pELP-ProG, pELP-ProL, and pELP- ProLG. [0087] Example 2 - Expression, Purification, & Characterization of Fusions

[0088] The supernatant of hybridoma cell culture (C1B7) was purchased from

Developmental Studies Hybridoma Bank (Iowa City, IA). IgGs and serums of mouse and rabbit were purchased from Sigma-Aldrich (Saint Louise, MO). Goat anti-mouse IgG- horseradish peroxidase (HRP) conjugate and human IgM-HRP conjugate were purchased from Pierce Biotechnology, Inc. (Rockford, IL). Goat anti-mouse IgG-alkaline phosphatase (AP) conjugate, AP reagent, and chloronaphthol were purchased from Bio-Rad (Hercules, CA).

[0089] E. coli strain BL21(DE3) containing each plasmid was inoculated from a single colony and grown at 37 ⁰C and 300 rpm in 25 mL of terrific broth. After 48 h, the culture was harvested and resuspended in 5 mL of phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7). Cells were then disrupted for 5 min by sonicator (Virtis, NY) and cell debris was removed by centrifugation for 15 min at 15,000 g.

[0090] Inverse temperature transition was used for the purification of ELP fusions.

NaCl was added to a final concentration of 1 M to the crude extract and the samples were heated to 37 °C for 10 min and centrifuged at 15,000 g at 37 °C for 15 min. The pellets containing ELP fusions were dissolved in ice-cold PBS and centrifuged at 15,000 g at 4 ⁰C for 15 min to remove undissolved proteins. This temperature transition cycle was repeated once more, and the pellets containing ELP fusions were finally dissolved in ice-cold PBS. The purity of the protein preparation was determined by silver staining (Bio-Rad) after SDS-PAGE electrophoresis. Western blot was performed using goat IgG-AP with the AP color reagent for G fusions and human IgM-HRP with HRP color reagent (filtered 10 mL of 50 mM pH 7.6 Tris buffer with 3 mg of chloronaphthol in 0.1 mL ethanol, containing 10 mL of 30 % H2O2) for L fusions.

[0091] The inverse transition profiles of the ELP fusions were determined spectrophotometrically in a 96-well microplate reader (POLARstar Optima, BMG Labtechnologies, INC., NC). Absorbance was measured at 620 nm with 0.1 mM of ELP78 and ELP fusions in 0.1 mL PBS containing 0.5 M NaCl during temperature increase from 25 to 40 ⁰C. As shown in Figure 2, the turbidity profile of ELPs with temperature is not substantially changed by fusion with proteins G and/or protein L.

[0092] Example 3 - Immobilization of antibodies by ELP fusions

[0093] To demonstrate the immobilization of antibodies via ELP fusions, 0.1 mg of each fusion protein in 0.1 mL of PBS was incubated for 30 min at 37 ⁰C on ELISA microplate wells. After discharging solutions in microplate wells, 1:5000 dilutions in PBS of 1 mg/mL of goat IgG-HRP, human IgM-HRP, and HRP were incubated for 30 min at 37 ⁰C. The plate was washed three times with 37 ⁰C PBST (0.5 % Tween-20 in PBS) and HRP activity was detected with 0.1 mL of substrate (20 mg of σ-phenylenediamine in 10 mL of 0.1 M pH 4.6 citrate-phosphate buffer, containing 4 μL of 30 % H₂O₂) at 490 nm.

[0094] The specific immobilization of antibodies on affinity ligands was confirmed with ELP-SpA and donkey IgG-HRP. Serial dilutions of ELP-SpA were immobilized onto microplate wells and donkey IgG-HRP was added. The relationship between ELP-SpA concentration and IgG binding was analyzed by HRP activity as described previously.

[0095] Example 4 - IgG purification

[0096] ELP-ligand fusions were used to capture and purify antibodies from several sources ranging from pre-purified antibodies to complex blood serum sources.

[0097] For example, 0.5 mg of purified mouse-IgG or rabbit-IgG in 0.5 mL of PBS was mixed with 1 mg of ELP-ProG for capture. The mixture was incubated for 30 min at room temperature for the binding between protein G and IgG. To recover ELP-ProG-IgG complex, 0.1 mL of 5 M NaCl was added to the sample and incubated at 37 ⁰C for 5 min. After centrifugation at 15,000 g while maintaining 37°C, the pellet containing ELP-ProG- IgG complex was resolubilized with 0.1 mL of ice-cold PBS. For elution, 0.4 mL of 0.1 M sodium citrate (pH 2.6) was added and the sample was stored on ice for 10 min. The elution fraction was recovered by inverse temperature transition with 0.1 mL of 5 M NaCl. Each fraction was analyzed by native SDS-PAGE electrophoresis, followed by silver staining. See Figure 4.

[0098] For IgG purification from hybridoma cell culture, a supernatant (C1B7) was used as IgG source. 1 mg of ELP-ProG was mixed with C1B7 (450 μL, relevant to 19.35 μg of IgG) in the final volume of 0.5 mL of PBS. By using inverse temperature transition described above, the elution fraction was recovered. Each fraction was signalized by western blot using goat anti-mouse IgG-HRP conjugate and the enhanced chemiluminescence kit (ECL) (Amershampharmacia biotech, Piscataway, NJ). See Figure 5.

[0099] IgG purification from serums was demonstrated using mouse and rabbit serums. Insoluble proteins in serums were separated by centrifugation for 5 min at 10,000 g. 0.1 mL of serum supernatants were mixed with 1 mg of ELP-ProG in 0.4 mL of PBS. After recovering elution fraction by inverse temperature transition, each fraction was analyzed by silver staining, as shown in Figure 6.

[0100] Feasibility of repeated ELP-ProG usage was demonstrated. IgG purifications from rabbit serum were repeated three times with same sample of ELP-ProG. After each elution step, ELP-ProG was recovered in cold-PBS buffer and used for another cycle of IgG purification. Silver stained SDS-PAGE gels of the replicate purifications are shown in Figure 7. The intensity of the protein bands was quantified using Bio-Rad Gel Doc 2000 Gel Documentation System and Quantity One software.

[0101] In conclusion, the temperature-triggered immobilization and purification of antibodies can be demonstrated by tunable ELP fusions with antibody-affinity ligands. Although the results reported here are restricted in some sources of antibodies, the ELP fusions with several types of ligands can be similarly applied and used for other antibodies. We believe that this technology will be useful as an economical and highly efficient tool for the purification and immobilization of antibodies.

[0102] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. For example, based on teachings of this specification, one skilled will appreciate that fusions of a variety of different ELP sequences with any of a variety of immunoglobulin-binding proteins can capture any appropriate type of immunoglobulin to enjoy the simple and efficient immobilizations and/or purifications of the invention. [0103] The C1B7 hybridoma was obtained from the Developmental Studies

Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We thank Dr. UIf Sjobring for providing the plasmid coding for protein L and G.

[0104] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, many of the techniques and apparatus described above can be used in various combinations.

[0105] AU publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A fusion protein for purifying immunoglobulins or antibodies comprising an elastin-like polypeptide fused to one or more antibody-affinity ligands.

2. The fusion protein of claim 1, wherein the elastin-like polypeptide comprises one or more pentapeptide VPGVG units.

3. The fusion protein of claim 1, wherein the one or more antibody-affinity ligands comprise an immunoglobulin-binding protein or an antigen.

4. The fusion protein of claim 3, wherein the immunoglobulin-binding protein comprises a wild-type protein or mutated protein.

5. The fusion protein of claim 4, wherein the innumoglobulin-binding protein is selected from the group consisting of: protein A from Staphylococcus aureus or a fragment thereof, protein G from the group G streptococci or a fragment thereof, protein L from Peptostreptococcus magnus or a fragment thereof, and a hybrid molecule comprising at least two of these proteins or fragments thereof.

6. The fusion protein of claim 5, wherein any of said fragments comprise a domain able to bind to an immunoglobulin chain.

7. The fusion protein of claim 1, wherein the one or more antibody-affinity ligands comprise two different antibody-affinity ligands or three or more different antibody-affinity ligands.

8. The fusion protein of claim 1, further comprising a spacer peptide between the elastin-like polypeptide and the one or more antibody-affinity ligands.

9. The fusion protein of claim 7, further comprising one or more spacer sequences separating the different antibody-affinity ligands from each other.

10. The fusion protein of claim 1, wherein the fusion protein is able to bind to an immunoglobulin selected from the group consisting of: a chain of IgG, a chain IgM, a chain IgA, a chain IgE, a chain IgD and fragments thereof.

11. A nucleic acid molecule with a nucleotide sequence encoding a fusion protein capable of binding and co-precipitating immunoglobulins or antibodies by inverse phase transition.

12. The nucleic acid molecule of claim 11, comprising a first nucleotide sequence encoding a elastin-like polypeptide which consists of one or more pentapeptide VPGVG units and one or more second nucleotide sequence functionally linked to the first nucleotide sequence and encoding an antibody-affinity ligand.

13. The nucleic acid molecule of claim 12, wherein the second nucleotide sequence encodes a wild-type or mutated immunoglobulin-binding protein or an antigen specific to a desired antibody.

14. The nucleic acid molecule of claim 12, wherein the second nucleotide sequence encodes protein A from Staphylococcus aureus or a fragment thereof, protein G from the group G streptococci or a fragment thereof, protein L from Peptostreptococcus magnus or a fragment thereof, or a hybrid protein comprising at least two of the these proteins or fragments thereof.

15. The nucleic acid molecule of claim 12, comprising at least a third nucleotide sequence encoding a spacer wherein the third nucleotide sequence is located between the first and second nucleotide sequences.

16. A vector comprising a nucleic acid having a nucleotide sequence encoding a fusion protein for purifying immunoglobulins or antibodies.

17. The vector according to claim 16, wherein the fusion protein is capable of binding and co-precipitating immunoglobulins or antibodies by inverse phase transition

18. The vector according to claim 16, wherein the encoded fusion protein is operably linked to one or more regulatory units which are able to drive the expression of the fusion protein from the nucleic acid in a suitable environment.

19. The vector according to claim 16, wherein the nucleotide sequence comprising a first nucleotide sequence encoding a elastin-like polypeptide which consists of one or more pentapeptide VPGVG units and one or more second nucleotide sequence functionally linked to the first nucleotide sequence and encoding an antibody-affinity ligand.

20. The vector according to claim 18, wherein the one or more regulatory units are selected from the group consisting of: a promoter, an enhancer, a transcriptional terminator and a ribosome-binding site.

21. A host cell carrying at least one vector of claim 16.

22. The host cell of claim 21, wherein the host cell is a eukaryotic or prokaryotic cell.

23. The host cell of claim 22, wherein the eukaryotic cell is a mammalian cell.

24. A kit for immobilization and purification of antibodies comprising a component selected from the group consisting of: at least one fusion protein of claim 1, at least one nucleic acid encoding a fusion protein of claim 11, at least one vector of claim 16 and at least one host cell of claim 21.

25. A method for isolating or purifying antibodies from a sample containing an antibody of interest, the method comprising: mixing a fusion protein of claim 1 in a liquid medium sample containing the antibody; incubating the mixture for a time sufficient to allow binding of the antibody to the fusion protein thus forming a complex between them; separating the complex from the rest of the sample by precipitation or hydrophobic interaction at a temperature ranging from about 25°C to about 4O⁰C; solubilizing the separated complex at a temperature ranging from about O⁰C to about 10⁰C; incubating the recovered complex under conditions that cause the antibody to become unbound from the fusion protein; and, separating the fusion protein from the antibody by precipitation or hydrophobic interaction of the fusion protein at a temperature ranging from about 25⁰C to about 40⁰C.

26. The method of claim 25, wherein the precipitation temperature is about 37⁰C.

27. The method of claim 25, wherein the solubilizing temperature is about 4⁰C.

28. The method of claim 25, wherein any of said separating comprises centrifugation.

29. The method of claim 25, wherein the antibody is a monoclonal antibody, polyclonal antibody, chimeric antibody or a fragment thereof.