WO1998015293A2

WO1998015293A2 - Targeted addition of soluble polymers to recombinant proteins

Info

Publication number: WO1998015293A2
Application number: PCT/US1997/018104
Authority: WO
Inventors: Brian Seed; Wen Shao; John Seed
Original assignee: Edge Biosystems, Inc.
Priority date: 1996-10-07
Filing date: 1997-10-07
Publication date: 1998-04-16
Also published as: EP1011730A2; AU4672297A; WO1998015293A3

Abstract

Proteins are modified with soluble polymers to change their biological properties, such as to render them less immunogenic and longer-lived in the body. The modification is by a strong, but non-covalent complexation between a metal chelate and an oligohistidine tag which is engineered into the protein. This method of protein modification leads to homogeneous preparations which can be readily prepared in large quantities.

Description

TARGETED ADDITION OF SOLUBLE POLYMERS TO RECOMBJTNAΓVT PROTEINS

TECHNICAL FIELD F THE INVENTION

This invention is related to modification of recombinant proteins to increase their favorable characteristics for use as therapeutic agents.

BACKGROUND OF THE INVENTION

Polyethylene glycol (PEG), a nontoxic, nonimmunogenic water soluble polyether, has been used for many years to confer immunological privilege and prolonged serum half life on foreign proteins modified by it (Abuchowski et al., 1977; Abuchowski et al., 1977; Wie et al., 1981). Although the mechanism by which PEG modification frustrates immune system recognition is formally not understood, it appears to be due in part to the ability of PEG to change the bulk or colligative properties of modified proteins in such a way as to decrease the strength of their interaction with antibody combining sites. Often this is visualized as a fundamentally steric effect, in which the PEG physically obtrudes between antibody and epitope, preventing recognition. However a complete explanation for the longstanding observation that PEG modification (often referred to as PEGylation) reduces immunogenicity has not been advanced, and it may be that PEGylation also has an effect on the fate of proteins internalized by surface antibody on B cells, thereby compromising processing of the antigen for MHC class II presentation and preventing subsequent recognition by T cells. Qualitatively different behaviors have been shown to attend very high degrees of PEG modification, and highly modified proteins appear to be able to induce tolerance (Wie et al. , 1981; Holford et al. , 1982; Wilkinson et al., 1987; Wilkinson et al., 1987; Maiti et al., 1988; Chen et al., 1992; Bitoh et al., 1995).

In addition to decreased immunogenicity, an important consequence of PEGylation of proteins of low and intermediate molecular mass is the decrease in the rate at which the modified proteins are excreted by renal filtration, thereby increasing their serum persistence (Abuchowski et al. , 1977; Kamisaki et al., 1981; Savoca et al., 1984; Abuchowski et al., 1984; Viau et al., 1986). Again, although a detailed mechanism has not been established, it appears likely that the increased hydrodynamic radius of PEGylated proteins decreases the rate at which they are shunted from plasma to urine. The plausibility of this explanation follows from the well known dependence of renal filtration on molecular mass, and from the observation that linear polymers in good solvents have large effective radii of gyration (Stokes radius).

Because of these effects, modification of proteins with PEG has come to be an established approach for the creation of longer-lived, less immunogenic versions of recombinant proteins. The actual practice of PEGylation, however, can be complex. Typically proteins which are to be modified are reacted with a capped form of PEG, i.e., with a methoxy-terminated PEG (mPEG), which bears a reactive group at the opposite end. Although early experiments (Abuchowski et al., 1977; Abuchowski et al., 1977) relied on the use of cyanuric chloride (2,4,6 trichloro-s-triazine), a heterocycle with three reactive groups, a more common form in recent applications has been the N- hydroxysuccinimide, which is used to form amide bonds between an activated acid and a primary amine. In either case, the targeted primary amine is typically the epsilon amino group of a lysine side chain, and the reaction cannot be steered to a particular lysyl group, but rather occurs with equal facility with all of the available side chains. Targeting of the mPEG can be performed to a limited extent by increasing the density of reactive groups in the protein of interest, for example by providing a short oligo-lysyl tag element engineered into the protein, or by the addition of cysteine residues which can react with relatively cysteine-specific reactive groups on derivatized mPEG, such as maleimide or haloacetyl groups (Goodson and Katre, 1990; Benhar et al., 1994). However, although some specificity can be engendered in this way, neither maleimides nor iodo- or bromoacetyl groups are absolutely specific for sulfhydryls, and coupling to lysine side chains by Michael addition or SN2 substitution occurs with reasonable frequency. Because there are typically a relatively large number of lysines and few cysteines, the net result is frequently a heterogeneous coupling. In addition, inclusion of cysteines can affect protein global structure through the formation of unwanted or unintended disulfide linkages. Natural sites for N- or O-linked glycan addition also afford the potential for targeting the PEG modification to nonpeptide constituents of the protein. However, not all secreted proteins bear these substituents and the reaction conditions for scission of carbohydrates to generate reactive aldehydes are relatively oxidizing.

Other soluble polymers have been identified which also confer immunologic privilege and enhanced serum half life on proteins. These include, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Ficoll^® and albumin. Protein modification with these soluble polymers frequendy requires the same or similar approaches as were described for PEGylation. Thus, the problems of broad and heterogeneous coupling, protein oxidation and unintended changes to protein global structure apply here as well.

Therefore there is a need in the art for new methods of coupling PEG and other soluble polymers to proteins which will achieve greater specificity and therefore homogeneity and biological activity of the product. STπVfM ARV OF THF INVENTION

It is an object of the present invention to provide a method for modifying recombinant proteins with soluble polymers.

It is another object of the present invention to provide a molecular complex comprising a recombinant protein and a soluble polymer.

These and other objects of the invention are achieved by one or more of the following embodiments. In one embodiment a method of modifying a recombinant protein with a soluble polymer is provided. The method comprises the step of: mixing in an aqueous solution to form a complex comprising the protein and a soluble polymer:

(1) a recombinant protein comprising an oligohistidine tag with

(2) a metal chelate of a conjugate of (a) the soluble polymer and (b) a cheland.

In another embodiment of the invention a molecular complex is provided. The complex comprises a recombinant protein and a soluble polymer, wherein the recombinant protein comprises an oligohistidine tag and the soluble polymer is conjugated to a metal chelate, wherein the molecular complex is formed by interaction of the metal chelate with the oligohistidine tag.

These and other embodiments of the invention provide the art with a method for modifying the physical and immunological properties of recombinant proteins using gentle conditions which are not harmful to the protein.

ETAΠ ED DESCRIPTION

It is a discovery of the present invention that recombinant proteins can be modified with soluble polymers to alter their physical and immunological properties using oligohistidine tags and metal chelates. The modification employs gentle conditions which do not harm the protein's biochemical properties. In addition, the method is specifically targeted to one portion of the protein, so that unwanted modifications do not occur, and so that the product is relatively homogeneous. In addition, the method provides a high yield of the reaction products.

The method of the present invention exploits precise targeting to predetermined residues through very high affinity noncovalent bonds. The targeting allows the soluble polymers to be added to the protein under very mild conditions and the reaction allows convenient purification of the modified protein. In one particular embodiment of the invention a derivatized methoxypolyethyleneglycol (mPEG) which bears a metal ion in a tetradentate cheland is complexed with a recombinant protein engineered to comprise an oligohistidine tag. The very high affinity of cheland-His tag complexes (estimated K ι about 10^~13 M) allows the complex to persist over the biological lifetime of the protein.

Oligohistidine tags are known in the art and can be introduced into a protein by introducing an oligonucleotide which encodes oligohistidine into a protein-encoding gene. This will preferably be done at either the amino terminal or carboxy terminal or both so as to minimize disruption of the protein's structure. Preferably the tag will be between 6 and 20 amino acids in length. More preferably the tag will be between 6 and 12 amino acids in length.

Any metal ion can be used to form the chelate. These include but are not limited to transistion metals such as nickel, copper, zinc, and iron, lanthanides such as lanthanum, terbium and ytterbium, and actinides such as uranium. Preferably the coordination number of the metal ion is greater than the number of coordination sites of the cheland. The extra coordination sites are used for binding to the histidines in the oligohistidine tag. Any soluble polymer can be used to modify the physical, immunological, or other biological properties of the recombinant protein. Particularly preferred polymers are methoxypolyethyleneglycol, and dextran. Others as are known in the art and which impart desirable properties to the modified protein can be used.

Any metal cheland as is known in the art can be used in the practice of the present invention. They may be tetradentate, octadentate, hexadentate, etc. One particularly preferred cheland is N-(5 -amino- 1-carboxypentyl)- iminodiacetic acid (NT A). Standard methods for conjugating the metal cheland to the soluble polymer can be used. See Example 2, which does not limit the scope of the invention but is merely exemplary.

Metal-cheland-directed soluble polymer conjugation represents an improved method for polymer modification of proteins and more complex biological structures, such as viral vectors for gene delivery, and intact cells. In the setting of gene therapy, the ability to use very mild reaction conditions and to achieve precise molecular targeting represents an opportunity which previous methods for PEGylation could not attain. The immunogenicity of adenoviral vectors, for example, is a particularly important problem which has stymied their full development as gene delivery agents. Many workers in the field have focused on the tendency of these vectors to provoke cellular immunity, i.e. , the tendency of the host to mount cytotoxic T cell responses against cells treated with the vectors. An equally important but relatively underaddressed problem is the ability of the host to mount a protective humoral response, leading to the formation of neutralizing antibodies which prevent the reuse of the vector (Yang et al., 1994; Kay et al., 1995; Yang et al., 1995). Since there are relatively few applications of in vivo gene therapy which are likely to require only a single use of the genetic agent, the ability to prevent or decrease antibody recognition of the viral vector is important. Although early experiments with mPEG modification were carried out at very high degrees of modification (up to 40% or so of available lysine side groups), potent biological effects have been observed with molecules bearing as few as one or two mPEG chains per molecule. For mPEG chains of approximately 5 Kd there appears to be a qualitatively different behavior between highly modified and weakly modified proteins. The former act as tolerogens, and can be used to desensitize IgE-dependent allergic responses, both in animals and humans (Wie et al. , 1981; Mosbech et al., 1989; Mosbech et al., 1990; Bitoh et al., 1993; Bitoh et al., 1995). Less highly modified proteins retain the features of immunological privilege, but do not actively function as tolerogens (Brumeanu et al. , 1995). On the other hand, relatively weak modification, on the order of three mPEG molecules per protein, has been shown to induce tolerance to hapten-coupled ovalbumin when the mPEG molecular weight is 10 or 20 Kd (Wie et al., 1981). Thus there may be a requirement for a certain mass ratio of mPEG to protein which can be satisfied by the use of either a large number of short chains or a smaller number of longer chains. In general, longer chain polymers (up to about 10⁵ KD) are preferred over shorter chain polymers.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of N-(5-aιnino-l-carboxypentyl)iιnijτιodiacetic acid (1)

We have prepared (S)-l-aminopentyl N,N,diacetylaminoacetic acid (1) in two steps from epsilon amino carbobenzoxylysine and bromoacetic acid according to the scheme of Hochuli and coworkers (Hochuli et al., 1987), and have reacted this compound with methoxypolyethylene glycol activated with carbonyl diimidazole (2) to generate the substituted carbamate (3).

N-(5-amino-l-carboxypentyl)iminodiacetic acid was synthesized following a modification of a previously reported procedure (Hochuli et al. , 1987). A prechilled 13.5 ml aliquot of 2M NaOH containing N-e- carbobenzyloxylysine (8.6 mmole, 2.4 g) was added dropwise to 10.8 ml of 2M NaOH containing bromoacetic acid (17.2 mmole, 2.4 g) at 0°C. The resulting solution was stirred for two hours at 0°C, then allowed to warm to room temperature with continuous stirring overnight. The reaction proceeded for another two hours at 55°C. The consumption of unreacted primary and secondary amine was monitored by ninhydrin test. At the end of the incubation 30 ml of 1 M HCL was added to the solution with stirring. The mixture was cooled to room temperature and the resulting white crystals were filtered off and washed with 0.05 M HCL. The crystals were dissolved in 15 ml of 1M NaOH and 30 ml of 1M HCL was again added. The resulting crystals were washed once with 0.05 M HC1 and once with water. The final product was dried under vacuum at room temperature and stored in a desiccator. The yield of N-(5- benzyloxycarrκ>nyl-ammo-l-carboxypentyl)iminodiacetic acid was 75%. Purity and structure of the compound were confirmed by proton NMR and FT-IR. NMR in perdeuterated DMSO gave the following peaks: δ 2.3 (m, 4H), 2.6(m, 2H), 3.0(m, 2H), 3.3-3.6(m, 5H), 5.0 (s, 1H), 7.2-7.4(m, 5H). In the FT-IR spectrum, peaks at 1265 cm- , 1720 cnrl and broad peaks around 2500-3500 cm"l are associated with carboxy groups. The N-(5-benzyloxycarbonylamino-l- carrx)xypentyl)iminodiacetic acid was deprotected by hydrogenolysis. 2.4 g of N-(5-benzyloxycarbonylamino-l-carboxypentyl)iminodiacetic acid dissolved in 15 ml of 1M NaOH was hydrogenated with addition of 0.5 g of 5 % Pd/C. The progress of the reaction was monitored by detection of by-product carbon dioxide with Ba(OH)₂ solution and exposure of the amino group with ninhydrin reagent. The vacuum dried product, N-(5-amino-l-carboxypentyl)iminodiacetic acid, weighed 1.9 grams. The complete deprotection was confirmed by proton NMR of a sample dissolved in D₂O.

F.XAMPT 7 Synthesis of mPEG-NTA-Ni²+

Methoxypolyethylene glycol carbonyl imidazolide (2, nominal MW 5000 or 20,000; 0.04 mmole) dissolved in chloroform was added dropwise to a 0.1 M Na2CO3 solution of N-(5-amino-l-carboxypentyl)iminodiacetic acid (0.4 mmole) with vigorous stirring. The aqueous phase was removed and chloroform phase was dried with anhydrous sodium sulfate. The dried solution was added dropwise to dry ether. The precipitate which formed was collected by filtration, dissolved in a small amount of chloroform and reprecipitated in ether. The crude product was dried under vacuum. N-(5-amino-l-carboxypentyl)imino diacetic acid terminated methoxypolyethylene glycol (3) was purified by ion exchange chromatography on DEAE sephadex A-25 with a 0.1 - 1.0 M gradient of triethylammonium bicarbonate as an eluent. The conjugation was confirmed by proton NMR, FT-IR and ninhydrin assay. mPEG-NTA was dissolved in 1 % NiSO4 solution and dialyzed against distilled water to remove free nickel ions.

EXAMPLE S Preparation of GFP target proteins

In principle, any proteins bearing His tags are suitable for using the His- directed PEGylation scheme. We have focused on one easily monitored protein, the green fluorescent protein (GFP) of the jellyfish Aequorea ictoria, which bears an integral fluorophore formed by oxidative coupling of three adjacent residues, serine, tyrosine and glycine. The fluorophore is formed in a variety of organisms, including E. coli. A completely synthetic gene for green fluorescent protein has been prepared which replaces the naturally occurring codons with those chosen for optimal mammalian expression (Haas et al., 1996). The incorporation of two additional mutations, F64L (B. Cormack, pers. comm.) and S65T (Heim et al. , 1995) result in a more soluble GFP which can be excited at 490 nm and emits at about 520 nm, making the conditions for detection very similar to those for fluorescein. The synthetic gene was inserted into a prokaryotic expression vector bearing a six histidine amino terminal tag upstream of a clotting factor Xa cleavage site (IEGR) and a consensus protein kinase A (PKA) phosphorylation site (RRAS). The latter allows the convenient in vitro radiolabeling of purified protein using commercially available protein kinase A and 32p or ^5S γ-labeled ATP. The carboxyl terminus was either left unmodified or extended by an additional six histidine residues as shown in Figure 2. The expression vector relies on the regulated induction of transcription by IPTG. The expressed protein is harvested from bacterial cells by EDTA treatment of the harvested cells to remove the outer lipopolysaccharide leaflet of the outer membrane (thereby destabilizing the outer membrane) and then washing to remove EDTA, which would otherwise interfere with the subsequent chromatographic step. Following lysozyme treatment and lysis with a mild nonionic detergent (Triton X-100) in the presence of spermidine to compact the bacterial chromatin, the supernatant from a high speed spin was purified on immobilized nickel nitrilotriacetic acid columns using imidazole step elution (50 mM wash, 250 mM elution). The imidazole was removed by dialysis and the resulting GFP analyzed by SDS polyacrylamide gel electrophoresis. Synthesis of mPEG-NTA-Ni²+ GFP (green fluorescent protein)

Green fluorescent proteins with one or two histidine tags were mixed with mPEG-NTA-Ni²+ in aqueous solution. Unreacted mPEG-NTA-Ni²⁺ was removed by dialysis using membranes with a nominal molecular weight cutoff of 10 Kd. Gel electrophoresis confirmed the formation of the complex.

Rfiferenres

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Abuchowski, A., Kazo, G.M., Verhoest, C.J., Van, E.T. , Kafkewitz, D., Nucci, M.L., Viau, A.T. and Davis, F.F. (1984). Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol- asparaginase conjugates. Cancer Biochem. Biophys. 7, 175-86.

Benhar, I., Wang, Q.C., FitzGerald, D. and Pastan, I. (1994). Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site- specific manner. J. Biol. Chem. 269, 13398-404.

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Claims

CT ATMS

1. A method of modifying a recombinant protein with a soluble polymer comprising the step of: mixing in an aqueous solution to form a complex comprising the protein and PEG:

(1) a recombinant protein comprising an oligohistidine tag with

2. A molecular complex comprising: a recombinant protein and a soluble polymer, wherein the recombinant protein comprises an oligohistidine tag and the soluble polymer is conjugated to a metal chelate, wherein the molecular complex is formed by interaction of the metal chelate with the oligohistidine tag.

3. The method of claim 1 or 2 wherein the cheland is tetradentate.

4. The method of claim 1 or 2 wherein the cheland is octadentate.

5. The method of claim 1 or 2 wherein the cheland is hexadentate.

6. The method of claim 1 or 2 wherein the metal is nickel.

7. The method of claim 1 wherein the cheland is N-(5-amino-l- carboxypentyl)-iminodiacetic acid.

8. The method of claim 1 or 2 wherein the soluble polymer is methoxypolyethyleneglycol.

9. The method of claim 1 or 2 wherein the soluble polymer is polyethyleneglycol.

10. The method of claim 1 or 2 wherein the soluble polymer is hyaluronic acid.