Methods for chemically synthesizing immunoglobulin chimeric proteins
http://www.pharmcast.com/Patents200/Yr2008/June200 [2008-7-15]
Tag : amide resin
Abstract
The invention provides methods of chemically synthesizing chimericproteins comprising at least a portion of an immunoglobulinconstant region and a biologically active molecule.
Description of the Invention
SUMMARY OF THE INVENTION
The invention provides a method of chemically synthesizing chimericproteins comprising combining at least one biologically activemolecule and a portion of an immunoglobulin constant region suchthat a bond forms between the biologically active molecule and theportion of an immunoglobulin constant region where the biologicallyactive molecule comprises a first functional group or moiety andthe portion of an immunoglobulin constant region comprises a secondfunctional group or moiety, and where the first and secondfunctional group or moiety are capable of reacting with each otherto form a chemical bond. In certain embodiments, the inventionprovides a method of chemically synthesizing chimeric proteins byperforming native ligation (U.S. Pat. No. 6,184,344) such that abond forms between at least one biologically active molecule and aportion of an immunoglobulin constant region.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins comprising combining at least onebiologically active molecule and at least a portion of animmunoglobulin constant region, wherein a) the portion of animmunoglobulin constant region comprises an amino (N) terminuscysteine and b) the biologically active molecule comprises afunctional group capable of reacting with an N terminus cysteine toform a bond. In certain embodiments, the functional group is athioester. In certain embodiments, e.g., where the biologicallyactive molecule is a polypeptide, the thioester may be a carboxy(C) terminus thioester. In other embodiments, the thioester is nota carboxy thioester. In certain embodiments, the functional groupis an aldehyde. In certain embodiments, e.g., where thebiologically active molecule is a polypeptide, the aldehyde may bea carboxy (C) terminus aldehyde. In other embodiments, the aldehydeis not a C terminus aldehyde.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins comprising the steps of a)recombinantly expressing a fusion protein comprising at least aportion of an immunoglobulin constant region and a splicing proteincapable of forming a C terminus thioester on the portion of animmunoglobulin constant region; b) adding a thiol cofactor to thefusion protein of a); c) adding at least one biologically activemolecule having an N terminal cysteine, thereby synthesizing thechimeric protein. In some embodiments, the splicing protein isintein or a mutant form of intein which is defective in completionof the splicing reaction, but is still capable of thioesterintermediate formation. In some embodiments, the fusion protein isfurther comprised of a chitin binding domain.
In certain embodiments, the chemical synthesis is performed insolution. In some embodiments, at least one of the reactants islinked to a solid support. The biologically active molecule may belinked to the solid support. The portion of an immunoglobulinconstant region may be linked to a solid support. The fusionprotein comprising the portion of an immunoglobulin and thesplicing protein may be linked to a solid support.
DESCRIPTION OF THE EMBODIMENTS
Synthesis of Chimeric Proteins
Chimeric proteins comprising at least a portion of animmunoglobulin constant region and a biologically active moleculecan be synthesized using techniques well known in the art. Forexample, the chimeric proteins of the invention may be synthesizedrecombinantly in cells (see e.g. Sambrook et al. 1989, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.and Ausubel et al. 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).Alternatively, the chimeric proteins of the invention may besynthesized using known synthetic methods such as native ligation(U.S. Pat. No. 6,326,468) or solid phase synthesis (see e.g.Merrifield, 1973, Chemical Polypeptides, (Katsoyannis and Panayotiseds.) pp. 335-61; Merrifield 1963, J. Am. Chem. Soc. 85:2149; Daviset al. 1985, Biochem. Intl. 10:394; Finn et al. 1976, The Proteins(3d ed.) 2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257;U.S. Pat. No. 3,941,763. Alternatively, the chimeric proteins ofthe invention may be synthesized using a combination of recombinantand synthetic methods. In certain applications, it may bebeneficial to use either a recombinant method or a combination ofrecombinant and synthetic methods.
Combining recombinant and chemical synthesis allows for the rapidscreening of biologically active molecules and linkers to optimizedesired properties of the chimeric protein of the invention, e.g.,viral inhibition, hemostasis, production of red blood cells,biological half-life, stability, binding to serum proteins or someother property of the chimeric protein. The method also allows forthe incorporation of non-natural amino acids into the chimericprotein of the invention which may be useful for optimizing adesired property of the chimeric protein of the invention.
1. Chemical Synthesis
In certain embodiments, the invention provides a method ofsynthesizing a chimeric protein of the invention comprising atleast one biologically active molecule and at least a portion of animmunoglobulin constant region, or fragment thereof, where one ofeither the biologically active molecule or the portion of animmunoglobulin constant region may comprise an N terminus cysteineand the other comprises a functional group capable of reactingspecifically with the N terminal cysteine. The biologically activemolecule may include a polypeptide. The biologically activemolecule may include a small organic molecule or a small inorganicmolecule. The biologically active molecule include a nucleic acid.
In one embodiment, the N terminal cysteine is on the portion of animmunoglobulin constant region. In one embodiment, the portion ofan immunoglobulin constant region is an Fc fragment. The Fcfragment can be recombinantly produced to form cysteine-Fc (Cys-Fc)and reacted with at least one biologically active moleculeexpressing a thioester to make a chimeric protein of the invention,e.g., monomer-dimer hybrid. In another embodiment, an Fc-thioesteris made and reacted with at least one biologically active moleculeexpressing an N terminus cysteine.
In one embodiment, the N-terminal cysteine may be on the portion ofan immunoglobulin constant region, e.g., an Fc fragment. An Fcfragment can be generated with an N-terminal cysteine by takingadvantage of the fact that a native Fc has a cysteine at positions220, 226 and 229 (see Kabat et al. 1991, Sequences of Proteins ofImmunological Interest, U.S. Department of Public Health, Bethesda,Md.). Any of these cysteines may be used to generate an Fc fragmentfor use in the methods described herein. Additionally, recombinanttechniques can be used to generate Fc fragments having at least onenon-native (i.e. engineered by humans) N terminus cysteine. Forexample, cysteines can be added on at positions higher than 226, orlower than 220, of the EU numbering system.
In a specific embodiment, an Fc fragment is expressed with thehuman .alpha. interferon signal peptide adjacent to the Cys atposition 226. When a construct encoding this polypeptide isexpressed in CHO cells, the CHO cells cleave the signal peptide attwo distinct positions (at Cys 226 and at Val within the signalpeptide 2 amino acids upstream in the N terminus direction). Thisgenerates a mixture of two species of Fc fragments (one with anN-terminal Val and one with an N-terminal Cys). This in turnresults in a mixture of dimeric species (homodimers with terminalVal, homodimers with terminal Cys and heterodimers where one chainhas a terminal Cys and the other chain has a terminal Val) (FIG. 4A(see Original Patent)). The Fc fragments can be reacted with atleast one biologically active molecule having a C terminalthioester and the resulting monomer-dimer hybrid or dimer can beisolated from the mixture (e.g. by size exclusion chromatography).It is contemplated that when other signal peptide sequences areused for expression of Fc fragments in CHO cells a mixture ofspecies of Fc fragments with at least two different N termini willbe generated.
Cys-Fc may be recombinantly expressed. In one embodiment, the Fcfragment is expressed in a prokaryotic cell, e.g., E. coli. Thesequence encoding the Fc portion beginning with Cys 226 (EUnumbering) can be placed immediately following a sequence encodinga signal peptide, e.g., OmpA, PhoA, STII. The prokaryotic cell canbe osmotically shocked to release the recombinant Fc fragment. Inanother embodiment, the Fc fragment is produced in a eukaryoticcell, e.g., a CHO cell, a BHK cell. The sequence encoding the Fcportion fragment can be placed directly following a sequenceencoding a signal peptide, e.g., mouse IgK light chain or MHC class1 Kb signal sequence, such that when the recombinant chimericprotein is synthesized by a eukaryotic cell, the signal sequencewill be cleaved, leaving an N terminal cysteine which can than beisolated and chemically reacted with a molecule bearing a thioester(e.g. a C terminal thioester if the molecule is comprised of aminoacids).
The N terminal cysteine on an Fc fragment can also be generatedusing an enzyme that cleaves its substrate at its N terminus, e.g.,Factor X.sup.a, enterokinase, and the product isolated and reactedwith a molecule with a thioester.
In some embodiments, a recombinantly produced Cys-Fc can form ahomodimer. The homodimer may be reacted with peptide that has abranched linker on the C terminus, wherein the branched linker hastwo C terminal thioesters that can be reacted with the Cys-Fc, thusforming a dimerically linked monomer dimer hybrid (FIG. 4 (seeOriginal Patent)). In another embodiment, the biologically activemolecule may have a single non-terminal thioester that can bereacted with Cys-Fc.
In some embodiments, the branched linker can have two C terminalcysteines that can be reacted with an Fc thioester. In anotherembodiment, the branched linker has two functional groups that canbe reacted with the Fc thioester, e.g., 2-mercaptoamine.
Where the portion of the immunoglobulin constant region has an Nterminus cysteine, the functional group on the biologically activemolecule may be an aldehyde. If necessary, the aldehydefunctionality can be chemically synthesized for example byoxidation of an N-terminal serine with periodate (see, e.g.,Georghagen et al., 1992, Bioconjugate Chem. 3:138). Alternatively,where the biologically active molecule is a DNA molecule, it maylabeled with an aldehyde group by first coupling the DNA, duringthe last round of synthesis, with phosphoramidite thus generating aprotected amine. After deprotection, the DNA molecule labeled witha free amine may be generated. The amine could be coupled tosuccinimidyl 4-formylbenzoate (as shown below) to generate thealdehyde. This could be done on either its 3' or 5' end.
Another way of introducing an aldehyde into an oligonucleotide isdescribed in Zatsepin et al., 2002, Bioconjugate Chem, 13:822. Aprotected 1,2-diol may be incorporated in the 2' position. Afterthe synthesis of the oligo, the protecting groups can be removed toreveal the free 1,2-diol, which can then be oxidized with periodateto give an aldehyde.
Aldehydes are known to react with N-terminal cysteines (in effect,a 1,2-amino thiol moiety) to form thiazolidines. (See Botti, 1996,J. Am Chem. Soc., 118:10018; Zhang et al., 1998, Proc. Nat. Acad.Sci. USA, 95:9184). In one embodiment, the portion of animmunoglobulin constant region is an Fc fragment with an N terminuscysteine (CysFc). CysFc can thus react selectively with anyaldehyde to form this linkage site-specifically at the N-terminus.
In another embodiment, where the immunoglobulin constant region hasan N terminus cysteine, the functional group on the biologicallyactive molecule may be a thioester. In certain embodiments, thethioester may be a C terminus thioester. When the biologicallyactive molecule comprising the thioester is combined with theportion of an immunoglobulin comprising an N terminus cysteinenucleophilic substitution occurs and yields a thioester-linkedintermediate which spontaneously undergoes rearrangement to form anative amide bond at the ligation site.
Nucleic acids, e.g., DNA, RNA may be chemically synthesized toprovide one thioester, see McPherson et al. 1999, Synlett. S1:978.The nucleic acid can be coupled with a thiophosphate at the 5' endand then reacted with bromo-acetylated thioester to generate a 5'thioester. The 5' thioester could in turn be reacted with a portionof an immunoglobulin constant region comprising an N terminuscysteine, e.g., Cys-Fc.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins which combines chemical synthesiswith recombinant synthesis (described below). Thus, thebiologically active molecule may be synthesized chemically orrecombinantly. Similarly, the portion of the immunoglobulin may besynthesized chemically or recombinantly. Once both individualcomponents are synthesized they may be combined chemically tosynthesize the chimeric protein of the invention.
A combination of chemical and recombinant synthesis may be usedwhere it is desirable to link a biologically active molecule to theC terminus of a portion of an immunoglobulin. In this embodiment,the portion of an immunoglobulin constant region, e.g., the Fcregion, is expressed as a fusion protein comprising a proteinsplicer which forms a thioester intermediate, e.g., intein, linkedto the C terminus of the portion of an immunoglobulin constantregion. Commercially available vectors (PCYB2-IMPACT) (New EnglandBiolabs, Beverly, Mass.) provide a cloning cite adjacent to amutant form of intein which is upstream of a chitin binding domain.The mutant intein does not splice the fusion protein, but does formthe thioester intermediate. In one embodiment, the portion of theimmunoglobulin with the intein linked to its C terminus can beexpressed in a prokaryotic cell. In another embodiment, the portionof the immunoglobulin with the intein linked to its C terminus canbe expressed in a eukaryotic cell. The fusion protein may beisolated using chitin linked to a solid support. Addition of athiol cofactor, such as thiophenol or MESNA, and a biologicallyactive molecule with a N-terminus cysteine allows for the linkageof a biologically active molecule to the C terminus thioester ofthe portion of an immunoglobulin. The biologically active moleculeand portion of an immunoglobulin may be reacted together such thatnucleophilic rearrangement occurs and the biologically activemolecule is covalently linked to the portion of an immunoglobulinvia an amide bond. (Dawsen et al. 2000, Annu. Rev. Biochem.69:923).
Where the biological molecule of interest is a nucleic acidmolecule, e.g., a DNA molecule, phosphoramidite can be used tointroduce a terminal cysteine residue to the DNA. The DNA can thenbe linked to the Fc-thioester generated as described above.
Chemical synthesis may be used to synthesize any of the chimericproteins of the invention, including monomer-dimer hybrids, dimersand dimerically linked monomer-dimer hybrids. Chemical synthesismay be used to synthesize a chimeric protein of the inventioncomprising any biologically active molecule including apolypeptide, a nucleic acid, or a small molecule. In someembodiments, chemical synthesis may be used to synthesize achimeric protein comprising an Fc fragment of an immunoglobulin.
In one embodiment, the portion of an immunoglobulin constant regionligated to the biologically active molecule will form homodimers.The homodimers may be isolated. Alternatively, the homodimers canbe disrupted by exposing the homodimers to denaturing and reducingconditions (e.g. beta-mercaptoethanol and 8 M urea) and thensubsequently combined with a portion of an immunoglobulin constantregion not linked to a biologically active molecule to formmonomer-dimer hybrids. The monomer-dimer hybrids are then renaturedand refolded by dialyzing into PBS and isolated, e.g., by sizeexclusion or affinity chromatography.
In another embodiment, the portion of an immunoglobulin constantregion will form homodimers before being linked to a biologicallyactive molecule. In this embodiment, reaction conditions forlinking the biologically active molecule to the homodimer can beadjusted such that linkage of the biologically active molecule toonly one chain of the homodimer is favored (e.g. by adjusting themolar equivalents of each reactant).
The chimeric protein chemically synthesized can optionally includea linker peptide between the portion of an immunoglobulin and thebiologically active molecule. Any linker known in the art may beused. The linker may for example be linked to the N terminus of thebiologically active molecule, where the biologically activemolecule is a polypeptide. Linkers can include peptides and/ororganic molecules (e.g. polyethylene glycol and/or short amino acidsequences). The linker may be a branching molecule that facilitatesthe bonding of multiple copies of the biologically active moleculeto the portion of an immunoglobulin constant region. Alternatively,the linker may be a branching molecule that facilitates the bondingof one biologically active molecule to more than one portion of animmunoglobulin constant region.
Any of the chemical synthesis techniques described herein can beperformed by linking at least one of the biologically activemolecule and the portion of an immunoglobulin constant region to asolid support. As an example an amino-Spherilose.TM. (Isco,Lincoln, Nebr.) may be derivatized with Boc-aminooxyacetic acid.Other resins which may be used as the solid support include EAHSepharose (Pharmacia, NY, N.Y.), Amino PEGA (Novabiochem, SanDiego, Calif.), CLEAR base resin (Peptides International,Louisville, Ky.), long chain alkylamine controlled pore glass(Sigma, St. Louis, Mo.), HCl.PEG polystyrene (PerSeptiveBiosystems, Waltham, Mass.), Lysine Hyper D resin (Biosepra,Freemont, Calif.), ArgoGel Base resin (Argonaut Technologies,Foster City, Calif.). These resins are available inamino-derivatized form or are readily converted to anamino-derivatized form to facilitate coupling.
2. Recombinant Synthesis
Nucleic acids encoding a biologically active molecule can bereadily synthesized using recombinant techniques well known in theart. Alternatively, the peptides themselves can be chemicallysynthesized. Nucleic acids of the invention may be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch,Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al.1988, Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotidescan be prepared by use of controlled pore glass polymer supports asdescribed in Sarin et al. 1988, Proc. Natl. Acad. Sci. USA 85:7448.Additional methods of nucleic acid synthesis are known in the art.(see e.g. U.S. Pat. Nos. 6,015,881; 6,281,331; 6,469,136).
DNA sequences encoding immunoglobulin constant regions, orfragments thereof, may be cloned from a variety of genomic or cDNAlibraries known in the art. The techniques for isolating such DNAsequences using probe-based methods are conventional techniques andare well known to those skilled in the art. Probes for isolatingsuch DNA sequences may be based on published DNA sequences (see,e.g., Hieter et al. 1980, Cell 22:197-207). The polymerase chainreaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No.4,683,195) and Mullis (U.S. Pat. No. 4,683,202) may be used. Thechoice of library and selection of probes for the isolation of suchDNA sequences is within the level of ordinary skill in the art.Alternatively, DNA sequences encoding immunoglobulins or fragmentsthereof can be obtained from vectors known in the art to containimmunoglobulins or fragments thereof.
For recombinant production, a first polynucleotide sequenceencoding a portion of the chimeric protein of the invention (e.g. aportion of an immunoglobulin constant region) and a secondpolynucleotide sequence encoding a portion of the chimeric proteinof the invention (e.g. a portion of an immunoglobulin constantregion and a biologically active molecule) are inserted intoappropriate expression vehicles, i.e. vectors which contains thenecessary elements for the transcription and translation of theinserted coding sequence, or in the case of an RNA viral vector,the necessary elements for replication and translation. The nucleicacids encoding the chimeric protein are inserted into the vector inproper reading frame.
The expression vehicles are then transfected or co-transfected intoa suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are notlimited to, calcium phosphate precipitation (Wigler et al. 1978,Cell 14:725) and electroporation (Neumann et al. 1982, EMBO, J.1:841), and liposome based reagents. A variety of host-expressionvector systems may be utilized to express the chimeric proteinsdescribed herein including both prokaryotic or eukaryotic cells.These include, but are not limited to, microorganisms such asbacteria (e.g. E. coli) transformed with recombinant bacteriophageDNA or plasmid DNA expression vectors containing an appropriatecoding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing anappropriate coding sequence; insect cell systems infected withrecombinant virus expression vectors (e.g. baculovirus) containingan appropriate coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g. cauliflower mosaic virusor tobacco mosaic virus) or transformed with recombinant plasmidexpression vectors (e.g. Ti plasmid) containing an appropriatecoding sequence; or animal cell systems, including mammalian cells(e.g. CHO, Cos, HeLa cells).
When the chimeric protein of the invention is recombinantlysynthesized in a prokaryotic cell it may be desirable to refold thechimeric protein. The chimeric protein produced by this method canbe refolded to a biologically active conformation using conditionsknown in the art, e.g., reducing conditions and then dialyzedslowly into PBS.
Depending on the expression system used, the expressed chimericprotein is then isolated by procedures well-established in the art(e.g. affinity chromatography, size exclusion chromatography, ionexchange chromatography).
The expression vectors can encode for tags that permit for easypurification of the recombinantly produced chimeric protein.Examples include, but are not limited to vector pUR278 (Ruther etal. 1983, EMBO J. 2:1791) in which the chimeric protein describedherein coding sequences may be ligated into the vector in framewith the lac z coding region so that a hybrid protein is produced;pGEX vectors may be used to express chimeric proteins of theinvention with a glutathione S-transferase (GST) tag. Theseproteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution inthe presence of free glutathione. The vectors include cleavagesites (thrombin or Factor Xa protease or PreScission Protease.TM.(Pharmacia, Peapack, N.J.)) for easy removal of the tag afterpurification.
To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the chimeric protein of theinvention separated by enzymatic cleavage sites. The resultingpolypeptide can be cleaved (e.g. by treatment with the appropriateenzyme) in order to recover the polypeptide units. This canincrease the yield of polypeptides driven by a single promoter.When used in appropriate viral expression systems, the translationof each polypeptide encoded by the mRNA is directed internally inthe transcript; e.g., by an internal ribosome entry site, IRES.Thus, the polycistronic construct directs the transcription of asingle, large polycistronic mRNA which, in turn, directs thetranslation of multiple, individual polypeptides. This approacheliminates the production and enzymatic processing of polypeptidesand may significantly increase yield of polypeptide driven by asingle promoter.
Vectors used in transformation will usually contain a selectablemarker used to identify transformants. In bacterial systems, thiscan include an antibiotic resistance gene such as ampicillin orkanamycin. Selectable markers for use in cultured mammalian cellsinclude genes that confer resistance to drugs, such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. One amplifiable selectable marker isthe DHFR gene. Another amplifiable marker is the DHFR cDNA(Simonsen and Levinson 1983, Proc. Natl. Acad. Sci. USA 80:2495).Selectable markers are reviewed by Thilly (Mammalian CellTechnology, Butterworth Publishers, Stoneham, Mass.) and the choiceof selectable markers is well within the level of ordinary skill inthe art.
Selectable markers may be introduced into the cell on a separateplasmid at the same time as the gene of interest, or they may beintroduced on the same plasmid. If on the same plasmid, theselectable marker and the gene of interest may be under the controlof different promoters or the same promoter, the latter arrangementproducing a dicistronic message. Constructs of this type are knownin the art (for example, U.S. Pat. No. 4,713,339).
The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may beused in the expression vector. For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophage.lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the likemay be used; when cloning in insect cell systems, promoters such asthe baculovirus polyhedron promoter may be used; when cloning inplant cell systems, promoters derived from the genome of plantcells (e.g. heat shock promoters; the promoter for the smallsubunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g. the 35S RNA promoter of CaMV;the coat protein promoter of TMV) may be used; when cloning inmammalian cell systems, promoters derived from the genome ofmammalian cells (e.g. metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that containmultiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.
In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the chimericproteins of the invention may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S RNA and 19SRNA promoters of CaMV (Brisson et al. 1984, Nature 310:511-514), orthe coat protein promoter of TMV (Takamatsu et al. 1987, EMBO J.6:307-311) may be used; alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al. 1984, EMBO J. 3:1671-1680;Broglie et al. 1984, Science 224:838-843) or heat shock promoters,e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. 1986, Mol.Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plantvirus vectors, direct DNA transformation, microinjection,electroporation, etc. For reviews of such techniques see e.g.Weissbach & Weissbach 1988, Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.
In one insect expression system that may be used to produce thechimeric proteins of the invention, Autographa californica nuclearpolyhidrosis virus (AcNPV) is used as a vector to express theforeign genes. The virus grows in Spodoptera frugiperda cells. Acoding sequence may be cloned into non-essential regions (forexample, the polyhedron gene) of the virus and placed under controlof an AcNPV promoter (for example, the polyhedron promoter).Successful insertion of a coding sequence will result ininactivation of the polyhedron gene and production of non-occludedrecombinant virus (i.e. virus lacking the proteinaceous coat codedfor by the polyhedron gene). These recombinant viruses are thenused to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see e.g. Smith et al. 1983, J. Virol. 46:584;U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Ausubel et al., eds. 1989, Current Protocolsin Molecular Biology, Vol. 2, Greene Publish. Assoc. & WileyInterscience.
Another system which can be used to express the chimeric proteinsof the invention is the glutamine synthetase gene expressionsystem, also referred to as the "GS expression system"(Lonza Biologics PLC, Berkshire UK). This expression system isdescribed in detail in U.S. Pat. No. 5,981,216.
In mammalian host cells, a number of viral based expression systemsmay be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric genemay then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of theviral genome (e.g. region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing peptide in infectedhosts (see e.g. Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA81:3655). Alternatively, the vaccinia 7.5 K promoter may be used(see e.g. Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415;Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc.Natl. Acad. Sci. USA 79:4927).
In cases where an adenovirus is used as an expression vector, acoding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoterand tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viralgenome (e.g. region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing peptide in infected hosts(see e.g. Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA81:3655). Alternatively, the vaccinia 7.5 K promoter may be used(see e.g. Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415;Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc.Natl. Acad. Sci. USA 79:4927).
Host cells containing DNA constructs of the chimeric protein aregrown in an appropriate growth medium. As used herein, the term"appropriate growth medium" means a medium containingnutrients required for the growth of cells. Nutrients required forcell growth may include a carbon source, a nitrogen source,essential amino acids, vitamins, minerals and growth factors.Optionally the media can contain bovine calf serum or fetal calfserum. In one embodiment, the media contains substantially no IgG.The growth medium will generally select for cells containing theDNA construct by, for example, drug selection or deficiency in anessential nutrient which is complemented by the selectable markeron the DNA construct or co-transfected with the DNA construct.Cultured mammalian cells are generally grown in commerciallyavailable serum-containing or serum-free media (e.g. MEM, DMEM).Selection of a medium appropriate for the particular cell line usedis within the level of ordinary skill in the art.
The recombinantly produced chimeric protein of the invention can beisolated from the culture media. The culture medium fromappropriately grown transformed or transfected host cells isseparated from the cell material, and the presence of chimericproteins is demonstrated. One method of detecting the chimericproteins, for example, is by the binding of the chimeric proteinsor portions of the chimeric proteins to a specific antibodyrecognizing the chimeric protein of the invention. An anti-chimericprotein antibody may be a monoclonal or polyclonal antibody raisedagainst the chimeric protein in question. For example, the chimericprotein contains at least a portion of an immunoglobulin constantregion. Antibodies recognizing the constant region of manyimmunoglobulins are known in the art and are commerciallyavailable. An antibody can be used to perform an ELISA or a westernblot to detect the presence of the chimeric protein of theinvention.
The chimeric protein of the invention can be synthesized in atransgenic animal, such as a rodent, cow, pig, sheep, or goat. Theterm "transgenic animals" refers to non-human animalsthat have incorporated a foreign gene into their genome. Becausethis gene is present in germline tissues, it is passed from parentto offspring. Exogenous genes are introduced into single-celledembryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438).Methods of producing transgenic animals are known in the art,including transgenics that produce immunoglobulin molecules (Wagneret al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; McKnight et al.1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie etal. 1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology59:831; Robl et al. 2003, Theriogenology 59:107; Malassagne et al.2003, Xenotransplantation 10(3):267).
D. Improvements Offered by Certain Embodiments of the Invention
Recombinant technology provides a fast and relatively inexpensiveway to produce large quantities of chimeric proteins, however thetechnology is not without its limitations. For example largemulti-domain proteins can be difficult to express recombinantly.Recombinant expression of chimeric proteins often results in aheterogenous product requiring extensive purification. Somechimeric proteins may be toxic to cells making their expression,difficult, if not impossible. Moreover, recombinantly expressedproteins can only be comprised of the naturally occurring 20 aminoacids. Thus, only L-configuration amino acids are possible usingrecombinant methods. Expressing chimeric proteins comprised ofnon-naturally occurring amino acids, provides a way to generateanalogs useful in studying protein function and inhibitingundesirable metabolic pathways. Alternatively, analogs comprisingnon-naturally occurring amino acids may be used in some cases toenhance the activity of desirable metabolic pathways. Lastly,chimeric proteins comprising both amino acids and anotherbiologically active molecules, e.g., nucleic acids, smallmolecules, are impossible to express using recombinant technologyalone.
Many of the limitations described above may be overcome usingchemical synthesis alone or a combination of recombinant techniquesand chemical synthesis. A number of traditional techniques forchemically synthesizing proteins, such as solid phase synthesis areknown in the art, see, e.g., Merrifield, 1973, ChemicalPolypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61;Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985,Biochem. Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.)2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257; U.S. Pat.No. 3,941,763.
Recent improvements in the chemical synthesis of proteins includethe advent of native chemical ligation. As initially described,native ligation provides for the rapid synthesis of largepolypeptides with a natural peptide backbone via the nativechemical ligation of two or more unprotected peptide segments. Innative ligation none of the reactive functionalities on the peptidesegments need to be temporarily masked by a protecting group.Native ligation also allows for the solid phase sequential chemicalligation of peptide segments in an N-terminus to C-terminusdirection, with the first solid phase-bound unprotected peptidesegment bearing a C-terminal alpha-thioester that reacts withanother unprotected peptide segment containing an N-terminalcysteine. Native chemical ligation also permits the solid-phaseligation in the C- to N-terminus direction, with temporaryprotection of N-terminal cysteine residues on an incoming (second)peptide segment (see, e.g., U.S. Pat. No. 6,326,468; WO 02/18417).Native ligation may also be combined with recombinant technologyusing intein linked to a chitin binding domain (Muir et al., 1998,Proc. Natl. Acad. Sci. USA, 95:6705).
The invention provides for chimeric proteins (monomer-dimerhybrids) comprising a first and a second polypeptide chain, whereinsaid first chain comprises a biologically active molecule and atleast a portion of an immunoglobulin constant region, and saidsecond chain comprises at least a portion of an immunoglobulinconstant region without any biologically active molecule orvariable region of an immunoglobulin. FIG. 1 (see Original Patent)contrasts traditional fusion protein dimers with one example of themonomer-dimer hybrid of the invention. In this example, thebiologically active molecule is EPO and the portion of animmunoglobulin is IgG Fc region.
Like other chimeric proteins comprised of at least a portion of animmunoglobulin constant region, the invention provides for chimericproteins which afford enhanced stability and increasedbioavailability of the chimeric protein compared to thebiologically active molecule alone. Additionally, however, becauseonly one of the two chains comprises the biologically activemolecule, the chimeric protein has a lower molecular weight than achimeric protein wherein all chains comprise a biologically activemolecule and while not wishing to be bound by any theory, this mayresult in the chimeric protein being more readily transcytosedacross the epithelium barrier, e.g., by binding to the FcRnreceptor thereby increasing the half-life of the chimeric protein.In one embodiment, the invention thus provides for an improvednon-invasive method (e.g. via any mucosal surface, such as, orally,buccally, sublingually, nasally, rectally, vaginally, or viapulmonary or occular route) of administering a therapeutic chimericprotein of the invention. The invention thus provides methods ofattaining therapeutic levels of the chimeric proteins of theinvention using less frequent and lower doses compared topreviously described chimeric proteins (e.g. chimeric proteinscomprised of at least a portion of an immunoglobulin constantregion and a biologically active molecule, wherein all chains ofthe chimeric protein comprise a biologically active molecule).
In another embodiment, the invention provides an invasive method,e.g., subcutaneously, intravenously, of administering a therapeuticchimeric protein of the invention. Invasive administration of thetherapeutic chimeric protein of the invention provides for anincreased half life of the therapeutic chimeric protein whichresults in using less frequent and lower doses compared topreviously described chimeric proteins (e.g. chimeric proteinscomprised of at least a portion of an immunoglobulin constantregion and a biologically active molecule, wherein all chains ofthe chimeric protein comprise a biologically active molecule).
Yet another advantage of a chimeric protein wherein only one of thechains comprises a biologically active molecule is the enhancedaccessibility of the biologically active molecule for its targetcell or molecule resulting from decreased steric hindrance,decreased hydrophobic interactions, decreased ionic interactions,or decreased molecular weight compared to a chimeric proteinwherein all chains are comprised of a biologically active molecule.
Claim 1 of 26 Claims
1. A method of producing a chimeric protein comprising combining(a) at least a portion of an immunoglobulin constant regioncomprising an FcRn binding site and having a naturally occurringcysteine from the interchain region of the immunoglobulin as theN-terminal amino acid; and (b) a biologically active moleculecomprising a functional group capable of reacting with an Nterminal cysteine; wherein the combining of (a) and (b) producesthe chimeric protein.
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Abstract
The invention provides methods of chemically synthesizing chimericproteins comprising at least a portion of an immunoglobulinconstant region and a biologically active molecule.
Description of the Invention
SUMMARY OF THE INVENTION
The invention provides a method of chemically synthesizing chimericproteins comprising combining at least one biologically activemolecule and a portion of an immunoglobulin constant region suchthat a bond forms between the biologically active molecule and theportion of an immunoglobulin constant region where the biologicallyactive molecule comprises a first functional group or moiety andthe portion of an immunoglobulin constant region comprises a secondfunctional group or moiety, and where the first and secondfunctional group or moiety are capable of reacting with each otherto form a chemical bond. In certain embodiments, the inventionprovides a method of chemically synthesizing chimeric proteins byperforming native ligation (U.S. Pat. No. 6,184,344) such that abond forms between at least one biologically active molecule and aportion of an immunoglobulin constant region.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins comprising combining at least onebiologically active molecule and at least a portion of animmunoglobulin constant region, wherein a) the portion of animmunoglobulin constant region comprises an amino (N) terminuscysteine and b) the biologically active molecule comprises afunctional group capable of reacting with an N terminus cysteine toform a bond. In certain embodiments, the functional group is athioester. In certain embodiments, e.g., where the biologicallyactive molecule is a polypeptide, the thioester may be a carboxy(C) terminus thioester. In other embodiments, the thioester is nota carboxy thioester. In certain embodiments, the functional groupis an aldehyde. In certain embodiments, e.g., where thebiologically active molecule is a polypeptide, the aldehyde may bea carboxy (C) terminus aldehyde. In other embodiments, the aldehydeis not a C terminus aldehyde.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins comprising the steps of a)recombinantly expressing a fusion protein comprising at least aportion of an immunoglobulin constant region and a splicing proteincapable of forming a C terminus thioester on the portion of animmunoglobulin constant region; b) adding a thiol cofactor to thefusion protein of a); c) adding at least one biologically activemolecule having an N terminal cysteine, thereby synthesizing thechimeric protein. In some embodiments, the splicing protein isintein or a mutant form of intein which is defective in completionof the splicing reaction, but is still capable of thioesterintermediate formation. In some embodiments, the fusion protein isfurther comprised of a chitin binding domain.
In certain embodiments, the chemical synthesis is performed insolution. In some embodiments, at least one of the reactants islinked to a solid support. The biologically active molecule may belinked to the solid support. The portion of an immunoglobulinconstant region may be linked to a solid support. The fusionprotein comprising the portion of an immunoglobulin and thesplicing protein may be linked to a solid support.
DESCRIPTION OF THE EMBODIMENTS
Synthesis of Chimeric Proteins
Chimeric proteins comprising at least a portion of animmunoglobulin constant region and a biologically active moleculecan be synthesized using techniques well known in the art. Forexample, the chimeric proteins of the invention may be synthesizedrecombinantly in cells (see e.g. Sambrook et al. 1989, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.and Ausubel et al. 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).Alternatively, the chimeric proteins of the invention may besynthesized using known synthetic methods such as native ligation(U.S. Pat. No. 6,326,468) or solid phase synthesis (see e.g.Merrifield, 1973, Chemical Polypeptides, (Katsoyannis and Panayotiseds.) pp. 335-61; Merrifield 1963, J. Am. Chem. Soc. 85:2149; Daviset al. 1985, Biochem. Intl. 10:394; Finn et al. 1976, The Proteins(3d ed.) 2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257;U.S. Pat. No. 3,941,763. Alternatively, the chimeric proteins ofthe invention may be synthesized using a combination of recombinantand synthetic methods. In certain applications, it may bebeneficial to use either a recombinant method or a combination ofrecombinant and synthetic methods.
Combining recombinant and chemical synthesis allows for the rapidscreening of biologically active molecules and linkers to optimizedesired properties of the chimeric protein of the invention, e.g.,viral inhibition, hemostasis, production of red blood cells,biological half-life, stability, binding to serum proteins or someother property of the chimeric protein. The method also allows forthe incorporation of non-natural amino acids into the chimericprotein of the invention which may be useful for optimizing adesired property of the chimeric protein of the invention.
1. Chemical Synthesis
In certain embodiments, the invention provides a method ofsynthesizing a chimeric protein of the invention comprising atleast one biologically active molecule and at least a portion of animmunoglobulin constant region, or fragment thereof, where one ofeither the biologically active molecule or the portion of animmunoglobulin constant region may comprise an N terminus cysteineand the other comprises a functional group capable of reactingspecifically with the N terminal cysteine. The biologically activemolecule may include a polypeptide. The biologically activemolecule may include a small organic molecule or a small inorganicmolecule. The biologically active molecule include a nucleic acid.
In one embodiment, the N terminal cysteine is on the portion of animmunoglobulin constant region. In one embodiment, the portion ofan immunoglobulin constant region is an Fc fragment. The Fcfragment can be recombinantly produced to form cysteine-Fc (Cys-Fc)and reacted with at least one biologically active moleculeexpressing a thioester to make a chimeric protein of the invention,e.g., monomer-dimer hybrid. In another embodiment, an Fc-thioesteris made and reacted with at least one biologically active moleculeexpressing an N terminus cysteine.
In one embodiment, the N-terminal cysteine may be on the portion ofan immunoglobulin constant region, e.g., an Fc fragment. An Fcfragment can be generated with an N-terminal cysteine by takingadvantage of the fact that a native Fc has a cysteine at positions220, 226 and 229 (see Kabat et al. 1991, Sequences of Proteins ofImmunological Interest, U.S. Department of Public Health, Bethesda,Md.). Any of these cysteines may be used to generate an Fc fragmentfor use in the methods described herein. Additionally, recombinanttechniques can be used to generate Fc fragments having at least onenon-native (i.e. engineered by humans) N terminus cysteine. Forexample, cysteines can be added on at positions higher than 226, orlower than 220, of the EU numbering system.
In a specific embodiment, an Fc fragment is expressed with thehuman .alpha. interferon signal peptide adjacent to the Cys atposition 226. When a construct encoding this polypeptide isexpressed in CHO cells, the CHO cells cleave the signal peptide attwo distinct positions (at Cys 226 and at Val within the signalpeptide 2 amino acids upstream in the N terminus direction). Thisgenerates a mixture of two species of Fc fragments (one with anN-terminal Val and one with an N-terminal Cys). This in turnresults in a mixture of dimeric species (homodimers with terminalVal, homodimers with terminal Cys and heterodimers where one chainhas a terminal Cys and the other chain has a terminal Val) (FIG. 4A(see Original Patent)). The Fc fragments can be reacted with atleast one biologically active molecule having a C terminalthioester and the resulting monomer-dimer hybrid or dimer can beisolated from the mixture (e.g. by size exclusion chromatography).It is contemplated that when other signal peptide sequences areused for expression of Fc fragments in CHO cells a mixture ofspecies of Fc fragments with at least two different N termini willbe generated.
Cys-Fc may be recombinantly expressed. In one embodiment, the Fcfragment is expressed in a prokaryotic cell, e.g., E. coli. Thesequence encoding the Fc portion beginning with Cys 226 (EUnumbering) can be placed immediately following a sequence encodinga signal peptide, e.g., OmpA, PhoA, STII. The prokaryotic cell canbe osmotically shocked to release the recombinant Fc fragment. Inanother embodiment, the Fc fragment is produced in a eukaryoticcell, e.g., a CHO cell, a BHK cell. The sequence encoding the Fcportion fragment can be placed directly following a sequenceencoding a signal peptide, e.g., mouse IgK light chain or MHC class1 Kb signal sequence, such that when the recombinant chimericprotein is synthesized by a eukaryotic cell, the signal sequencewill be cleaved, leaving an N terminal cysteine which can than beisolated and chemically reacted with a molecule bearing a thioester(e.g. a C terminal thioester if the molecule is comprised of aminoacids).
The N terminal cysteine on an Fc fragment can also be generatedusing an enzyme that cleaves its substrate at its N terminus, e.g.,Factor X.sup.a, enterokinase, and the product isolated and reactedwith a molecule with a thioester.
In some embodiments, a recombinantly produced Cys-Fc can form ahomodimer. The homodimer may be reacted with peptide that has abranched linker on the C terminus, wherein the branched linker hastwo C terminal thioesters that can be reacted with the Cys-Fc, thusforming a dimerically linked monomer dimer hybrid (FIG. 4 (seeOriginal Patent)). In another embodiment, the biologically activemolecule may have a single non-terminal thioester that can bereacted with Cys-Fc.
In some embodiments, the branched linker can have two C terminalcysteines that can be reacted with an Fc thioester. In anotherembodiment, the branched linker has two functional groups that canbe reacted with the Fc thioester, e.g., 2-mercaptoamine.
Where the portion of the immunoglobulin constant region has an Nterminus cysteine, the functional group on the biologically activemolecule may be an aldehyde. If necessary, the aldehydefunctionality can be chemically synthesized for example byoxidation of an N-terminal serine with periodate (see, e.g.,Georghagen et al., 1992, Bioconjugate Chem. 3:138). Alternatively,where the biologically active molecule is a DNA molecule, it maylabeled with an aldehyde group by first coupling the DNA, duringthe last round of synthesis, with phosphoramidite thus generating aprotected amine. After deprotection, the DNA molecule labeled witha free amine may be generated. The amine could be coupled tosuccinimidyl 4-formylbenzoate (as shown below) to generate thealdehyde. This could be done on either its 3' or 5' end.
Another way of introducing an aldehyde into an oligonucleotide isdescribed in Zatsepin et al., 2002, Bioconjugate Chem, 13:822. Aprotected 1,2-diol may be incorporated in the 2' position. Afterthe synthesis of the oligo, the protecting groups can be removed toreveal the free 1,2-diol, which can then be oxidized with periodateto give an aldehyde.
Aldehydes are known to react with N-terminal cysteines (in effect,a 1,2-amino thiol moiety) to form thiazolidines. (See Botti, 1996,J. Am Chem. Soc., 118:10018; Zhang et al., 1998, Proc. Nat. Acad.Sci. USA, 95:9184). In one embodiment, the portion of animmunoglobulin constant region is an Fc fragment with an N terminuscysteine (CysFc). CysFc can thus react selectively with anyaldehyde to form this linkage site-specifically at the N-terminus.
In another embodiment, where the immunoglobulin constant region hasan N terminus cysteine, the functional group on the biologicallyactive molecule may be a thioester. In certain embodiments, thethioester may be a C terminus thioester. When the biologicallyactive molecule comprising the thioester is combined with theportion of an immunoglobulin comprising an N terminus cysteinenucleophilic substitution occurs and yields a thioester-linkedintermediate which spontaneously undergoes rearrangement to form anative amide bond at the ligation site.
Nucleic acids, e.g., DNA, RNA may be chemically synthesized toprovide one thioester, see McPherson et al. 1999, Synlett. S1:978.The nucleic acid can be coupled with a thiophosphate at the 5' endand then reacted with bromo-acetylated thioester to generate a 5'thioester. The 5' thioester could in turn be reacted with a portionof an immunoglobulin constant region comprising an N terminuscysteine, e.g., Cys-Fc.
In certain embodiments, the invention provides a method ofsynthesizing chimeric proteins which combines chemical synthesiswith recombinant synthesis (described below). Thus, thebiologically active molecule may be synthesized chemically orrecombinantly. Similarly, the portion of the immunoglobulin may besynthesized chemically or recombinantly. Once both individualcomponents are synthesized they may be combined chemically tosynthesize the chimeric protein of the invention.
A combination of chemical and recombinant synthesis may be usedwhere it is desirable to link a biologically active molecule to theC terminus of a portion of an immunoglobulin. In this embodiment,the portion of an immunoglobulin constant region, e.g., the Fcregion, is expressed as a fusion protein comprising a proteinsplicer which forms a thioester intermediate, e.g., intein, linkedto the C terminus of the portion of an immunoglobulin constantregion. Commercially available vectors (PCYB2-IMPACT) (New EnglandBiolabs, Beverly, Mass.) provide a cloning cite adjacent to amutant form of intein which is upstream of a chitin binding domain.The mutant intein does not splice the fusion protein, but does formthe thioester intermediate. In one embodiment, the portion of theimmunoglobulin with the intein linked to its C terminus can beexpressed in a prokaryotic cell. In another embodiment, the portionof the immunoglobulin with the intein linked to its C terminus canbe expressed in a eukaryotic cell. The fusion protein may beisolated using chitin linked to a solid support. Addition of athiol cofactor, such as thiophenol or MESNA, and a biologicallyactive molecule with a N-terminus cysteine allows for the linkageof a biologically active molecule to the C terminus thioester ofthe portion of an immunoglobulin. The biologically active moleculeand portion of an immunoglobulin may be reacted together such thatnucleophilic rearrangement occurs and the biologically activemolecule is covalently linked to the portion of an immunoglobulinvia an amide bond. (Dawsen et al. 2000, Annu. Rev. Biochem.69:923).
Where the biological molecule of interest is a nucleic acidmolecule, e.g., a DNA molecule, phosphoramidite can be used tointroduce a terminal cysteine residue to the DNA. The DNA can thenbe linked to the Fc-thioester generated as described above.
Chemical synthesis may be used to synthesize any of the chimericproteins of the invention, including monomer-dimer hybrids, dimersand dimerically linked monomer-dimer hybrids. Chemical synthesismay be used to synthesize a chimeric protein of the inventioncomprising any biologically active molecule including apolypeptide, a nucleic acid, or a small molecule. In someembodiments, chemical synthesis may be used to synthesize achimeric protein comprising an Fc fragment of an immunoglobulin.
In one embodiment, the portion of an immunoglobulin constant regionligated to the biologically active molecule will form homodimers.The homodimers may be isolated. Alternatively, the homodimers canbe disrupted by exposing the homodimers to denaturing and reducingconditions (e.g. beta-mercaptoethanol and 8 M urea) and thensubsequently combined with a portion of an immunoglobulin constantregion not linked to a biologically active molecule to formmonomer-dimer hybrids. The monomer-dimer hybrids are then renaturedand refolded by dialyzing into PBS and isolated, e.g., by sizeexclusion or affinity chromatography.
In another embodiment, the portion of an immunoglobulin constantregion will form homodimers before being linked to a biologicallyactive molecule. In this embodiment, reaction conditions forlinking the biologically active molecule to the homodimer can beadjusted such that linkage of the biologically active molecule toonly one chain of the homodimer is favored (e.g. by adjusting themolar equivalents of each reactant).
The chimeric protein chemically synthesized can optionally includea linker peptide between the portion of an immunoglobulin and thebiologically active molecule. Any linker known in the art may beused. The linker may for example be linked to the N terminus of thebiologically active molecule, where the biologically activemolecule is a polypeptide. Linkers can include peptides and/ororganic molecules (e.g. polyethylene glycol and/or short amino acidsequences). The linker may be a branching molecule that facilitatesthe bonding of multiple copies of the biologically active moleculeto the portion of an immunoglobulin constant region. Alternatively,the linker may be a branching molecule that facilitates the bondingof one biologically active molecule to more than one portion of animmunoglobulin constant region.
Any of the chemical synthesis techniques described herein can beperformed by linking at least one of the biologically activemolecule and the portion of an immunoglobulin constant region to asolid support. As an example an amino-Spherilose.TM. (Isco,Lincoln, Nebr.) may be derivatized with Boc-aminooxyacetic acid.Other resins which may be used as the solid support include EAHSepharose (Pharmacia, NY, N.Y.), Amino PEGA (Novabiochem, SanDiego, Calif.), CLEAR base resin (Peptides International,Louisville, Ky.), long chain alkylamine controlled pore glass(Sigma, St. Louis, Mo.), HCl.PEG polystyrene (PerSeptiveBiosystems, Waltham, Mass.), Lysine Hyper D resin (Biosepra,Freemont, Calif.), ArgoGel Base resin (Argonaut Technologies,Foster City, Calif.). These resins are available inamino-derivatized form or are readily converted to anamino-derivatized form to facilitate coupling.
2. Recombinant Synthesis
Nucleic acids encoding a biologically active molecule can bereadily synthesized using recombinant techniques well known in theart. Alternatively, the peptides themselves can be chemicallysynthesized. Nucleic acids of the invention may be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch,Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al.1988, Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotidescan be prepared by use of controlled pore glass polymer supports asdescribed in Sarin et al. 1988, Proc. Natl. Acad. Sci. USA 85:7448.Additional methods of nucleic acid synthesis are known in the art.(see e.g. U.S. Pat. Nos. 6,015,881; 6,281,331; 6,469,136).
DNA sequences encoding immunoglobulin constant regions, orfragments thereof, may be cloned from a variety of genomic or cDNAlibraries known in the art. The techniques for isolating such DNAsequences using probe-based methods are conventional techniques andare well known to those skilled in the art. Probes for isolatingsuch DNA sequences may be based on published DNA sequences (see,e.g., Hieter et al. 1980, Cell 22:197-207). The polymerase chainreaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No.4,683,195) and Mullis (U.S. Pat. No. 4,683,202) may be used. Thechoice of library and selection of probes for the isolation of suchDNA sequences is within the level of ordinary skill in the art.Alternatively, DNA sequences encoding immunoglobulins or fragmentsthereof can be obtained from vectors known in the art to containimmunoglobulins or fragments thereof.
For recombinant production, a first polynucleotide sequenceencoding a portion of the chimeric protein of the invention (e.g. aportion of an immunoglobulin constant region) and a secondpolynucleotide sequence encoding a portion of the chimeric proteinof the invention (e.g. a portion of an immunoglobulin constantregion and a biologically active molecule) are inserted intoappropriate expression vehicles, i.e. vectors which contains thenecessary elements for the transcription and translation of theinserted coding sequence, or in the case of an RNA viral vector,the necessary elements for replication and translation. The nucleicacids encoding the chimeric protein are inserted into the vector inproper reading frame.
The expression vehicles are then transfected or co-transfected intoa suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are notlimited to, calcium phosphate precipitation (Wigler et al. 1978,Cell 14:725) and electroporation (Neumann et al. 1982, EMBO, J.1:841), and liposome based reagents. A variety of host-expressionvector systems may be utilized to express the chimeric proteinsdescribed herein including both prokaryotic or eukaryotic cells.These include, but are not limited to, microorganisms such asbacteria (e.g. E. coli) transformed with recombinant bacteriophageDNA or plasmid DNA expression vectors containing an appropriatecoding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing anappropriate coding sequence; insect cell systems infected withrecombinant virus expression vectors (e.g. baculovirus) containingan appropriate coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g. cauliflower mosaic virusor tobacco mosaic virus) or transformed with recombinant plasmidexpression vectors (e.g. Ti plasmid) containing an appropriatecoding sequence; or animal cell systems, including mammalian cells(e.g. CHO, Cos, HeLa cells).
When the chimeric protein of the invention is recombinantlysynthesized in a prokaryotic cell it may be desirable to refold thechimeric protein. The chimeric protein produced by this method canbe refolded to a biologically active conformation using conditionsknown in the art, e.g., reducing conditions and then dialyzedslowly into PBS.
Depending on the expression system used, the expressed chimericprotein is then isolated by procedures well-established in the art(e.g. affinity chromatography, size exclusion chromatography, ionexchange chromatography).
The expression vectors can encode for tags that permit for easypurification of the recombinantly produced chimeric protein.Examples include, but are not limited to vector pUR278 (Ruther etal. 1983, EMBO J. 2:1791) in which the chimeric protein describedherein coding sequences may be ligated into the vector in framewith the lac z coding region so that a hybrid protein is produced;pGEX vectors may be used to express chimeric proteins of theinvention with a glutathione S-transferase (GST) tag. Theseproteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution inthe presence of free glutathione. The vectors include cleavagesites (thrombin or Factor Xa protease or PreScission Protease.TM.(Pharmacia, Peapack, N.J.)) for easy removal of the tag afterpurification.
To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the chimeric protein of theinvention separated by enzymatic cleavage sites. The resultingpolypeptide can be cleaved (e.g. by treatment with the appropriateenzyme) in order to recover the polypeptide units. This canincrease the yield of polypeptides driven by a single promoter.When used in appropriate viral expression systems, the translationof each polypeptide encoded by the mRNA is directed internally inthe transcript; e.g., by an internal ribosome entry site, IRES.Thus, the polycistronic construct directs the transcription of asingle, large polycistronic mRNA which, in turn, directs thetranslation of multiple, individual polypeptides. This approacheliminates the production and enzymatic processing of polypeptidesand may significantly increase yield of polypeptide driven by asingle promoter.
Vectors used in transformation will usually contain a selectablemarker used to identify transformants. In bacterial systems, thiscan include an antibiotic resistance gene such as ampicillin orkanamycin. Selectable markers for use in cultured mammalian cellsinclude genes that confer resistance to drugs, such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. One amplifiable selectable marker isthe DHFR gene. Another amplifiable marker is the DHFR cDNA(Simonsen and Levinson 1983, Proc. Natl. Acad. Sci. USA 80:2495).Selectable markers are reviewed by Thilly (Mammalian CellTechnology, Butterworth Publishers, Stoneham, Mass.) and the choiceof selectable markers is well within the level of ordinary skill inthe art.
Selectable markers may be introduced into the cell on a separateplasmid at the same time as the gene of interest, or they may beintroduced on the same plasmid. If on the same plasmid, theselectable marker and the gene of interest may be under the controlof different promoters or the same promoter, the latter arrangementproducing a dicistronic message. Constructs of this type are knownin the art (for example, U.S. Pat. No. 4,713,339).
The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may beused in the expression vector. For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophage.lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the likemay be used; when cloning in insect cell systems, promoters such asthe baculovirus polyhedron promoter may be used; when cloning inplant cell systems, promoters derived from the genome of plantcells (e.g. heat shock promoters; the promoter for the smallsubunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g. the 35S RNA promoter of CaMV;the coat protein promoter of TMV) may be used; when cloning inmammalian cell systems, promoters derived from the genome ofmammalian cells (e.g. metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that containmultiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.
In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the chimericproteins of the invention may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S RNA and 19SRNA promoters of CaMV (Brisson et al. 1984, Nature 310:511-514), orthe coat protein promoter of TMV (Takamatsu et al. 1987, EMBO J.6:307-311) may be used; alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al. 1984, EMBO J. 3:1671-1680;Broglie et al. 1984, Science 224:838-843) or heat shock promoters,e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. 1986, Mol.Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plantvirus vectors, direct DNA transformation, microinjection,electroporation, etc. For reviews of such techniques see e.g.Weissbach & Weissbach 1988, Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.
In one insect expression system that may be used to produce thechimeric proteins of the invention, Autographa californica nuclearpolyhidrosis virus (AcNPV) is used as a vector to express theforeign genes. The virus grows in Spodoptera frugiperda cells. Acoding sequence may be cloned into non-essential regions (forexample, the polyhedron gene) of the virus and placed under controlof an AcNPV promoter (for example, the polyhedron promoter).Successful insertion of a coding sequence will result ininactivation of the polyhedron gene and production of non-occludedrecombinant virus (i.e. virus lacking the proteinaceous coat codedfor by the polyhedron gene). These recombinant viruses are thenused to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see e.g. Smith et al. 1983, J. Virol. 46:584;U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Ausubel et al., eds. 1989, Current Protocolsin Molecular Biology, Vol. 2, Greene Publish. Assoc. & WileyInterscience.
Another system which can be used to express the chimeric proteinsof the invention is the glutamine synthetase gene expressionsystem, also referred to as the "GS expression system"(Lonza Biologics PLC, Berkshire UK). This expression system isdescribed in detail in U.S. Pat. No. 5,981,216.
In mammalian host cells, a number of viral based expression systemsmay be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric genemay then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of theviral genome (e.g. region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing peptide in infectedhosts (see e.g. Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA81:3655). Alternatively, the vaccinia 7.5 K promoter may be used(see e.g. Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415;Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc.Natl. Acad. Sci. USA 79:4927).
In cases where an adenovirus is used as an expression vector, acoding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoterand tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viralgenome (e.g. region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing peptide in infected hosts(see e.g. Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA81:3655). Alternatively, the vaccinia 7.5 K promoter may be used(see e.g. Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415;Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc.Natl. Acad. Sci. USA 79:4927).
Host cells containing DNA constructs of the chimeric protein aregrown in an appropriate growth medium. As used herein, the term"appropriate growth medium" means a medium containingnutrients required for the growth of cells. Nutrients required forcell growth may include a carbon source, a nitrogen source,essential amino acids, vitamins, minerals and growth factors.Optionally the media can contain bovine calf serum or fetal calfserum. In one embodiment, the media contains substantially no IgG.The growth medium will generally select for cells containing theDNA construct by, for example, drug selection or deficiency in anessential nutrient which is complemented by the selectable markeron the DNA construct or co-transfected with the DNA construct.Cultured mammalian cells are generally grown in commerciallyavailable serum-containing or serum-free media (e.g. MEM, DMEM).Selection of a medium appropriate for the particular cell line usedis within the level of ordinary skill in the art.
The recombinantly produced chimeric protein of the invention can beisolated from the culture media. The culture medium fromappropriately grown transformed or transfected host cells isseparated from the cell material, and the presence of chimericproteins is demonstrated. One method of detecting the chimericproteins, for example, is by the binding of the chimeric proteinsor portions of the chimeric proteins to a specific antibodyrecognizing the chimeric protein of the invention. An anti-chimericprotein antibody may be a monoclonal or polyclonal antibody raisedagainst the chimeric protein in question. For example, the chimericprotein contains at least a portion of an immunoglobulin constantregion. Antibodies recognizing the constant region of manyimmunoglobulins are known in the art and are commerciallyavailable. An antibody can be used to perform an ELISA or a westernblot to detect the presence of the chimeric protein of theinvention.
The chimeric protein of the invention can be synthesized in atransgenic animal, such as a rodent, cow, pig, sheep, or goat. Theterm "transgenic animals" refers to non-human animalsthat have incorporated a foreign gene into their genome. Becausethis gene is present in germline tissues, it is passed from parentto offspring. Exogenous genes are introduced into single-celledembryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438).Methods of producing transgenic animals are known in the art,including transgenics that produce immunoglobulin molecules (Wagneret al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; McKnight et al.1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie etal. 1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology59:831; Robl et al. 2003, Theriogenology 59:107; Malassagne et al.2003, Xenotransplantation 10(3):267).
D. Improvements Offered by Certain Embodiments of the Invention
Recombinant technology provides a fast and relatively inexpensiveway to produce large quantities of chimeric proteins, however thetechnology is not without its limitations. For example largemulti-domain proteins can be difficult to express recombinantly.Recombinant expression of chimeric proteins often results in aheterogenous product requiring extensive purification. Somechimeric proteins may be toxic to cells making their expression,difficult, if not impossible. Moreover, recombinantly expressedproteins can only be comprised of the naturally occurring 20 aminoacids. Thus, only L-configuration amino acids are possible usingrecombinant methods. Expressing chimeric proteins comprised ofnon-naturally occurring amino acids, provides a way to generateanalogs useful in studying protein function and inhibitingundesirable metabolic pathways. Alternatively, analogs comprisingnon-naturally occurring amino acids may be used in some cases toenhance the activity of desirable metabolic pathways. Lastly,chimeric proteins comprising both amino acids and anotherbiologically active molecules, e.g., nucleic acids, smallmolecules, are impossible to express using recombinant technologyalone.
Many of the limitations described above may be overcome usingchemical synthesis alone or a combination of recombinant techniquesand chemical synthesis. A number of traditional techniques forchemically synthesizing proteins, such as solid phase synthesis areknown in the art, see, e.g., Merrifield, 1973, ChemicalPolypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61;Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985,Biochem. Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.)2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257; U.S. Pat.No. 3,941,763.
Recent improvements in the chemical synthesis of proteins includethe advent of native chemical ligation. As initially described,native ligation provides for the rapid synthesis of largepolypeptides with a natural peptide backbone via the nativechemical ligation of two or more unprotected peptide segments. Innative ligation none of the reactive functionalities on the peptidesegments need to be temporarily masked by a protecting group.Native ligation also allows for the solid phase sequential chemicalligation of peptide segments in an N-terminus to C-terminusdirection, with the first solid phase-bound unprotected peptidesegment bearing a C-terminal alpha-thioester that reacts withanother unprotected peptide segment containing an N-terminalcysteine. Native chemical ligation also permits the solid-phaseligation in the C- to N-terminus direction, with temporaryprotection of N-terminal cysteine residues on an incoming (second)peptide segment (see, e.g., U.S. Pat. No. 6,326,468; WO 02/18417).Native ligation may also be combined with recombinant technologyusing intein linked to a chitin binding domain (Muir et al., 1998,Proc. Natl. Acad. Sci. USA, 95:6705).
The invention provides for chimeric proteins (monomer-dimerhybrids) comprising a first and a second polypeptide chain, whereinsaid first chain comprises a biologically active molecule and atleast a portion of an immunoglobulin constant region, and saidsecond chain comprises at least a portion of an immunoglobulinconstant region without any biologically active molecule orvariable region of an immunoglobulin. FIG. 1 (see Original Patent)contrasts traditional fusion protein dimers with one example of themonomer-dimer hybrid of the invention. In this example, thebiologically active molecule is EPO and the portion of animmunoglobulin is IgG Fc region.
Like other chimeric proteins comprised of at least a portion of animmunoglobulin constant region, the invention provides for chimericproteins which afford enhanced stability and increasedbioavailability of the chimeric protein compared to thebiologically active molecule alone. Additionally, however, becauseonly one of the two chains comprises the biologically activemolecule, the chimeric protein has a lower molecular weight than achimeric protein wherein all chains comprise a biologically activemolecule and while not wishing to be bound by any theory, this mayresult in the chimeric protein being more readily transcytosedacross the epithelium barrier, e.g., by binding to the FcRnreceptor thereby increasing the half-life of the chimeric protein.In one embodiment, the invention thus provides for an improvednon-invasive method (e.g. via any mucosal surface, such as, orally,buccally, sublingually, nasally, rectally, vaginally, or viapulmonary or occular route) of administering a therapeutic chimericprotein of the invention. The invention thus provides methods ofattaining therapeutic levels of the chimeric proteins of theinvention using less frequent and lower doses compared topreviously described chimeric proteins (e.g. chimeric proteinscomprised of at least a portion of an immunoglobulin constantregion and a biologically active molecule, wherein all chains ofthe chimeric protein comprise a biologically active molecule).
In another embodiment, the invention provides an invasive method,e.g., subcutaneously, intravenously, of administering a therapeuticchimeric protein of the invention. Invasive administration of thetherapeutic chimeric protein of the invention provides for anincreased half life of the therapeutic chimeric protein whichresults in using less frequent and lower doses compared topreviously described chimeric proteins (e.g. chimeric proteinscomprised of at least a portion of an immunoglobulin constantregion and a biologically active molecule, wherein all chains ofthe chimeric protein comprise a biologically active molecule).
Yet another advantage of a chimeric protein wherein only one of thechains comprises a biologically active molecule is the enhancedaccessibility of the biologically active molecule for its targetcell or molecule resulting from decreased steric hindrance,decreased hydrophobic interactions, decreased ionic interactions,or decreased molecular weight compared to a chimeric proteinwherein all chains are comprised of a biologically active molecule.
Claim 1 of 26 Claims
1. A method of producing a chimeric protein comprising combining(a) at least a portion of an immunoglobulin constant regioncomprising an FcRn binding site and having a naturally occurringcysteine from the interchain region of the immunoglobulin as theN-terminal amino acid; and (b) a biologically active moleculecomprising a functional group capable of reacting with an Nterminal cysteine; wherein the combining of (a) and (b) producesthe chimeric protein.
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