Patent Application
20160032344
This application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [DATE], is named [.txt] and is [#######] bytes in size.
The disclosure herein generally relates to the field of glycobiology and protein engineering. More specifically, the embodiments described herein relate to oligosaccharide compositions and production of therapeutic glycoproteins in recombinant hosts.
Protein and peptide drugs have had a huge clinical impact and constitute a $70 billion market. Unfortunately, the efficacy of protein drugs is often compromised by limitations arising from proteolytic degradation, uptake by cells of the reticuloendothelial system, renal removal, and immunocomplex formation. This can lead to elimination from the blood before effective concentrations are reached, and can result in unacceptably short therapeutic windows. The predominant factors that contribute to these pharmacokinetic limitations are stability and immunogenicity. Efforts have been made to address these problems, including changing the primary structure, conjugating glycans or polymers to the protein, or entrapping the protein in nanoparticles to improve residence time and reduce immunogenicity. The most popular approach to date has been conjugation to monomethoxy poly(ethyleneglycol) (mPEG) commonly referred to as PEGylation. PEGylation can endow protein and peptide drugs with longer circulatory half-lives and reduce immunogenicity. A number of PEGylated drugs are now used clinically (e.g., asparaginase, interferon α, tumor necrosis factor and granulocyte-colony stimulating factor). However, PEG is not biodegradable via normal detoxification mechanisms and the administration of PEGylated proteins has been found to elicit anti-PEG antibodies.
PEGylation is a well-accepted approach to enhance stability and reduce immunogenicity, whereby protein is conjugated to poly(ethyleneglycol) (PEG) [1]. Such PEGylation involves the covalent attachment of either linear or branched chains of PEG via a chemically reactive side-chain, such as a hydroxysuccinimidylester or an aldehyde group, for linking to either the α or ε amino groups on the protein [2]. PEGylation can endow protein and peptide drugs with longer circulatory half-lives and reduced immunogenicity, as PEG is water-soluble and increases the size of the protein and reduces proteolytic cleavage by occluding cleavage sites [1]. The value of PEGylation was demonstrated for several proteins, including: (i) asparaginase [3], an enzyme used in the treatment of leukemia, and (ii) adenosine deaminase [4], which participates in purine metabolism. PEGylation was also used to enhance the activity of immunological factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) [5], tumor necrosis factor (TNF), interferon α-2a (IFN α-2a) and IFN α-2b [1]. While PEGylation is a chemical modification that can enhance pharmacokinetic properties, it is not without drawbacks. First, the heterogeneity of PEGylation yields many different isoforms of varying biological activity. This is primarily a result of the polydisperse nature of the polymer. Second, concerns have been raised about introducing a synthetic polymer into the human body that does not appear to be completely biodegradable [6]. Third, the extended half-life of PEGylated proteins that is often observed can be accompanied by reduced biological activity related to the structural change in the molecules as a result of conjugation [2]. Fourth, the process of PEGylation is expensive and requires several in vitro chemical reactions and multiple purifications [7]. Thus, while PEGylation has been clinically proven as a method to increase circulatory half-lives and reduce immunogenicity, clearly it is not the optimal solution.
An emerging clinical alternative to PEGylation is polysialylation which involves attachment of a polymer of natural N-acetylneuraminic acid (polysialic acid or PSA) to the protein. PSA is highly hydrophilic with similar hydration properties to PEG, is inconspicuous to the innate and adaptive immune systems, and is naturally synthesized and displayed on human cells. PSA has recently been developed for clinical use with polysialylated versions of insulin and erythropoietin each displaying improved tolerance and pharmacokinetics. Unfortunately, as with PEGylation, the PSA conjugation process is technically challenging and expensive making the final product cost prohibitive to the healthcare consumer. PSA conjugation requires the separate production and purification of the target protein and PSA, as well as the in vitro reductive amination of the nonreducing end of PSA to allow chemical linkage to primary amine groups on the protein.
PSA conjugation has proven to be a very effective method to increase the active life of therapeutic proteins and prevent them from being recognized by the immune system. PSA conjugation has several performance advantages over PEGylation and is currently being tested in the clinic.
Molecules that are inconspicuous to the innate and adaptive immune systems are likely to survive for prolonged periods in circulation. Polysialic acid (PSA; polymers of N-acetylneuraminic (sialic) acid) is one such molecule and offers a natural alternative to PEG as a conjugate that can modify serum persistence of proteins. PSA is a human polymer found almost exclusively on neural cell adhesion molecule (NCAM) where it has an antiadhesive function in brain development [8]. When used for protein and therapeutic peptide drug delivery, conjugated PSA provides a protective microenvironment. This increases the active life of the therapeutic protein in circulation and prevents it from being recognized by the immune system. Unlike PEG, PSA is metabolized as a natural sugar molecule by tissue sialidases [9]. The highly hydrophilic nature of PSA results in similar hydration properties to PEG, giving it a high apparent molecular weight in the blood. This increases circulation time since no receptors with PSA specificity have been identified to date [10].
While PSA is naturally found in the human body, it is also synthesized as a capsule by bacteria such as Neisseria meningitidis and certain strains of E. coli [11]. These polysialylated bacteria use molecular mimicry to evade the defense systems of the human body. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, and is chemically identical to PSA in the human body to the extent that PSA has been developed for clinical use. Reductive amination of the nonreducing end of oxidized PSA allows in vitro chemical conjugation via primary amine groups on proteins, and the therapeutic benefits of PSA conjugation have been demonstrated with asparaginase [12] and insulin [13] for the treatment of leukemia and diabetes, respectively. Recent clinical data from trials with polysialylated insulin and erythropoietin showed that these biopharmaceuticals were well tolerated with enhanced pharmacokinetics [14]. Recently, two exciting discoveries have increased enthusiasm for PSA conjugation. First, it was observed that chemically polysialylated antitumor Fab fragments resulted in a 5-fold increase in bioavailability with a corresponding 3-fold increase in tumor uptake compared to unmodified Fab [15]. Second, site-specific (rather than random) coupling of PSA to engineered C-terminal thiols lead to antibody fragments with full immunoreactivity, increased blood half-life, higher tumor uptake, and improved specificity ratios [14]. PSA conjugation may add significant therapeutic value and polysialylated antibody fragments may be a viable alternative to whole IgGs by improving serum half-life and ameliorating concerns associated with Fc-domains.
Unfortunately, even PSA conjugation is not without its drawbacks. While effective in a therapeutic context, the production process of PSA conjugation is intensive and comes with a significant capital and processing cost. Currently, production involves a laborious eight-step process including: (i) fermentation of E. coli K1 and (ii) purification of its capsular coating, (iii) fermentation of E. coli expressing therapeutic protein and (iv) purification of therapeutic protein, (v) chemical cleavage of PSA from membrane anchor, (vi) purification of PSA, (vii) chemical crosslinking PSA to primary amine groups on the therapeutic protein by reductive amination of the nonreducing end of oxidized PSA, and (viii) purification of PSA-conjugated protein. This eight-step process requires two fermentations, two in vitro chemical reactions, and four purifications. The process is further complicated by the fact that standard amine-directed chemical conjugation of PSA results in random attachment patterns of undesirable heterogeneity [14]. To address this problem, site-specific, thiol-directed chemical conjugation can be used. However, this requires the addition of multiple C-terminal thiols, which are problematic to express in E. coli fermentation and require a mammalian expression system [14].
Accordingly, what is needed, therefore, is a method and composition for recombinant production of therapeutic proteins linked to an oligosaccharide composition that is structurally homogeneous and human-like produced in a controlled, rapid and cost-effective manner.
Described herein are methods and compositions for the recombinant production of human or human-like glycans including polysialic acid and blood group antigens on proteins. The present invention provides methods and compositions for the recombinant production of human or human-like glycans including A antigen, H antigen, B antigen, T antigen, sialyl T antigen, Lewis×antigen and polysialylated antigen. The methods further provide for the production of non-native carbohydrates containing human glycans in prokaryotic host cells and attaching them as N-linked glycans to proteins. Various host cells are engineered to express proteins required to produce the necessary sugar nucleotides and glycosyltransferase activities required to synthesize specified oligosaccharide structures.
In certain aspects, methods and compositions are provided for producing an oligosaccharide composition comprising: culturing a recombinant host cell to express GalNAc transferase activity (EC 2.4.1.-) (EC 2.4.1.290) and galactosyltransferase activity (EC 2.4.1.-) (EC 2.4.1.309); wherein the host cell produces an oligosaccharide composition comprising one or more GalNAc, galactose or galactose-GalNAc residues linked to a lipid carrier.
Additional embodiments provide expression of one or more enzyme activities selected from fucosyltransferase (EC 2.4.1.69); sialyltransferase (EC 2.4.99.4, EC 2.4.99.-, EC 2.4.99.8); and N-acetylglucosaminyl transferase (EC 2.4.1-) for the production of an oligosaccharide composition comprising at least one fucose, sialic acid or GlcNAc residues linked to a lipid carrier.
Certain embodiments provide expression of one or more activities selected from UndP N-acetylglucosaminyl transferase (EC 7.8.33), UndPP GalNAc epimerase (EC 5.1.3.c) and UndP bacillosamine transferase (EC 2.7.8.36).
Certain embodiments provide expression of α1,3-N-acetylgalactosamine transferase activity (EC 2.4.1-, EC 2.4.1.306).
Certain embodiments provide expression of one or more activities selected from β1,3 galactosyltransferase (EC 2.4.1-) β1,4 galactosyltransferase (EC2.4.1.22) and α1,3 galactosyl transferase activity (EC 2.4.1.309).
Certain embodiments provide expression of one or more activities selected from α1,2 fucosyltransferase (EC 2.4.1.69), α1,3 fucosyltransferase (EC 2.4.1.152), and α1,3/1,4 fucosyltransferase (EC 2.4.1.65).
Certain embodiments provide expression of β1,3N-acetylglucosaminyl transferase activity (EC 2.4.1.101).
Certain embodiments provide expression of one or more activities selected from α2,3 NeuNAc transferase (EC 2.4.99.4), α2,6 NeuNAc transferase (EC 2.4.99.1), bifunctional α2,3 α2,8 NeuNAc transferase (EC 2.4.99.-, EC 2.4.99.4, EC 2.4.99.8) and α2,8 polysialyltransferase (EC 2.4.99.8).
Certain embodiments provide expression of undecaprenyl-phosphate α-N-acetylglucosaminyltransferase activity (EC 2.7.8.33).
Certain embodiments provide expression of N-acetyl-α-D-glucosaminyl-diphospho-ditrans, octacis-undecaprenol 4-epimerase activity (EC 5.1.3.c).
Certain embodiments provide expression of undecaprenyl phosphate N,N′-diacetylbacillosamine 1-phosphate transferase activity (EC 2.7.8.36).
Additional embodiments provide an attenuation in at least one of the enzyme activities selected from N-acetylneuraminate lyase (EC 4.1.3.3), undecaprenyl-phosphate glucose phosphotransferase (EC 2.7.8.-)(EC 2.7.8.31) and O-antigen ligase activity.
Certain embodiments provide expression of one or more activities selected from N-acetylneuraminate synthase (EC2.5.1.56), N-acetylneuraminate cytidylyltransferase (EC 2.7.7.43), UDP-N-acetylglucosamine 2-epimerase (EC 5.1.3.14) and N-acetylneuraminate acetyltransferase (EC 2.3.1.45).
Certain embodiments provide expression of GalNAc epimerase activity (EC 5.1.3.2).
Certain embodiments provide expression of Gal epimerase activity (EC 5.1.3.2).
Certain embodiments provide expression of one or more enzyme activities selected from GDP-mannose 4,6 dehydratase (EC 4.2.1.47), GDP-fucose synthetase (EC 1.1.1.271), GDP-mannose mannosyl hydrolase (EC 3.2.1.42), mannose-1-phosphate guanyltransferase (EC 2.7.7.13) and phosphomannomutase (EC 5.4.2.8).
Certain embodiments provide expression of one or more enzyme activities selected from UDP-N-acetylbacillosamine N-acetyltransferase (EC 2.3.1.203), UDP-N-acetylglucosamine 4,6 dehydratase (EC 4.2.1.135) and UDP-N-acetylbacillosamine transaminase (EC2.6.1.34).
In one embodiment, the invention provides a glycoprotein composition comprising an N-linked sialic acid residue on the glycoprotein. Preferably, the glycoprotein composition comprising the N-linked sialic acid residue comprises one of following glycoforms: (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc α1,3-GlcNAc; (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAc; (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc, and (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-(GalNAc α1,3). Alternatively, enzyme activities that convert UDP-GlcNAc to CMP-NeuNAc are introduced and expressed in a select host system. For instance, Neu enzyme activities that convert UDP-GlcNAc to CMP-NeuNAc comprise NeuB (synthase), NeuC (epimerase), and NeuA (synthase). In addition, enzyme activities required to synthesize polysialic acid and/or an acetylated form including NeuE, NeuS (polysialyltransferase), NeuD (acyltransferase family), NeuO (PSA O-acetyltransferase), and KpsCS are expressed. In certain embodiments, PSA is produced using minimal genes neuES and kpsCS to produce [α(2→3)Neu5Ac]n; [α(2→6)Neu5Ac]n; [α(2→8)Neu5Ac]n [α(2→9)Neu5Ac]n, or [α(2→8)Neu5Ac-α(2→9)Neu5Ac]n, or a combination thereof. In yet further embodiments, the glycoprotein composition has a defined degree of polymerization from about 1 to about 500, preferably between 2 and 125 sialic acid residues.
In various other aspects of the invention, a combination of glycosyltransferase enzymes are expressed to produce glycans containing, for example, H-antigen (Fuc α1,2-Galβ1,3-GalNAc α1,3-GlcNAc); T-antigen (Galβ1,3-GalNAc α1,3-GlcNAc; Galβ1,3-GalNAc α1,3-GalNAc β) and Sialyl T-antigen (Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAc).
While various host cells can be engineered to produce oligosaccharides and glycoprotein compositions, a preferred expression system involves prokaryotic host cells. Prokaryotic host cells further comprise an oligosaccharyl transferase activity (EC 2.4.1.119) capable of transferring the oligosaccharide composition onto an N-glycosylation acceptor site of the protein of interest.
In preferred aspects, the invention provides methods and host cells comprising a heterologous protein of interest. In certain embodiments, the protein of interest comprises desired oligosaccharide composition. Accordingly, the invention provides various oligosaccharide compositions produced as described herein.
In other preferred aspects, the glycoprotein compositions produced by the host cell are described herein. In more preferred aspects, the glycoproteins enhance pharmacokinetic properties such as improved serum half-life, enhanced stability, reduced immunogenicity or non-immunogenic or illicit a desired immune response.
Still in other aspects, cell cultures comprising the host cell are provided. Additionally, various methods for producing an oligosaccharide composition comprising culturing recombinant prokaryotic host cells are provided. Various methods for producing a glycoprotein composition comprising culturing recombinant prokaryotic host cells are also provided.
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a cell” includes one or a plurality of such cells. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.
EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (available at http://www.chem.qmul.ac.uk/iubmb/enzyme/). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of March 2013.
The accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. Unless otherwise indicated, the accession numbers are as provided in the database as of March 2013.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).
The term “claim” in the provisional application is synonymous with embodiments or preferred embodiments.
The term “human-like” with respect to a glycoprotein refers to proteins having attached either N-acetylglucosamine (GlcNAc) residue or N-acetylgalactosamine (GalNAc) residue linked to the amide nitrogen of an asparagine residue (N-linked) in the protein, that is similar or even identical to those produced in humans.
“N-glycans” or “N-linked glycans” refer to N-linked saccharide structures. The N-glycans can be attached to proteins or synthetic glycoprotein intermediates, which can be manipulated further in vitro or in vivo. The predominant sugars found on glycoproteins are are glucose (Glu), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetyl-neuraminic acid (Neu5Ac, NeuAc, NeuNA, NeuNAc, Sia or NANA). Hexose (Hex) refers to mannose or galactose.
The term “blood group antigens”, “BGA” or “human antigen” are used interchangeably and comprise an oligosaccharide moiet(ies).
The term “polysialic acid”, or “PSA” refers to an oligosaccharide structure that comprises at least two NeuNAc residues.
Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
An “isolated” or “substantially pure” nucleic acid or polynucleotide (e.g., RNA, DNA, or a mixed polymer) or glycoprotein is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated. The term embraces a nucleic acid, polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “isolated” or “substantially pure” also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems.
However, “isolated” does not necessarily require that the nucleic acid, polynucleotide or glycoprotein so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed “isolated” if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “isolated” because it is separated from at least some of the sequences that naturally flank it.
A nucleic acid is also considered “isolated” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “isolated” if it contains an insertion, deletion, or a point mutation introduced artificially, e.g., by human intervention. An “isolated nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome. Moreover, an “isolated nucleic acid” can be substantially free of other cellular material or substantially free of culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized.
The term “binding affinity” refers to a protein binding to a target receptor. The binding affinity of a glycosylated protein or peptide can range from about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about 2% to about 10% of the binding affinity of the corresponding aglycosylated protein or peptide. Binding affinity of a glycosylated protein or peptide can be increased or reduced at least about 3-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 12-fold, or at least about 15-fold, or at least about 17-fold, or at least about 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold less binding affinity compared to the aglycosylated protein or peptide.
The term “serum persistence” as applied to proteins or peptides refers to the ability of the proteins or peptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by as shown in Example 20.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.
In various aspects, the present invention provides glycoengineered host cells to recombinatly produce oligosaccharides such as BGA-conjugated or PSA-conjugated proteins in a single fermentation without the added step for in vitro chemical modification. Advantageously, glycoengineered host expression technology enables control of the location and stoichiometry of attached polysaccharides and eliminates the need for excess thiols and in vitro chemical reactions. Accordingly, in certain embodiments, the present invention provides methods and compositions for producing an oligosaccharide composition comprising: culturing a recombinant host cell to express GalNAc transferase activity (EC 2.4.1.-) (EC 2.4.1.290) and galactosyltransferase activity (EC 2.4.1.-) (EC 2.4.1.309); wherein the host cell produces an oligosaccharide composition comprising one or more GalNAc, galactose or galactose-GalNAc residues linked to a lipid carrier.
Methods and compositions are also provided to express one or more activities selected from UndP N-acetylglucosaminyl transferase (EC 7.8.33), UndPP GalNAc epimerase activity (EC 5.1.3.c) and UndP bacillosamine transferase activity (EC 2.7.8.36).
Human T Antigen
In exemplary embodiments, the invention provides methods to recombinantly express the genetic machinery needed for the production of various BGAs. A preferred method to produce the human T antigen comprises the recombinant expression of a GalNAc transferase activity (EC 2.4.1.-) (EC 2.4.1.290) that catalyzes the transfer of a UDP-GalNAc residue onto an acceptor substrate β1,4GlcNAc. The host cell further expresses a galactosyltransferase enzyme activity (EC 2.4.1.-) (EC 2.4.1.309), which caps the GalNAc acceptor oligosaccharide resulting in a human T antigen.
Human Sialyl T Antigen
In another aspect of the invention, a method is provided to produce the human sialyl T antigen, which comprises the recombinant expression of a GalNAc transferase activity (EC 2.4.1.-) (EC 2.4.1.290), a galactosyltransferase enzyme activity (EC 2.4.1.-) (EC 2.4.1.309) and a 2,3 NeuNAc transferase activity (EC 2.4.99.4, EC 2.4.99.-, EC 2.4.99.8).
In more preferred embodiments, an improved level of a glycoform is produced by expressing one or more of the enzyme activities selected from sialic acid biosynthesis protein, N-acetylneuraminate synthase (EC 2.5.1.56), N-acetylneuraminate cytidylyltransferase (EC 2.7.7.43), UDP-N-acetylglucosamine 2-epimerase (EC 5.1.3.14) and N-acetylneuraminate acetyltransferase (EC 2.3.1.45) e.g., neuDBAC.
In additional embodiments, α2,6 sialyl T glycoform is produced by expression of one or more α2,6 NeuNAc transferase (EC 2.4.99.1). A glucagon peptide comprising a linkage other than the α2,3 linkage, e.g., α2,6 sialyl T glycoform is shown in
Polysialic Acid
In other exemplary embodiments, the present invention provides a method for producing an oligosaccharide composition comprising: culturing a recombinant host cell to express one or more of the enzymes comprising: GalNAc transferase activity (EC 2.4.1.-) that transfers a GalNAc residue onto an acceptor substrate; galactosyltransferase enzyme activity (EC 2.4.1.-); fucosyltransferase enzyme activity (EC 2.4.1.69); and sialyltransferase enzyme activity (EC 2.4.99.4, EC 2.4.99.-, EC 2.4.99.8), wherein the host cell produces a polysialic acid.
Evidence of PSA on the cell wall is shown in
In select embodiments, the invention provides methods to recombinantly express the genetic machinery needed for the PSA production. As described in Example 12, the genes representing the capsular biosynthetic loci harboring the kps and neu genes of E. coli K1 and K92 are cloned into plasmid pACYC 184 for transformation of a preferred strain of E. coli.
In other select embodiments, the N-linked oligosaccharide compositions comprise or consists of [α(2→3)Neu5Ac]n; [α(2→6)Neu5Ac]n; [α(2→8)Neu5Ac]n; [α(2→9)Neu5Ac]n or a combination thereof.
Also disclosed are genes for producing the desired PSA oligosaccharide compositions. In certain embodiments, one or more Neu activity such as NeuDBACES and Kps activity such as KpsSCUDEF are expressed. In yet other embodiments, one or more genes encoding KpsMT is attenuated. The invention provides a method for producing an N-linked sialic acid on a glycoprotein comprising: culturing a host cell to produce CMP-Neu5Ac from UDP-GlcNAc; PSA from CMP-NeuNAc; and expressing an OST activity; wherein the OST activity transfers the sialic acid onto an acceptor asparagine of the resulting glycoprotein.
Preferably the oligosaccharide structure is N-linked to a protein, comprises a terminal sialic acid residue and is more preferably a polysialic acid that is a polysaccharide comprising at least 2 sialic acid residues joined to one another through α2-8 or α2-9 linkages. A suitable polysialic acid has a weight average molecular weight in the range 2 to 100 kDa, preferably in the range 1 to 35 kDa. The most preferred polysialic acid has a molecular weight in the range of 10-20 kDa, typically about 14 kDa.
More preferably, the N-linked PSA glycoprotein comprises about 2-125 sialic acid residues. Polymerized PSA can be transferred onto the glycoprotein, N-linked, some comprising 10-80 sialic acid residues, others 20-60 sialic acid residues, or 40-50 sialic acid residues. The preferred N-linked PSA glycoprotein composition has a defined degree of polymerization.
In additional embodiments, the glycoprotein composition further comprises a second N-linked oligosaccharide structure for example eukaryotic, human or human-like glycans such as Neu5Ac1-4Gal1-4GlcNAc1-5Man3GlcNAc2, Man3-5GlcNAc1-2, GlcNAc1-2, bacterial glycans such as GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]GalNAc-α1,4-GalNAc-a 1,4-GalNAc-α1,3-Bac-β1,N-Asn (GalNAc5GlcBac, where Bac is bacillosamine or 2,4-diacetamido-2,4,6-trideoxyglucose). A mixture of N-linked PSA and N-linked oligosaccharide composition is also contemplated.
Glycoengineered E. coli have been used to attach diverse lipid-linked O-antigen glycans to corresponding asparagines in acceptor proteins in vivo (Feldman M F et al, (2005) Engineering N-linked protein glycosylation with diverse 0 antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci USA. 2005 Feb. 22; 102(8):3016-21.). Enabling control of the location and stoichiometry of attached polysaccharides such as PSA may be critically important as amine-directed chemical conjugation of PSA is random and results in an unacceptably heterogeneous product. Favorable conjugation has only recently been achieved by site-specific, chemical coupling of PSA to engineered C-terminal thiols.
The PSA-conjugated protein is expected to improved circulating half-life and provide stability. Because PSA is a natural part of the human body, the recombinant PSA composition, which is chemically and immunologically similar to human PSA and (unlike PEG) is expected to be degraded or metabolized by tissue neuraminidases or sialidases to sialic acid residues. The recombinant PSA compositions are also immunologically invisible as a biodegrable polymer.
Additional advantages of the recombinant biosynthesis are as follows. While PSA conjugation requires several intricate in vitro chemical reactions and multiple purifications, direct recombinant production of PSA via host cell expression obviates the need for in vitro chemical reactions. There is no need to isolate PSA from E. coli K1 capsules prior to in vitro chemical crosslinking Random attachment patterns and undesirable heterogeneity resulting from the standard amine-directed chemical conjugation of PSA is also obviated. While site-specific, thiol-directed chemical conjugation can be used, this requires the appendage of multiple C-terminal thiols and expression from a mammalian host. Capital cost and production are kept low for efficient production and processing using the glycoengineered hosts. Therefore, in one aspect of the invention, the methods and host cells serve as a glycoprotein expression system for producing N-linked glycoproteins with structurally homogeneous human-like glycans and overcomes many of the above limitations and challenges. The host cells address the clear clinical demand for PSA-conjugated protein therapeutics.
Human H Antigen
In further exemplary embodiments, the present invention provides a method for producing an oligosaccharide composition comprising: culturing a recombinant host cell to express one or more of the enzymes comprising: GalNAc transferase activity that catalyzes a GalNAc residue onto an acceptor substrate (EC 2.4.1.-); galactosyltransferase enzyme activity (EC 2.4.1.-); and one or more activities selected from α1,2 fucosyltransferase (EC 2.4.1.69), α1,3 fucosyltransferase (EC 2.4.1.152) and α1,3/1,4 fucosyltransferase (EC 2.4.1.65). GDP-fucose transfer was confirmed with the treatment of the glycans with α1,2-fucosidase
Prokaryotic Expression System
In preferred aspects, the invention provides a glycoprotein production system that serves as an attractive solution for circumventing the significant hurdles associated with eukaryotic cell culture systems or in vitro chemical conjugation. The use of bacteria as a production vehicle is expected to yield structurally homogeneous glycoproteins while at the same time dramatically lowering the cost and time associated with protein drug development and manufacturing. Other key advantages include: (i) the massive volume of data surrounding the genetic manipulation of bacteria; (ii) the established track record of using bacteria for protein production—30% of protein therapeutics approved by the FDA since 2003 are produced in E. coli bacteria; and (iii) the existing infrastructure within numerous companies for bacterial production of protein drugs.
Previously, the ability to attach a foreign glycan to an acceptor protein in E. coli has been shown (Wacker et al 2002 N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 2002 Nov. 29; 298(5599):1790-3.). Also, the ability to attach foreign glycans to a recombinant protein in a site-directed, stoichiometric manner using our proprietary C-terminal GlycTag has been demonstrated (PCT/US2009/030110). Moreover, the ability to attach lipid-linked polysaccharides (e.g., poly-FucNAc) to acceptor proteins in E. coli have been described (Feldman 2005). Recently, Valderrama-Rincon, et. al. (Valderrama-Rincon, et. al. “An engineered eukaryotic protein glycosylation pathway in Escherichia coli,” Nat. Chem. Biol. AOP (2012)) disclosed a biosynthetic pathway for the biosynthesis and assembly of Man3GlcNAc2 on Und-PP in the cytoplasmic membrane of E. coli, however, to date, no studies have demonstrated the ability to recombinantly produce BGA or PSA-conjugated proteins directly from an expression platform in a simple fermentation and purification process.
Nucleic Acid Sequences
In select embodiments, the invention provides isolated nucleic acid molecules, variants thereof, expression optimized forms of the disclosed genes, and methods of improvement thereon.
In one embodiment is provided an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of glycosyltransferase gene homologs, variants and derivatives of the wild-type coding sequences. The invention provides nucleic acid molecules comprising or consisting of sequences which are structurally and functionally optimized versions of the wild-type genes. In a preferred embodiment, nucleic acid molecules and homologs, variants and derivatives comprising or consisting of sequences optimized for substrate affinity, specificity and/or substrate catalytic conversion rate, improved thermostability, activity at a different pH and/or optimized codon usage for improved expression in a host cell are provided.
In a further embodiment is provided nucleic acid molecules and homologs, variants and derivatives comprising or consisting of sequences which are variants of the glycosyltransferase genes having at least 60% identity. In a further embodiment provided nucleic acid molecules and homologs, variants and derivatives comprising or consisting of sequences which are variants having at least 62%, 65%, 68%, 70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 92%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type gene.
In another embodiment, the encoded polypeptides having at least 50%, preferably, at least 55%, 60%, 70%, 80%, 90% or 95%, more preferably, 98%, 99%, 99.9% or even higher identity to the wild-type gene.
Provided also are nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. As defined above, and as is well known in the art, stringent hybridizations are performed at about 25° C. below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions, where the Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Stringent washing can be performed at temperatures about 5° C. lower than the Tm for the specific DNA hybrid under a particular set of conditions.
The nucleic acid molecule includes DNA molecules (e.g., linear, circular, cDNA, chromosomal DNA, double stranded or single stranded) and RNA molecules (e.g., tRNA, rRNA, mRNA) and analogs of the DNA or RNA molecules of the described herein using nucleotide analogs. The isolated nucleic acid molecule of the invention includes a nucleic acid molecule free of naturally flanking sequences (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid molecule) in the chromosomal DNA of the organism from which the nucleic acid is derived. In various embodiments, an isolated nucleic acid molecule can contain less than about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 by of naturally flanking nucleotide chromosomal DNA sequences of the microorganism from which the nucleic acid molecule is derived.
The genes, as described herein, include nucleic acid molecules, for example, a polypeptide or RNA-encoding nucleic acid molecule, separated from another gene or other genes by intergenic DNA (for example, an intervening or spacer DNA which naturally flanks the gene and/or separates genes in the chromosomal DNA of the organism).
Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides.
In another embodiment, an isolated glycosyltransferase gene encoding nucleic acid molecule hybridizes to all or a portion of a nucleic acid molecule having the nucleotide sequence set forth in the sequence listings or hybridizes to all or a portion of a nucleic acid molecule having a nucleotide sequence that encodes a polypeptide having the amino acid sequence of any of amino acid sequences as set forth in the sequence listings. Such hybridization conditions are known to those skilled in the art (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995); Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). In another embodiment, an isolated nucleic acid molecule comprises a nucleotide sequence that is complementary to a neu or kps gene encoding nucleotide sequence as set forth herein.
The nucleic acid sequence fragments display utility in a variety of systems and methods. For example, the fragments may be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be either DNA or RNA. The target nucleic acid sequences may be fractionated (e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ. One of skill in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by Southern blotting) and in measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled, so that their specific hybridization to target sequences can be detected and optionally quantified. One of skill in the art will appreciate that the nucleic acid fragments may be used in a wide variety of blotting techniques not specifically described herein.
It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties. Analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, is a well-established utility for sequence fragments in the field of cell and molecular biology. Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of each of which is incorporated herein by reference in its entirety.
As is well known in the art, enzyme activities are measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically. Grubmeyer et al., J. Biol. Chem. 268:20299-20304 (1993). Alternatively, the activity of the enzyme is followed using chromatographic techniques, such as by high performance liquid chromatography. Chung and Sloan, J. Chromatogr. 371:71-81 (1986). As another alternative the activity is indirectly measured by determining the levels of product made from the enzyme activity. More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001). Current practice of gas chromatography—mass spectrometry. New York, N.Y: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix-Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G., R. O. Dunn, and M. O. Bagby. 1997. Biodiesel: The use of vegetable oils and their derivatives as alternative diesel fuels. Am. Chem. Soc. Symp. Series 666: 172-208), physical property-based methods, wet chemical methods, etc. are used to analyze the levels and the identity of the product produced by the organisms. Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art.
Another embodiment comprises mutant or chimeric nucleic acid molecules or genes. Typically, a mutant nucleic acid molecule or mutant gene is comprised of a nucleotide sequence that has at least one alteration including, but not limited to, a simple substitution, insertion or deletion. The polypeptide of said mutant can exhibit an activity that differs from the polypeptide encoded by the wild-type nucleic acid molecule or gene. Typically, a chimeric mutant polypeptide includes an entire domain derived from another polypeptide that is genetically engineered to be collinear with a corresponding domain. Preferably, a mutant nucleic acid molecule or mutant gene encodes a polypeptide having improved activity such as substrate affinity, substrate specificity, improved thermostability, activity at a different pH, improved soluability, improved expression, or optimized codon usage for improved expression in a host cell.
Isolated Polypeptides
In one embodiment, polypeptides encoded by nucleic acid sequences are produced by recombinant DNA techniques and can be isolated from expression host cells by an appropriate purification scheme using standard polypeptide purification techniques. In another embodiment, polypeptides encoded by nucleic acid sequences are synthesized chemically using standard peptide synthesis techniques.
Included within the scope of the invention are glycosyltransferase polypeptides or gene products that are derived polypeptides or gene products encoded by naturally-occurring bacterial genes. Further, included within the inventive scope, are bacteria-derived polypeptides or gene products which differ from wild-type genes, including genes that have altered, inserted or deleted nucleic acids but which encode polypeptides substantially similar in structure and/or function.
For example, it is well understood that one of skill in the art can mutate (e.g., substitute) nucleic acids which, due to the degeneracy of the genetic code, encode for an identical amino acid as that encoded by the naturally-occurring gene. This may be desirable in order to improve the codon usage of a nucleic acid to be expressed in a particular organism. Moreover, it is well understood that one of skill in the art can mutate (e.g., substitute) nucleic acids which encode for conservative amino acid substitutions. It is further well understood that one of skill in the art can substitute, add or delete amino acids to a certain degree to improve upon or at least insubstantially affect the function and/or structure of a gene product (e.g., glycosyltransferase activity) as compared with a naturally-occurring gene product, each instance of which is intended to be included within the scope of the invention. For example, the glycosyltransferase ctivity, enzyme/substrate affinity, enzyme thermostability, and/or enzyme activity at various pHs can be unaffected or rationally altered and readily evaluated using the assays described herein.
In various aspects, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules are provided. Preferably the isolated polypeptide has preferably 50%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to the sequences optimized for substrate affinity and/or substrate catalytic conversion rate.
According to other embodiments, isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids.
The polypeptides also include fusions between the above-described polypeptide sequences and heterologous polypeptides. The heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins. Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, alter the subcellular localization of the protein, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region.
Secretion Signal Sequences
In selected embodiments, the oligosaccharide-conjugated polypeptide is expressed with a secretion signal sequence. The secretion signal can be an amino terminal sequence that facilitates transit across a membrane. In those embodiments where the host organism is prokaryotic, secretion signal is a leader peptide domain of a protein that facilitates insertion into the membrane or transport through a membrane. The signal sequence is removed after crossing the inner membrane, and proteins may be retained in the periplasmic space.
Various secretion signals are used, for instance pelB. The predicted amino acid residue sequences of the secretion signal domain from two PelB gene product variants from Erwinia carotova are described in Lei et al., Nature, 331:543-546 (1988). The leader sequence of the PelB protein has previously been used as a secretion signal for fusion proteins (Better et al., Science, 240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci., USA, 86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci., USA, 87:8095-8099 (1990)). Amino acid residue sequences for other secretion signal polypeptide domains from E. coli useful in this invention include those described in Oliver, Escherichia coli and Salmonella Typhimurium, Neidhard, F. C. (ed.), American Society for Microbiology, Washington, D.C., 1:56-69 (1987).
Another typical secretion signal sequence is the gene III (gIII) secretion signal. Gene HI encodes Pill, one of the minor capsid proteins from the filamentous phage fd (similar to Ml 3 and rl). Pill is synthesized with an 18 amino acid, amino terminal signal sequence and requires the bacterial Sec system for insertion into the membrane.
Another typical secretion signal sequence is the SRP secretion signal. SRP secretion signals have been used, for example, to improve production of fusion protein for phage display (Steiner et al. Nat. Biotechnology, 24:823-831 (2006)). Most commonly used type II secretion signals, such as the PelB secretion signal, use the SecB pathway. Thus, secretion constructs presented herein for expression of human mAb heavy and light chains use an SRP secretion signal, namely the secretion signal of the E. coli dsbA gene. Other SRP secretion signals that can be used in the methods, polynucleotides and polypeptides provided herein include SfmC (chaperone), ToIB (translocation protein), and TorT (respiration regulator). The sequences of these signals are known in the art.
Secrection by the E. coli SecB mechanism involves attachment of a nascent polypeptide first to trigger factor, TF, and then to SecB. The ScB protein then directs attachment of the completed polypeptide to the Type II secretion complex which secretes the protein into the periplasm. Without being bound by theory, it is thought that some recombinant proteins may fold into forms which secrete poorly by this mechanism. In contrast, the SRP mechanism recognizes a different set of secretion signals and directs co-translation and secretion of nascent polypeptides through the Type II secretion complex into the periplasm. This mechanism can be used to avoid problems that could occur in secretion by the SecB pathway.
It will be apparent to one of ordinary skill in the art that any suitable secretion signal sequence may be used to facilitate secretion of expressed polypeptides.
Secretion of Proteins into Periplasm and Medium
To determine secretion of an active antibody into culture the medium, media samples collected during the expression analysis of the variousP constructs are assayed by ELISA for its antigen binding activity.
The polynucleotides or nucleic acid molecules of the present invention refer to the polymeric form of nucleotides of at least 10 bases in length. These include DNA molecules (e.g., linear, circular, cDNA, chromosomal, genomic, or synthetic, double stranded, single stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hair-pinned, circular, or in a padlocked conformation) and RNA molecules (e.g., tRNA, rRNA, mRNA, genomic, or synthetic) and analogs of the DNA or RNA molecules of the described as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native inter-nucleoside bonds, or both. The isolated nucleic acid molecule of the invention includes a nucleic acid molecule free of naturally flanking sequences (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid molecule) in the chromosomal DNA of the organism from which the nucleic acid is derived. In various embodiments, an isolated nucleic acid molecule can contain less than about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of naturally flanking nucleotide chromosomal DNA sequences of the microorganism from which the nucleic acid molecule is derived.
The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame. The preparation of the nucleic acid constructs can be carried out using standard cloning methods well known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory Press, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, also describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. Preparation of nucleic acid constructs can alternatively be prepared using homologous recombination in yeast as described by Shanks et al., AEM, 72, 2, (2006).
Suitable expression vectors include those which contain replicon and control sequences that are derived from species compatible with the host cell. For example, if E. coli is used as a host cell, plasmids such as pUC19, pUC18, or pBR322 may be used. Other suitable expression vectors are described in Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference in its entirety. Many known techniques and protocols for manipulation of nucleic acids, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., (1992), which is hereby incorporated by reference in its entirety.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation) and subsequently the amount of fusion protein that is displayed on the ribosome surface. Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase, and thereby promotes mRNA synthesis. Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is often desirable to use strong promoters to obtain a high level of transcription and, hence, expression and surface display. Therefore, depending upon the host system utilized, any one of a number of suitable promoters may also be incorporated into the expression vector carrying the deoxyribonucleic acid molecule encoding the protein of interest coupled to a stall sequence. For instance, when using E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals, which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
Host Cells
In accordance with the present invention, the host cell may be a prokaryote. Such cells serve as a host for expression of recombinant proteins for production of recombinant therapeutic proteins of interest. Exemplary host cells include E. coli and other Enterobacteriaceae, Escherichia sp., Campylobacter sp., Wolinella sp., Desulfovibrio sp. Vibrio sp., Pseudomonas sp. Bacillus sp., Listeria sp., Staphylococcus sp., Streptococcus sp., Peptostreptococcus sp., Megasphaera sp., Pectinatus sp., Selenomonas sp., Zymophilus sp., Actinomyces sp., Arthrobacter sp., Frankia sp., Micromonospora sp., Nocardia sp., Propionibacterium sp., Streptomyces sp., Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Acetobacterium sp., Eubacterium sp., Heliobacterium sp., Heliospirillum sp., Sporomusa sp., Spiroplasma sp., Ureaplasma sp., Erysipelothrix, sp., Corynebacterium sp. Enterococcus sp., Clostridium sp., Mycoplasma sp., Mycobacterium sp., Actinobacteria sp., Salmonella sp., Shigella sp., Moraxella sp., Helicobacter sp, Stenotrophomonas sp., Micrococcus sp., Neisseria sp., Bdellovibrio sp., Hemophilus sp., Klebsiella sp., Proteus mirabilis, Enterobacter cloacae., Citrobacter sp., Proteus sp., Serratia sp., Yersinia sp., Acinetobacter sp., Actinobacillus sp. Bordetella sp., Brucella sp., Capnocytophaga sp., Cardiobacterium sp., Eikenella sp., Francisella sp., Haemophilus sp., Kingella sp., Pasteurella sp., Flavobacterium sp. Xanthomonas sp., Burkholderia sp., Aeromonas sp., Plesiomonas sp., Legionella sp. and alpha-proteobacteria such as Wolbachia sp., cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria, Gram-negative cocci, Gram negative bacilli which are fastidious, Enterobacteriaceae-glucose-fermenting Gram-negative bacilli, Gram negative bacilli-non-glucose fermenters, Gram negative bacilli-glucose fermenting, oxidase positive.
In one embodiment of the present invention, the E. coli host strain C41(DE3) is used, because this strain has been previously optimized for general membrane protein overexpression (Miroux et al., “Over-production of Proteins in Escherichia coli: Mutant Hosts That Allow Synthesis of Some Membrane Proteins and Globular Proteins at High Levels,” J Mol Biol 260:289-298 (1996), which is hereby incorporated by reference in its entirety). Further optimization of the host strain includes deletion of the gene encoding the DnaJ protein (e.g., ΔdnaJ cells). The reason for this deletion is that inactivation of dnaJ is known to increase the accumulation of overexpressed membrane proteins and to suppress the severe cytotoxicity commonly associated with membrane protein overexpression (Skretas et al., “Genetic Analysis of G Protein-coupled Receptor Expression in Escherichia coli: Inhibitory Role of DnaJ on the Membrane Integration of the Human Central Cannabinoid Receptor,” Biotechnol Bioeng (2008), which is hereby incorporated by reference in its entirety). Applicants have observed this following expression of Alg1 and Alg2. Furthermore, deletion of competing sugar biosynthesis reactions may be required to ensure optimal levels of N-glycan biosynthesis. For instance, the deletion of genes in the E. coli O antigen biosynthesis pathway (Feldman et al., “The Activity of a Putative Polyisoprenol-linked Sugar Translocase (Wzx) Involved in Escherichia coli O Antigen Assembly is Independent of the Chemical Structure of the O Repeat,” J Biol Chem 274:35129-35138 (1999), which is hereby incorporated by reference in its entirety) will ensure that the bactoprenol-GlcNAc-PP substrate is available for other reactions. To eliminate unwanted side reactions, the following are representative genes that may be deleted from the E. coli host strain: wbbL, glcT, glf, gafT, wzx, wzy, waaL, nanA, wcaJ.
Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory Press, Cold Springs Harbor, N.Y. (1989). For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.
One aspect of the present invention is directed to a glycoprotein conjugate comprising a protein and at least one peptide comprising a D-X1-N-X2-T motif fused to the protein, wherein D is aspartic acid, X1 and X2 are any amino acid other than proline, N is asparagine, and T is threonine.
Various host cells can be used to recombinantly produce PSA. In select embodiments, host cells are genetically modified to remove the existing native glycosyltransferases and are engineered to express the glycosyltransferases of the invention for PSA production. To remove the existing glycosylation, e.g., eukaryotic host cells are engineered to express endoglycosidase or amidase that cleave between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. Since glycosylation is essential, one may not be able to entirely eliminate the native glycan. In other embodiments, sialic acid bearing glycans may be engineered in the host cell and used as substrates for polysialiation such as ST8Sia II, ST8Sia IV, or NeuS to transfer multiple α2-8 sialic acids to acceptor N-glycans.
In preferred aspects, the invention provides methods for recombinant production of various glycoproteins in vivo. In one embodiment, PSA-conjugated glucagon peptide is produced in glycoengineered E. coli. Using a glycosylation tag (GlycTag) [PCT/US2009/030110], glucagon peptide from glycoengineered E. coli harboring the PSA genetic machinery is expressed and purified. Conjugation of PSA is confirmed by Western blot analysis using commercially available anti-PSA antibodies.
Alternative Expression Systems
Use of eukaryotic expression systems such as mammalian, yeast, fungi, plant or insect cells can be employed to produce PSA-conjugated proteins. In these embodiments, native glycosylation pathways may be disrupted in order to reduce interference with the engineered glycan pathway.
Production of PSA Using Yeast or Fungal Systems
Expression of a sialyltransferase has been demonstrated in P. pastoris (Hamilton, et al, “Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins”, Science, vol. 313, pp. 1441-1443 (2006)). By amplifying the E. coli neuA, neuB and neuC genes, a pool of CMP-sialic acid was shown to accumulate in yeast. Yeast or other fungal systems are suitable expression hosts to express the various glycosyltransferases for the production of human antigens or PSA.
Expressing PSA Operon in Plant Cell, e.g., Tobacco, Lemna or Algae
As described in the U.S. Pat. No. 6,040,498, lemna (duckweed) can be transformed using both agrobacterium and ballistic methods. Using protocols described, lemna is transformed and the resulting oligosaccharide composition is transferred onto a target protein. Transgenic plants can be assayed for those that produce proteins with desired human antigens or PSA residues according to known screening techniques.
Production of PSA Using Insect Cell Systems
The present invention can also be applied to the metabolically transformed cell lines derived from Sf9 cells. Sf9 has been used as a production host for recombinant proteins such as interferons, IL-2, plasminogen activators among others, based on its relative ease at which proteins are cloned, expressed and purified in comparison to mammalian cells. Sf9 more readily accepts foreign genes coding for recombinant proteins than many vertebrate animal cells because it is very receptive to viral infection and replication [Bishop, D. H. L. and Possee, R. D., Adv. Gene Technol., 1, 55, (1990)]. Expression levels of recombinant proteins are extremely high in Sf9 and can approach 500 mg/liter [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990)]. The cell line performs a number of key post-translational modifications; however, they are not identical to those in vertebrates and, therefore, may alter protein function [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989)]. Despite this, the majority of recombinant proteins that undergo post-translational modification in insect cells are immunologically and functionally similar to their native counterparts [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989)]. In contrast to animal cell culture, Sf9 facilitates protein purification by expressing relatively low levels of proteases and having a high ratio of recombinant to native protein expression [Goswami, B. B. and Glazer, R. O. BioTechniques, 10, 626 (1991)].
Baculoviruses serve as expression systems for the production of recombinant proteins in insect cells. These viruses are pathogenic towards specific species of insects, causing cell lysis [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990)].
Recombinant protein expression in insect cells is achieved by viral infection or stable transformation. For the former, the desired gene is cloned into baculovirus at the site of the wild-type polyhedron gene [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990); Bishop, D. H. L. and Possee, R. D., Adv. Gene Technol., 1, 55, (1990)]. The polyhedron gene is nonessential for infection or replication of baculovirus. It is the principle component of a protein coat in occlusions which encapsulate virus particles. When a deletion or insertion is made in the polyhedron gene, occlusions fail to form. Occlusion negative viruses produce distinct morphological differences from the wild-type virus. These differences enable a researcher to identify and purify a recombinant virus. In baculovirus, the cloned gene is under the control of the polyhedron promoter, a strong promoter which is responsible for the high expression levels of recombinant protein that characterize this system. Expression of recombinant protein typically begins within 24 hours after viral infection and terminates after 72 hours when the Sf9 culture has lysed.
Stably-transformed insect cells provide an alternate expression system for recombinant protein production [Jarvis, D. L., Fleming, J.-A. G. W., Kovacs, G. R., Summers, M. D., and Guarino, L. A., Biotechnology, 8, 950 (1990); Cavegn, C., Young, J., Bertrand, M., and Bernard, A. R., in Animal Cell Technology: Products of Today, Prospects for Tomorrow, Spier, R. E., Griffiths, J. B., and Berthold, W., Eds. (Butterworth-Heinemann, Oxford, 1994, pp. 43-49)]. In these cells, the desired gene is expressed continuously in the absence of viral infection. Stable transformation is favored over viral infection when recombinant protein production requires cellular processes that are compromised by the baculovirus. This occurs, for example, in the secretion of recombinant human tissue plasminogen activator from Sf9 cells [Jarvis, D. L., Fleming, J.-A. G. W., Kovacs, G. R., Summers, M. D., and Guarino, L. A., Biotechnology, 8, 950 (1990)]. Viral infection is favored when the recombinant protein is cytotoxic since protein expression is transient in this system.
Insect cells for in vitro cultivation have been produced and several cell lines are commercially available. This process includes using insect cells capable of culture as described herein regardless of the source. The preferred cell line is Lepidoptera Sf9 cells. Other cell lines include Drosophila cells from the European Collection of Animal Cell Cultures (Salisbury, UK) or cabbage looper Trichoplusia ni cells including High Five available from Invitrogen Corp. (San Diego, Calif.) Sf9 insect cells from either Invitrogen Corporation or American Type Culture Collection (Rockville, Md.) are the preferred cell line and were cultivated in the bioreactor freely suspended in serum-free EX-CELL 401 Medium purchased from JRH Biosciences (Lenexa, Kans.) and maintained at 27° C.
Oligosaccharide Compositions
The prokaryotic system can yield homogenous glycans at a relatively high yield. In preferred embodiments, the oligosaccharide composition comprises or consists essentially of a single glycoform in at least 50, 60, 70, 80, 90, 95, 99 mole %. In further embodiments, the oligosaccharide composition consists essentially of two desired glycoforms of at least 50, 60, 70, 80, 90, 95, 99 mole %. In yet further embodiments, the oligosaccharide composition consists essentially of three desired glycoforms of at least 50, 60, 70, 80, 90, 95, 99 mole %. The present invention, therefore, provides stereospecific biosynthesis of a vast array of novel oligosaccharide compositions and N-linked glycoproteins including glycans for BGA and PSA. Methods for estimating glycan or glycoprotein homogeneity and yield may include Mass Spectrometry, NMR, Lectin blotting, fluorophore-assisted carbohydrate electrophoresis (FACE), or chromatography methods [16-18].
Select PSA oligosaccharide compositions include:
(Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc α1,3-GlcNAc; (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAc; (Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-(GalNAc α1,3)n-GlcNAc.
Select Sialyl T Antigen oligosaccharide compositions include:
Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAc; Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc α1,3; Sia α2,6-Galβ1,3-GalNAc α1,3-GlcNAc.
Select H Antigen oligosaccharide compositions include:
Fuc α1,2-Galβ1,3-GalNAc α1,3-GlcNAc; Fuc α1,2-Galβ1,3-GalNAc α1,3-GalNAc.
Select T Antigen oligosaccharide compositions include:
Galβ1,3-GalNAc α1,3-GlcNAc; and Galβ1,3-GalNAc α1,3-GalNAc α1,3.
Other select PSA oligosaacharide compositions include:
[βGlcNAc][αGalNAc][βGalNAc] Gal[β1,3][α(2→3)Neu5Ac]n; [α(2→6)Neu5Ac]n; [α(2→8)Neu5Ac]n; [α(2→8)Neu5Ac-α(2→9)Neu5Ac] or [α(2→9)Neu5Ac]n.
Various oligosaccharide compositions produced using the methods and compositions of the invention include but are not limited to the following:
(Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc α1,3-GlcNAcβ1-;
(Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
(Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAcα1-;
(Sia α2,8)n-Sia α2,8-Sia α2,3-Galβ1,3-GalNAc α1,3-Bacα1-;
Siaα2,8-Siaα2,3-Galβ1,3-GalNAcα1,3-GlcNAcβ1-;
Siaα2,8-Siaα2,3-Galβ1,3-GalNAcα1,3-GalNAcα1-;
Siaα2,8-Siaα2,3-Galβ1,3-GalNAcα1,3-Bacα1-;
Sia α2,3-Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
Sia α2,3-Galβ1,3-GalNAc α1,3-GalNAc1-;
Sia α2,3-Galβ1,3-GalNAc α1,3-Bacα1-;
Sia α2,6-Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
Sia α2,6-Galβ1,3-GalNAc α1,3-GalNAcα1-;
Sia α2,6-Galβ1,3-GalNAc α1,3-Bacα1-;
Fuc α1,2-Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
Fuc α1,2-Galβ1,3-GalNAc α1,3-GalNAcα1-;
Fuc α1,2-Galβ1,3-GalNAc α1,3-Bacα1-;
Galα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
Galα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-GalNAcα1-;
Galα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-Bacα1-;
GalNAcα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
GalNAcα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-GalNAcα1-;
GalNAcα1,3[Fuc α1,2] Galβ1,3-GalNAc α1,3-Bacα1-;
Galβ1,4[Fucα1-3]GlcNAcβ1,3-Galβ1,3-GlcNAcβ1-;
Galβ1,4[Fucα1-3]GlcNAcβ1,3-Galβ1,3-GalNAcα1-;
Galβ1,4[Fucα1-3]GlcNAcβ1,3-Galβ1,3-Bacα1-;
Galβ1,3-GalNAc α1,3-GlcNAcβ1-;
Galβ1,3-GalNAc α1,3-Bacα1-; and
Galβ1,3-GalNAc α1,3-GalNAc 1-.
Target Glycoproteins
Various examples of suitable target glycoproteins may be produced according to the invention, which include without limitation: cytokines such as interferons, G-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNase II, α-feto proteins, AAT, rhTBP-1 (aka TNF binding protein 1), TACI-Ig (transmembrane activator and calcium modulator and cyclophilin ligand interactor), FSH (follicle stimulating hormone), GM-CSF, glucagon, glucagon peptides, GLP-1 w/and w/o FC (glucagon like protein 1), GLP-1 receptor agonist e.g., exenatide, direct thrombin inhibitor e.g., bivalirudin, IGF-1 e.g., mecasermin, parathyroid hormone e.g., teriparatide, plasma kallikrein inhibitor e.g., ecallantide, IL-I receptor agonist, sTNFr (aka soluble TNF receptor Fc fusion), CTLA4-Ig (Cytotoxic T Lymphocyte associated Antigen 4-Ig), receptors, hormones such as human growth hormone, erythropoietin, peptides, stapled peptides, human vaccines, animal vaccines, serum albumin and enzymes such as ATIII, rhThrombin, glucocerebrosidase and asparaginase.
Antibodies, fragments thereof and more specifically, the Fab regions such as adalimumab, atorolimumab, fresolimumab, golimumab, lerdelimumab, metelimumab, morolimumab, sifalimumab, ipilimumab, tremelimumab, bertilimumab, briakinumab, canakinumab, fezakinumab, ustekinumab, adecatumumab, belimumab, cixutumumab, conatumumab, figitumumab, intetumumab, iratumumab, lexatumumab, lucatumumab, mapatumumab, necitumumab, ofatumamb, panitumumab, pritumumab, rilotumumab, robatumumab, votumumab, zalutumumab, zanolimumab, denosumab, stamulumab, efungumab, exbivirumab, foravirumab, libivirumab, rafivirumab, regavirumab, sevirumab, tuvirumab, nebacumab, panobacumab, raxibacumab, ramucirumab, gantenerumab.
Full-length monoclonal antibodies have traditionally been produced in mammalian cell culture due to their parental hybridoma source, the complexity of the molecule, and the desirability of glycosylation of the monoclonal antibodies. Generally, Escherichia coli is the host system of choice for the expression of antibody fragments such as Fv, scFv, Fab or F(ab′)2. These fragments can be made relatively quickly in large quantities with the retention of antigen binding activity. However, because antibody fragments lack the Fc domain, they do not bind the FcRn receptor and are cleared quickly. Full-length antibody chains can also be expressed in E. coli as insoluble aggregates and then refolded in vitro, but the complexity of this method limits its usefulness. Accordingly, the antibodies are produced in the periplasm.
In contrast to the widespread uses of bacterial systems for expressing antibody fragments, there have been few attempts to express and recover at high yield functional intact antibodies in E. coli. Because of the complex features and large size of an intact antibody, it is often difficult to achieve proper folding and assembly of the expressed light and heavy chain polypeptides, which results in poor yield of reconstituted tetrameric antibody. Furthermore, antibodies made in prokaryotes are not glycosylated. Since glycosylation is required for Fc receptor mediated activity, it is conventionally considered that E. coli would not be a useful system for making intact antibodies. (Pluckthun and Pack (1997) Immunotech 3:83-105; Kipriyanov and Little (1999) Mol. Biotech. 12:173-201.). Recombinant oligosaccharide synthesis changes this paradigm.
Recent developments in research and clinical studies suggest that in many instances, intact antibodies are preferred over antibody fragments. An intact antibody containing the Fc region tends to be more resistant to degradation and clearance in vivo, thereby having longer biological half life in circulation. This feature is particularly desirable where the antibody is used as a therapeutic agent for diseases requiring sustained therapies.
Currently, anti-TNF antibodies are produced in mammalian cells and are glycosylated. The cost of producing antibodies in mammalian cells (frequently in CHO cells) is high and the procedure is complex. Glycosylation of antibodies has two effects: first, it can increase the lifetime of the antibody in the blood serum, so that it circulates for many days or even weeks. This may be because of decreased kidney clearance or because of greater resistance to proteolysis. Second, as provided herein, glycosylation in the constant region of the antibody is important for activating the “effector functions” of the antibody, which are triggered when an antibody binds to a target that is attached to a cell surface. These functions are linked to activation of the immune system and can lead to natural killer (NK) mediated cell killing.
The present invention relates in part to glycoprotein compositions comprising peptides characterized as having enhanced pharmacokinetic properties such as improved serum half-life, enhanced stability, reduced immunogenicity or non-immunogenic or illicit a desired immune response. Example 19 provides recombinantly expressed human growth hormone placental variant (GH2) comprising a H antigen.
The invention further provides novel peptides characterized as having increased serum persistence as more fully described in Example 20. The in-vivo half-life assay in rat model provides evidence of increased serum persistence of GH2 comprising a H antigen as compared to the aglycosylated GH2 as evidenced in
Pharmaceutical Compositions and Pharmaceutical Administration
Another aspect of the invention is a composition as defined above which is a pharmaceutical composition and further comprises one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be in the form of an aqueous suspension. Aqueous suspensions contain the novel compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or homogeneous suspension. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
Pharmaceutical compositions may be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally, intradermal, topically or intratracheal for human or veterinary use.
The protein, peptide, antibody and antibody-portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the protein, peptide, antibody or antibody portion.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., protein, peptide, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
The protein, peptide, antibody and antibody-portions of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
In certain embodiments, an antibody or antibody portion of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The above disclosure generally describes the present invention. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the present invention. Changes in form and substitution of equivalents are contemplated as circumstances suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Plasmids in this study were constructed using standard homologous recombination in yeast (Shanks R M, Caiazza N C, Hinsa S M, Toutain C M, O'Toole G A: Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl Environ Microbiol 2006, 72(7):5027-5036.)). Plasmids were recovered from yeast and transferred to E. coli strain DH5a for confirmation via PCR and/or sequencing. The following list describes plasmids constructed during the course of this study. The plasmid name is followed by the inserted genes/sequences in order from 5′-3′ followed by the vector in parentheses. Glycan expression plasmids were constructed in vector pMW07 (Vaderrama-Rincon et al.). Protein expression plasmids were typically constructed in vector pTRCY. Sugar nucleotide synthesis plasmids were cloned in pTrcY, pMQ70.
In order of figures:
pMW07: (vector) pBAD, ChlorR, ura3, CEN ORI [19]
pDis-07: galE, pglB, pglA (pMW07)
pDisJ-07: galE, pglB, pglA, wbnJ (pMW07)
pTrcY: (vector) pTRC, AmpR, pBR322 ORI, 2μ
pMBP-hGH-Y: ssdsbA-malE (no signal sequence)-hexahistidine-TEV-hGH (pTrcY)
pTrc-spMBP-GT-MBP-GT: ssmalE-4×dqnat-malE-4×dqnat-hexahistidine (pTrc99a) [20]
pDisJD-07: galE, pglB, pglA, neuD, neuB, neuA, neuC, wbnJ (pMW07)
pTrc-spTorA-GFP-GT: sstorA-gfp-4×dqnat-hexahistidine (pTrc99a). [20]
pJDLST-07: galE, pglB, pglA, neuD, neuB, neuA, neuC, lst, wbnJ (pMW07)
pMG4×-Y: ssdsbA-malE-3×TEV-glucagon-4×dqnat hexahistidine (pTrcY)
pMG1×-Y: ssdsbA-malE-3×TEV-glucagon-1×dqnat-hexahistidine (pTrcY)
pMG1×D-Y: ssdsbA-malE-3×TEV-glucagon-1×dqnat-hexahistidine, neuDBAC (pTrcY)
pJDPdST6-07: galE, pglB, pglA, neuD, neuB, neuA, neuC, Pdst6 (pMW07)
pJCstIIS-07: galE, pglB, pglA, neuS, neuB, neuA, neuC, cstII260, wbnJ (pMW07)
pJLic3BS-07: galE, pglB, pglA, neuS, neuB, neuA, neuC, lic3B, wbnJ (pMW07)
pNeuD-Y: neuD (pTrcY)
pMBP4×-Y: ssdsbA-malE-4×GlycTag-hexahistadine (pTrcY)
pCstII*SiaD-Y: cstII1535260-siaD (pTrcY)
pCstIISiaD-Y: cstII260-siaD (pTrcY)
pJK-07: galE, pglB, pglA, wbnJK (pMW07)
pGNF-70: galE(Cj), galE(K12), gmd, fcl, gmm, cpsBG (pMQ70)
pTnfaFab4×-Y: tnfα light chain, tnfα heavy chain-4×dqnat-hexahistidine (pTrcY)
pMG1×GNF-Y: ssdsbA-malE-3×TEV-glucagon-1×dqnat-hexahistidine, galE (CJ), galE(K12), wbnK, gmd, fcl, gmm, cpsBG (pTrcY)
pMG1×KGF-Y: ssdsbA-malE-3×TEV-glucagon-1×dqnat-hexahistidine, galE(Ec), wbnK, gmd, fcl, gmm, cpsBG (pTrcY)
pG4-His-GNF-Y (ssdsbA-malE-1×TEV-hGHv-hexahistidine, galE Cj, galE Ec, gmd, fcl, gmm, cpsBG (pTrcY)
Strains (in order of figures)
MC4100
MC4100 ΔwaaL
MC4100 ΔwaaL ΔnanA
MC4100 ΔnanA
LPS1 ΔwaaL
LPS1
E. coli MC4100 was selected as a host for functional testing because it does not natively express glycan structures containing sialic acid and it has served as a functional host for glycosylation previously (Vaderrama-Rincon et al. “An engineered eukaryotic protein glycosylation pathway in E. coli,” Nat Chem Bio 8, 434-436 (2012)). The mutations in the waaL, and nanA genes were transduced from the corresponding mutant in the Keio collection. The kan cassette was later removed from the MC4100 ΔnanA strain. For surface expression of glycans, plasmids of interest were used to transform MC4100, MC4100ΔnanA, or MC4100ΔnanA ΔwaaL. Protein glycosylation experiments were performed in strains as indicated.
Media and Reagents
Antibiotic selection was maintained at: 100 μg/mL ampicillin (Amp), 25 μg/mL chloramphenicol (Chlor), 10 ug/mL tetracycline (Tet) and 50 μg/mL kanamycin (Kan). Routine growth of E. coli cultures was performed in LB medium supplemented with glucose at 0.2% (w/v) and antibiotics as necessary. For expression of PSA plasmids, LB medium was supplemented with sialic acid (Sigma or Millipore) at a final concentration of 0.25% (w/v) and the medium was adjusted to pH ˜7.5 and sterilized. Plasmids for glycan and protein expression were induced with the addition of L-arabinose at 0.2% or isopropyl β-d-thiogalactoside (IPTG) at 100 mM respectively. Yeast FY834 was maintained on YPD medium and synthetic defined-Uracil medium was used to select or maintain yeast plasmids.
Cell-Surface Glycan Detection
Dot blots were performed using 2.5 μl or 4 μl of overnight LB culture from strain indicated. Cells were spotted on a nitrocellulose membrane and PSA glycans were detected by immunoblot as below. For flow cytometry cultures were inoculated in LB supplemented with antibiotics as appropriate. Analysis was performed using lectins as indicated and a BD FACScalibur flow cytometer.
Protein Expression and Purification
Strains to be harvested for analysis of N-glycosylation were inoculated into LB with the appropriate antibiotics and incubated with shaking at 30° C. until the cultures reached an OD600 of 1.5-2. Plasmids for glycan expression were induced with the addition of arabinose and production of the acceptor protein was induced with IPTG. Cultures were harvested 16-18 h post induction. Cell lysis and purification of glycoproteins was performed using the Ni-NTA kit (Qiagen) for small scale cultures (50-100 mL). Larger preparations were in binding buffer (50 mM Tris, 30 mM Na2HPO4, 30 mM Imidazole, 500 mM NaCL pH=7.4) purified using HisTrap FF column (GE Healthcare) followed by elution with binding buffer containing a final concentration of 500 mM imidazole. Purification over a DEAE HiTrap FF column (GE Healthcare) typically followed using 20 mM Tris pH 6.8 as the binding buffer and elution with a gradient of 0-500 mM NaCl in the same buffer. For purification of glycoprotein containing the T antigen glycan, protein was exchanged to 10 mM HEPES pH 7.5, 0.15 M NaCl, 0.1 mM CaCl2, 0.01 mM MnCl2 and further separated using Peanut agglutinin (PNA)-agarose (Vector labs). Galactose was used to isolate glycoprotein.
Protein Analysis
Proteins were separated by SDS-polyacrylamide gels (Lonza), and Western blotting was performed as described previously (DeLisa M P, et al., Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway. Proc Natl Acad Sci USA 2003, 100(10):6115-6120.). Briefly, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and membranes were probed with one of the following: anti-6×-His antibodies conjugated to HRP (Sigma), anti-PSA-NCAM (Millipore), or PNA-Biotin (Vector Labs). In the case of the anti-PSA antiserum, anti-mouse IgG-HRP (Promega) was used as the secondary antibody. For PNA-Biotin, Streptavidin-HRP (Vector Labs) was used for secondary detection.
The T antigen glycan (T-antigen, Galβ1,3 GalNAcα-) is a structure found at the core of many human related human glycans. In order to assemble a glycan containing the human T antigen in E. coli, a plasmid was constructed for expression of the glycosyltransferase and sugar nucleotide epimerase activities necessary to produce this structure using the native UndPP-GlcNAc as a substrate. Plasmid pMW07 (Valderrama-Rincon et al.) was used as the vector because it contains a low copy number origin of replication (ORI), an inducible pBAD promoter, and a yeast ORI allowing for cloning via homologous recombination in Saccharomyces cerevisiae. The sequence of pMW07 is provided as SEQ ID NO: 1.
To generate a disaccharide glycan with the structure GalNAcα1,3 GlcNAc, a plasmid was constructed to express the C. jejuni GalNAc transferase PglA, and the epimerase GalE to promote synthesis of the UDP-GalNAc substrate. The gene encoding the OST PglB from C. jejuni was also included for use in glycosylation in the future. A PCR fragment including galE, pglB, and pglA along with linearized pMW07 was used to co-transform S. cerevisiae and cloning was performed by homologous recombination in yeast as previously described (Shanks et al.). The sequences of these genes are provided as SEQ ID NOs: 2, 3 and 4 respectively. Plasmid was isolated from colonies selected on synthetic defined-uracil medium and used to transform E. coli DH5a for confirmation of construct. The resulting plasmid was designated pDis-07.
The human Thomsen-Friedenreich or T-antigen glycan consists of Galβ1-3GalNAcα structure. Galactose transferase WbnJ from E. coli 086 was selected as the glycosyltransferase to incorporate the terminal galactose residue because it is reported to attach galactose in a β1,3 linkage to a GalNAc residue and is a native bacterial enzyme (Yi W, Shao J, Zhu L, Li M, Singh M, Lu Y, Lin S, Li H, Ryu K, Shen J et al: Escherichia coli O86 O-Antigen Biosynthetic Gene Cluster and Stepwise Enzymatic Synthesis of Human Blood Group B Antigen Tetrasaccharide. Journal of the American Chemical Society 2005, 127(7):2040-2041.). The wbnJ gene was amplified from a synthetic plasmid from Mr. Gene and homologous recombination in yeast was used to combine the resulting PCR product and linearized pDis-07 plasmid. The resulting plasmid is named pDisJ-07 and contains the following genes as a synthetic operon under control of a pBAD promoter: (5′-3′) galE, pglB, pglA, wbnJ. The sequence of wbnJ is included as SEQ ID NO: 5.
In their native context, the substrates for both glycosyltransferases PglA and WbnJ are saccharides assembled on the lipid undecaprenylpyrophosphate (UndPP). As part of the E. coli K12 LPS synthesis pathway, a GlcNAc residue is first added to UndPP via the activity of native WecA and the resulting GlcNAc is then transferred to the lipid A core oligosaccharide in the periplasm by the WaaL ligase. Finally, the lipid A moiety is transported to the outer membrane resulting in cell-surface display of the glycans. Cells carrying deletions in the waaL gene are unable to transport UndPP-linked glycans to the cell surface and thus, this mutation is useful for confirming that a glycan is linked to UndPP.
The waaL (rfaL) gene has been previously mutated as part of the Keio collection and the resulting strain rfaL734(del)::kan (JW3597-1) (Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko K A, Tomita M, Wanner B L, Mori H: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2.) was obtained from the Yale Coli Genetic Stock Center (CGSC). P1 vir phage was used to transduce the waaL mutation into an MC4100 recipient to make strain MC4100 ΔwaaL::kan. Plasmid pCP20 was used to then remove the kan cassette (Datsenko K A, Wanner B L: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 2000, 97(12):6640-6645.) resulting in strain MC4100 ΔwaaL.
Flow cytometry was used to analyze the cell surface glycans produced by E. coli MC4100 expressing pDisJ-07 to confirm the presence of a galactose-terminal structure compared to control plasmid pDis-07. Cultures were inoculated in 1.5 mL tubes containing 1000 μl LB supplemented with 25 μg/ml chloramphenicol and 0.2% arabinose (v/v). After a 24 hour incubation shaking at 30° C., the cultures were pelleted and resuspended in 200 μl PBS. 100 μl aliquots of each were heated at 95° C. for 10 minutes and cooled to room temperature. 400 μl PBS was added to each sample and 3 μl of fluorescein labeled Soy Bean Agglutinin (SBA, Vector laboratories) or Ricinus Communis Agglutinin I (RCA I, vector laboratories) which preferentially binds to galactose terminal glycans. Samples were incubated on a rocking platform at room temperature for 10 minutes in the dark prior to flow cytometry.
Flow cytometry with the RCA I lectin was suggests the presence of a galactose terminal glycan on the cell surface of MC4100 cells expressing pDisJ-07 but not pDis-07 (
The OST PglB is utilized to transfer UndPP-linked oligosaccharides to specific asparagine residues. This requires a target protein bearing the PglB recognition site consisting of the D/E X1 N X2 S/T sequon to be localized to the periplasm and the presence of an appropriate glycan substrate. For this study, we also constructed vector pTRCY for use in expression of glycoproteins.
pTRCY was cloned via homologous recombination in S. cerevisiae by adding the URA3 gene and the yeast 2 micron ORI to pTRC99a thus generating a novel vector capable of replicating in yeast. The URA3 gene and 2 micron ORI were amplified with primers containing homology to vector pTRC99a for insertion between the pBR322 ORI and lacI gene. The sequence of vector pTRCY is provided as SEQ ID NO: 6.
hGH was cloned as a c-terminal translational fusion following a signal peptide from E. coli DsbA, MBP, hexahistidine tag, and a tev cleavage site. The hGH gene was further modified to contain a single glycosylation acceptor site DQNAT and the final construct is named pMBP-hGH-Y. The sequence of the gene fusion is supplied as SEQ ID NO: 7.
Strains MC4100ΔnanAΔwaaL bearing plasmids pDisJ-07 and pMBP-hGH-Y or pMBP-hGH-Y alone were grown under ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml) or ampicillin (100 μg/ml) selection respectively. pDisJ-07 is induced with the addition of 0.2% (v/v) arabinose and IPTG (0.1 mM) after approximately 16 h to induce protein production. The protein was partially purified by nickel affinity chromatography and treated with TEV protease (Sigma) to release hGH prior to analysis by SDS-PAGE and Coomassie staining The visible mobility shift in the presence of the pDisJ-07 plasmid is consistent with glycosylation (
To further probe the identity of the glycan produced upon expression of pDisJ-07, we extracted the lipid-linked oligosaccharides and analyzed the released glycans by mass spectrometry. A 1:100 inoculum was use to seed 4 250 mL cultures containing LB supplemented with 25 μg/ml chloramphenicol. Cultures were grown at at 30° C. and induced when the ABS600 reached ˜2.0. Cells were harvested after ˜20 hours for isolation of lipid-linked oligosaccharides by the method of Gao and Lehrman (Gao N, Lehrman M: Non-radioactive analysis of lipid-linked oligosaccharide compositions by fluorophore-assisted carbohydrate electrophoresis. Methods Enzymol 2006, 415:3-20.). Briefly, pellet was resuspended in 10 mL methanol and lysed by sonication. Material was dried at 60° C. and subsequently resuspended in 1 mL 2:1 chloroform:methanol (v/v, CM) via sonication and material was washed two times in CM. The pellet was then washed in water then lipids were extracted with 10:10:3 chloroform:methanol:water (v/v/v, CMW) followed by methanol. The CMW and methanol extracts were combined and loaded onto a DEAE cellulose column. CMW was used to wash the column and lipid-linked oligosaccharides were eluted with 300 mM NH4OAc in CMW. The lipid-linked oligosaccharides were extracted with chloroform and dried.
To release the glycans from the lipids, the material was resuspended in 1.5 mL 0.1N HCl in 1:1 isopropanol:water (v/v). The solution was heated at 50° C. for 2 hours and then dried at 75° C. Residue was suspended in water saturated butanol and the aqueous phase containing the glycans was dried, resuspended in water, and purified with AG50W-H8(hydrogen atom) cation exchange resin followed by Ag1-X8 (formate form) anion exchange resin.
To confirm the identity of the terminal glycan, the sample was divided and half was treated with β1,3 galactosidase (NEB) and half with a water control. Samples were incubated at 37° C. for 48 hours. Mass spectrometry revealed a major peak (m/z 609) in the buffer control sample (
The human T antigen is frequently found to be abberently expressed in cancers and is thus known as a pancarcinoma antigen. It has been estimated that up to 90% of carcinomas carry the T antigen on the cell surface including carcinomas of the breast, colon, bladder, lung, prostate, liver, and stomach [21, 22]. Because of its specific expression in multiple cancers, the T antigen is of interest as a target of anti-cancer immunotherapy treatments.
To enable preparation of an immunogen bearing multiple T antigen glycans on a carrier protein in vivo, a plasmid was obtained (pTrc-spMBP-GT-MBP-GT) that encodes the MBP protein fused to a 4×GlycTag (bearing 4 DQNAT motifs) at both the N- and C-termini and a 6×-his tag for purification purposes [20]. A second plasmid (pJD-07) was constructed to express a uniform glycan terminating in the T antigen. pJD-07 was cloned using homologous recombination in yeast by insertion of the neuDBAC genes into pDisJ-07. pJD-07 contains the following genes as a synthetic operon under control of a pBAD promoter: (5′-3′) galE, pglB, pglA, neuD, neuB, neuA, neuC, and wbnJ.
E. coli strain MC4100 ΔwaaL was transformed with plasmids pTrc-spMBP-GT-MBP-GT and either pJD-07 for expression of target protein glycosylated with the T antigen glycan or pMW07 for expression of aglycosylated target protein. The target MBP protein expressed from plasmid pTrc-spMBP-GT-MBP-GT contains a total of 8 glycosylation sites (MBP8×DQNAT). Strains were cultured under selection with ampicillin (100 ug/mL) and chloramphenicol (25 μg/mL) at 30° C. and induced at an ABS600 of approximately 1.5 with 0.2% (v/v) arabinose and 0.1 mM IPTG for ˜16 hours. The MBP target protein was purified on a HisTrap FF column (GE Healthcare) followed by DEAE HiTrap FF column (GE Healthcare) and eluted with a NaCl gradient (0-500 mM) in 20 mM Tris pH 6.8. The glycosylated protein was affinity purified with Peanut agglutinin (PNA)-agarose (Vector labs) to isolate protein conjugated to the T antigen glycan. Resulting proteins were separated by PAGE and analyzed by Western blot with α6×-His (left), or biotin conjugated PNA (5 μg/mL, Vector labs) and peroxidase-conjugated streptavidin (1:3333, Vector labs, right) to confirm glycosylation. As expected, the MBP expressed with glycosylation plasmid pJD-07 migrated more slowly than the negative control (pMW07) and reacted with the PNA lectin consistant with glycosylation (
Female C3H mice at approximately 8-10 weeks of age were utilized for this study in groups of 5 with feed and water provided ad libitum. Aglycosylated MBP8×DQNAT and T-antigen-MBP8×DQNAT prepared as described above were adjusted to a concentration of 0.4 mg/mL in PBS. Immediately prior to use, proteins were mixed with an equal volume of Sigma Adjuvant System (Sigma) and mice were immunized through the intraperitoneal (IP) route with 20 μg of protein in a volume of 0.1 mL on days 0, 7, and 13. Serum samples were collected on day −1 (prior to immunization), and on days 14 and 21.
Analysis of Antibody Response
ELISA was used to determine the presence of specific antibodies in the resulting serum. To assess the immune response to the carrier protein, aglycosylated MBP8×DQNAT prepared above was adjusted to a concentration of 2 μg/mL in Coating Buffer (4.2 g/L NaHCO3, 1.78 g/L Na2CO3, pH 9.6) and 50 μL was applied in triplicate to the wells of a PolySorp microtiter plate (Nunc) and incubated overnight at 4° C. Wells were washed in triplicate with 200 μL PBS containing Tween-20 (PBST: 4 g/L NaCl, 0.1 g/L KCl, 0.72 g/L Na2HPO4, 0.12 g/L KH2PO4+0.05% v/v Tween-20) prior to blocking the wells with 200 μL 10% bovine serum albumin (BSA) in PBST for 60 minutes at room temperature. Serum samples were diluted 1:500 in 1% BSA in PBST, and 50 μL of each sample was applied in triplicate on coated wells and incubated at room temperature for 60 min. The plates were washed 4 times with 200 μL PBST then incubated for 60 minutes at room temperature with 50 μL of a 1:5000 dilution of either HRP conjugated anti-mouse IgM or HRP conjugated anti-mouse IgG specific secondary antibody (Jackson ImmunoResearch Laboratories). The microtiter plates were washed 7 times with 200 μL PBST and incubated for 10-30 min with 100 μL of 1-Step Ultra TMB-ELISA (Thermo) at room temperature in the dark. Reactions were stopped with the addition of 100 μL 2N HCl and absorbances were read at 450 nm (
A second ELISA was performed to determine the antibody response to the c-terminal portion of the immunogen using GFP modified with a similar tag. A plasmid was obtained (pTrc-spTorA-GFP-GT) [20] that expresses the GFP protein modified with a 4×GlycTag containing 4 iterations of the DQNAT motif, followed by a 6×-His tag (GFP4×GT). pTrc-spTorA-GFP-GT was used to cotransform MC4100ΔwaaL cells with pMW07 or pJD-07. Resulting strains were cultured under selection with ampicillin (100 ug/mL) and chloramphenicol (25 μg/mL) at 30° C. and induced at an ABS600 of approximately 1.5 with 0.2% v/v arabinose and 0.1 mM IPTG for ˜16 hours. The GFP target protein was purified on a HisTrap FF column (GE Healthcare) followed by DEAE HiTrap FF column (GE Healthcare) and eluted with a NaCl gradient (0-500 mM) in 20 mM Tris pH 6.8. The glycosylated protein was additionally affinity purified with Peanut agglutinin (PNA)-agarose (Vector labs) to isolate protein conjugated to the T antigen glycan.
Resulting T antigen-GFP4×GT, or aglycosylated GFP 4×GT was adjusted to a concentration of 2 μg/mL in Coating Buffer and 50 μL was applied in triplicate to the wells of a PolySorp microtiter plate (Nunc) and incubated overnight at 4° C. The wells were washed 3 times with 200 μL PBST prior to blocking the wells with 10% BSA in PBST for 60 min at room temperature. Serum samples as indicated by were diluted 1:500 in PBST with 1% BSA, and 50 μL was applied in triplicate on coated wells and incubated at room temperature for 60 min. Wells were washed 4 times with 200 μL PBST and incubated for 60 minutes at room temperature with 50 μL of a 1:5000 dilution of HRP conjugated anti-mouse secondary antibody (Promega). After 7 washes with PBST, reactions were developed by addition of 100 μL of 1-Step Ultra TMB-ELISA (Thermo) and incubated for 10-30 minutes in the dark. Reactions were stopped with addition of 100 μl 2N HCl and absorbances were read at 450 nm (
The human (2,3) sialyl-T antigen consists of the T antigen glycan modified with a terminal α2,3 Neuraminic acid (NeuNAc) residue resulting in the following structure: NeuNAcα2,3 Gal β1,3 GalNAcα-. To generate a glycan terminating with the sialyl T antigen structure in an E. coli host, the plasmid described above expressing genes required to synthesize the T-antigen glycan (pDisJ-07) was modified to include a gene encoding a sialyltransferase, and genes whose products comprise the cytidine 5′monophospho-N-acetylneuraminic acid (CMP-NeuNAc) synthesis pathway in E. coli K1.
A region of DNA was amplified from the E. coli K1 genome including the genes neuB, neuA, and neuC using PCR. These encode a Neu5Ac synthase, CMP-Neu5Ac synthetase, and UDP-GlcNAc2-epimerase respectively. The neuD gene was also included as it may help to stabilize the neuB gene product (Daines D A, Wright L F, Chaffin D O, Rubens C E, Silver R P: NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1. FEMS microbiology letters 2000, 189(2):281-284.). The lst gene encoding the N. meningitidis α2,3 sialyltransferase was also amplified and both PCR products along with linearized pDisJ-07 were used to co-transform S. cerevisiae to make resulting plasmid pJDLST-07 by homologous recombination. The sequences of neuB, neuA, neuC, and neuD are provided as Seq ID NOs: 8-11 and the sequence of the lst gene is provided as SEQ ID NO: 12. Plasmid pJDLST-07 contains a synthetic operon under control of the pBAD promoter with genes in the following order: galE, pglB, pglA, neuD, neuB, neuA, neuC, lst, wbnJ.
For use in expressing sialylated glycans, a strain was constructed in which the nanA gene encoding the sialic acid aldolase NanA was targeted for disruption. Deletion of the nanA gene prevents degradation of sialic acid from external sources (Vimr E R, Troy F A: Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J Bacteriol 1985, 164(2):845-853.). The ΔnanA::kan mutation was introduced into MC4100 E. coli via P1 vir phage transduction from the corresponding mutant generated as part of the Keio collection (CGSC #10423, Yale genetic stock center)(Baba et al.). The kanamycin cassette was removed by the method of Datsenko and Wanner (Datsenko et al.). To promote glycosylation, the ΔwaaL::kan mutation was subsequently introduced and cured of kanamycin resistance by the same method as described above.
To permit analysis of sialylated glycopeptide by Mass spectrometry, a Glucagon peptide modified with a 1×GlycTag containing a DQNAT motif was cloned. To construct this plasmid, the DsbA signal peptide sequence and the malE gene (which encodes MBP) were amplified with primers containing homology to vector pTRCY and the sequence for the TEV protease sites. Similarly, glucagon was amplified from a synthetic oligonucleotide with primers containing sequence encoding the TEV protease site or the sequence for the 4×GlycTag and 6×-His tag followed by homology to pTRCY. These PCR products were used with linearized pTRCY to co-transform S. cerevisiae for cloning by homologous recombination to generated plasmid pMG4×-Y. The related plasmid pMG1×-Y is a derivative of pMG4×-Y made by replacing the 4×GlycTag with a 1×GlycTag. Briefly, pMG4×-Y was linearized and an oligonucleotide encoding the 1×GlycTag was used to replace the 4×GlycTag by homologous recombination in S. cerevisiae. The sequence encoding proteins MBP-3TEV-GLUC-4×GlycTag-6H and MBP-3TEV-GLUC-1×GlycTag-6H are provided as SEQ ID NOs: 13 and 14.
In order to generate glycoprotein in vivo containing the human sialyl-T antigen, strain MC4100ΔnanA ΔwaaL described above was used to promote periplasmic accumulation of sialylated glycans. This strain was co-transformed with plasmid pMG1×-Y encoding a glycosylation acceptor protein and pJDLST-07 which expresses the machinery necessary to synthesize the sialyl-T antigen glycan.
An overnight culture consisting of MC4100ΔnanA ΔwaaL pMG1×-Y and pJDLST-07 was used to inoculate a 50 mL culture in LB with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. When the ABS600 reached approximately 1.5 the culture was induced with arabinose to 0.2% and IPTG to 0.1 mM and the cells were harvested by centrifugation approximately 19 hours post-induction. Following cell lysis, protein was purified on a NiNTA column and TEV protease was used to cleave 30 μl of the resulting eluate. The sample was incubated at 30° C. for 3 h and an aliquot was analyzed by mass spectrometry on an AB SCIEX TOF/TOF mass spectrometer using dihydroxybenzoic acid (DHB) as the matrix.
Mass spectrometry revealed major peaks consistent with the expected size of glucagon modified with the sialyl-T antigen (m/z 6251) and the expected size of glycosylated Glucagon bearing the T antigen terminal glycan (m/z 5960) (
One potential strategy for improving sialylation in this system is to increase the intracellular availability of CMP-NeuNAc. Although the necessary biosynthetic genes are present on plasmid pJDLST-07, it was hypothesized that additional copies could improve sialylation. The genes neuDBAC were amplified as a single PCR product and inserted into pMG1×-Y downstream of the glucagon fusion protein using homologous recombination in Saccharomyces cerevisiae. This resulted in creation of plasmid pMG1×D-Y.
Plasmid pMG1×D-Y was combined with pJDLST-07 in strain MC4100ΔnanA ΔwaaL to test glycosylation in 50 mL cultures as described above. Mass spectrometry of the TEV-cleaved peptide product reveals a major peak consistent with the expected size of glucagon modified with the (2,3) sialyl-T antigen containing glycan (m/z 6250). A second smaller peak consistent with the expected size of glucagon modified with the T antigen glycan (m/z 5959) is also detected (
To validate the sialylation of the glucagon peptide a neuramindse treatment was performed. Strain MC4100ΔnanA Δwaal carrying plasmids pMG1×D-Y and pJDLST-07 is grown in a 50 mL culture in LB with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol and induced with 0.2% arabinose and 0.1 mM IPTG for approximately 16 h. The recombinant protein is purified from the lysate with nickel affinity chromatography and the eluate is buffer exchanged in 50 mM Tris pH 8.0 100 mM NaCl and concentrated prior to incubation for 3 h at 30° C. with TEV protease. The protein is divided and incubated with α2,3 neuraminidase (NEB) or a buffer control for 2 hours at 37° C. prior to analysis by Mass spectrometry (
Glycosylation is a well-known strategy for improving the stability of a protein and is a rational approach for improving both in vivo or in vitro persistence. In order to determine if N-glycosylation in bacteria could be utilized for this purpose, the (2,3) sialyl-T antigen was conjugated to conjugated to glucagon for analysis.
Plasmid pMG1×D-Y was combined with pJDLST-07 in strain MC4100ΔnanAΔwaaL to generate sialylated glucagon and resulting cells were used to inoculate a 100 mL culture containing LB medium supplemented with 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. To generate aglycosylated glucagon, Origami2 ΔnanA ΔwaaL Δgmd::kan harboring plasmid pMG1×MCB-07 was used to inoculate a 100 mL culture containing LB medium and 100 μg/mL ampicillin. This strain was selected based on our ability to detect the aglycosylated peptide. Both cultures were grown with shaking at 30° C. until an ABS600 of ˜2.3 was reached then were induced with 0.1 mM IPTG (both) and arabinose 0.2% v/v (glycosylated only). Cultures were maintained at 30° C. overnight. The glucagon fusion protein was isolated by Ni affinity (NiNTA, Qiagen) and the eluate was concentrated. 1 μl TEV protease was added to 50 μl of glycosylated or aglycosylated glucagon and the reaction was incubated at 30° C. for 3 hours then transferred to 37° C. Presence of glucagon was monitored over time by MALDI TOF mass spectrometry. The aglycosylated glucagon was no longer detected after 21 hours of incubation whereas a peak at the expected m/z of glucagon bearing the (2,3) sialyl T antigen was still the most prominent. (
The human sialyl-T antigen consists of the T antigen glycan modified with a terminal α2,3 Neuraminic acid (NeuNAc) residue resulting in the following structure: NeuNAcα2,3 Gal β1,3 GalNAcα-. A related glycan was also explored differing only in the linkage of the terminal NeuNAc residue: NeuNAcα2,6 Galβ1,3 GalNAcα. To generate a glycan terminating with the 2,6 sialylated T antigen structure in an E. coli host, the plasmid described above expressing genes required to synthesize the 2,3 sialyl T-antigen glycan (pJDLST-07) was modified by replacing the lst gene with the a gene encoding a 2,6 siayltransferase.
To express the structure NeuNAcα2,6 Galβ1,3 GalNAcα1,3 GlcNAc, a codon-optimized version of Pdst6 encoding a 2,6 sialyltransferase from Photobacterium damselae JT0160 was synthesized by Mr. Gene and amplified by PCR. The Pdst6 gene was cloned in place of the lst gene in pJDLST-07 by homologous recombination in yeast to create pJDPdST6fl-07. The sequence of the PdST6 gene is provided as SEQ ID NO 15. Plasmid pJDPdST6fl-07 contains a synthetic operon under control of the pBAD promoter with genes in the following order: galE, pglB, pglA, neuD, neuB, neuA, neuC, Pdst6, wbnJ.
In vivo synthesis of proteins carrying an N-glycan terminating in 2,6 Sialic acid
In order to generate glycoprotein in vivo containing the 2,6 sialylated-T antigen, strain MC4100ΔnanA ΔwaaL described above was used to promote periplasmic accumulation of sialylated glycans. This strain was co-transformed with plasmid pMG1×D-Y encoding a glycosylation acceptor protein and pJDPdST6fl-07 which expresses the machinery necessary to synthesize the 2,6 sialic acid-terminal glycan.
An overnight culture consisting of MC4100ΔnanA ΔwaaL pMG1×D-Y and pJDPdST6fl-07 was used to inoculate a 50 mL culture in LB with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. When the ABS600 reached approximately 1.5 the culture was induced with arabinose to 0.2% and IPTG to 0.1 mM, and the cells were harvested by centrifugation approximately 19 hours post-induction. Following cell lysis, protein was purified on a NiNTA column and TEV protease was used to cleave 30 μl of the resulting eluate. The sample was incubated at 30° C. for 3 h and an aliquot was analyzed by mass spectrometry on an AB SCIEX TOF/TOF mass spectrometer using dihydroxybenzoic acid (DHB) as the matrix.
Mass spectrometry revealed major peaks consistent with the expected size of glucagon modified with the 2,6 sialylated T antigen (m/z 6257) and the expected size of glycosylated Glucagon bearing the T antigen terminal glycan (m/z 5964) (
To confirm that the glycan produced from plasmid pJDPdST6fl-07 does not in fact terminate in the 2,3 sialyl T antigen, the glycopeptide generated above was treated with neuraminidases with different specificities. The sialylated glucagon peptide was divided and incubated for 30 minutes at 37° C. with α2,3 Neuraminidase (NEB) or Neuraminidase (NEB) which is reported to hydrolyze α2,3-, α2,6-, and α2,8-linked sialic acid from glycans or glycoprotein. The resulting peptides were analyzed by Maldi TOF Mass spectrometry (
There are several bacteria known to produce polysialic acid (PSA) glycans including E. coli K1 and strains of Neisseria meningitidis. In these strains PSA forms a protective capsular polysaccharide. The PSA capsule is well-studied in E. coli K1 but the lipid substrate for PSA synthesis has not been identified. In order to adapt PSA for N-glycosylation, it is likely necessary to direct its synthesis on a substrate appropriate for the OST and provide the necessary disialic acid ‘primer’ required for the PSA polymerase to extend sialylation. The glycan described herein terminating in the human T antigen is a good candidate for polysialylation because it is efficiently used in glycosylation in this system. To elaborate the T antigen with a disialic acid motif, the genes cstII from C. jejuni and lic3B from H. influenza were selected based on their reported bifunctional 2,3 and 2,8 sialyltransferase activities. For polymerization the neuS gene was chosen for successive 2,8 sialylation because it is an E. coli gene.
To clone plasmids use in exploring polysialylation, synthetic versions of the cstII and lic3b genes were obtained (Mr. Gene). The sequences of cstII and lic3b are supplied as SEQ ID NOs: 16 and 17.
A truncated version of the gene cstII encoding the first 260 amino acids of the bifunctional α2,3 α2,8 sialyltransferase was cloned with neuBAC, the E. coli K1 polysialyltransferase neuS (SEQ ID NO: 18), and the genes to synthesize the T antigen glycan using homologous recombination in Saccharomyces cerevisiae. The full length bifunctional α2,3 α2,8 sialyltransferase lic3b was also cloned in the same manner. The resulting plasmids are called pJCstIIS-07 and pJLic3bS-07.
Plasmid pJCstIIS-07 was used to transform MC4100 ΔnanA and MC4100 ΔnanAΔwaaL for functional testing. A single colony is used to inoculate 1 mL of LB medium containing 0.25% NeuNAc (w/v), 25 μg/ml chloramphenicol and 0.2% (v/v) arabinose. Cultures were grown approximately 18 hours at 30° C. in a 1.5 mL tube and pelleted. After washing with PBS, cultures are normalized by optical density, heated for 10 min at 95° C., and the whole cells are spotted on nitrocellulose when cooled. The membrane is blotted with an anti-PSA antibody followed by anti-mouse-horseradish peroxidase (
To test the putative PSA-terminal glycan in a glycosylation reaction, the MC4100ΔwaalΔnanA strain was transformed with pMG4×-Y encoding a glycosylation acceptor protein. The resulting strain was transformed with plasmid pDisJ-07 or pJLlc3B-07. Resulting strains were grown in 50 mLs LB+/−0.25% NeuNAc and appropriate antibiotics. Cultures are induced at an approximate optical density between 2-4 with 0.2% arabinose and 0.1 mM IPTG. Proteins were purified by nickel affinity chromatography, concentrated, and treated with TEV protease prior to analysis by Western blot (
Detection with the αPSA antibody (
The neuD gene is part of the genetic locus for PSA synthesis in E. coli K1 and other strains that produce sialylated glycans although there are conflicting assignments of NeuD function. In order to confirm the importance of NeuD in the sialylation platform it was cloned as an individual gene into vector pTRCY using homologous recombination in Saccharomyces cerevisiae. The resulting plasmid containing NeuD under the control of the Trc promoter is called pNeuD-Y.
To test pNeuD-Y, this plasmid was used with pJLic3BS-07 to cotransform strain MC4100ΔnanA. A single colony is used to inoculate 1 mL of LB medium containing 25 μg/ml chloramphenicol and 0.2% arabinose. LB medium was made with or without sialic acid at a final concentration of 0.25% (w/v) and was adjusted for pH and filter sterilized. Cultures are grown approximately 18 hours at 30° C. in a 1.5 mL tube and the cultures are pelleted. After washing with PBS, cultures are normalized by optical density and heated for 10 min at 95° C. and the whole cells are spotted on nitrocellulose when cooled. The membrane is blotted with an anti-PSA antibody followed by anti-mouse-horseradish peroxidase (
Reactivity with the PSA antibody suggests the presence of a cell surface PSA glycan in the presence of pNeuD-Y or NeuNAc. This result suggests the importance of NeuD in production of sialylated compounds in laboratory E. coli (
As an alternative method to confirm the functionality of polysialyltransferases in laboratory E. coli, an ex vivo method for polysialylation was utilized. For this method a lysate is generated from a strain expressing a polysialyltransferase and it is combined with CMP-NeuNAc and an acceptor protein produced in a separate strain. MBP was selected for use as the acceptor protein because it is expressed and glycosylated efficiently in this system.
To prepare the acceptor protein plasmid, the coding sequence for MBP modified with the DsbA signal peptide and a 4×GlycTag and hexahistidine motif was subcloned from pTRC99-MBP 4×DQNAT (Fisher A C, Haitjema C H, Guarino C, celik E, Endicott C E, Reading C A, Merritt J H, Ptak A C, Zhang S, DeLisa M P: Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli. Applied and Environmental Microbiology 2011, 77(3):871-881.). The resulting plasmid is termed pMBP4×GT-Y. CstII was also cloned as a translation fusion to the Neisserial polysialyltransferase SiaD (obtained from Genwiz) to make a self-priming polysialyltransferase as described by Willis et al (Willis L M, Gilbert M, Karwaski M-F, Blanchard M-C, Wakarchuk W W: Characterization of the α-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology 2008, 18(2):177-186.). Two versions were cloned using homologous recombination in Saccharomyces cerevisiae resulting in plasmids pCstII-SiaD-Y and pCstII153S-SiaD-Y, the latter of which includes a mutation of isoleusine 53 to cysteine which is reported to improve the α2,8 sialyltransferase activity. The sequence of siaD is provides as SEQ ID NO: 19.
An acceptor glycoprotein was first prepared by addition of the T antigen-containing glycan to the MBP4×GT protein. Plasmids pMBP4×GT-Y and pDisJ-07 were used to transform strain MC4100ΔwaaL. The resulting strain was used to inoculate a 1 L culture containing LB, ampicillin (100 ug/ml), and chloramphenicol (25 ug/ml). The culture was incubated at 30° C. until the optical density reached OD 1.5 and then both glycan and glycoprotein production are induced with 0.2% arabinose and 0.1 mM IPTG respectively. The pellet was harvested after 16 hours and the his-tagged protein purified by nickel affinity chromatography. Eluted protein is buffer exchanged into ex vivo sialylation buffer containing 50 mM Tris 7.5, 10 mM MgCl2 and concentrated.
To prepare the polysialyltransferase lysates, strains MC4100ΔwaaL containing plasmid pTRCY, pCstII-SiaD-Y, or pCstII153S-SiaD-Y were grown in 50 mL cultures containing LB and ampicillin. When the optical density reached 1-5-1.9, protein expression is induced with the addition of IPTG to a final concentration of 0.1 mM and induction is carried out at 20° C. for approximately 16 hours. Pellets were harvested and resuspended in ex vivo sialylation buffer. Following cell lysis, the material is centrifuges at 1000×g for 11 minutes and the supernatant was retained.
For the ex vivo reaction, 20 μl of the MBP glycoprotein was combined with 30 μl of the polysialylation or control lysate and CMP-NeuNAc. Reactions are incubated at 37° C. for 45 minutes prior to analysis by SDS-PAGE and Western blot (
The human blood group O determinant or H-antigen consists of a fucosylated glycan that is similar to the human T antigen. The type III H-antigen structure consists of Fucose α1,2 Galactose β1,3 GalNAc α-. To synthesize a glycan in E. coli terminating in the human H-antigen structure, the genes from the plasmid described above expressing genes required to synthesize the T-antigen glycan were combined with a gene encoding a fucosyltransferase.
Fucosyltransferase WbnK from E. coli O86 was selected because it is a bacterial enzyme that fucosylates a glycan with similar structure in its native context. The sequence of wbnK is provides as SEQ ID NO: 20. A PCR product containing the wbnJ and wbnK genes was generated using a synthetic template from Genewiz. The PCR product was combined with linear pDis-07 plasmid using homologous recombination in yeast to generate plasmid pDisJK-07. The resulting plasmid, pDisJK-07, contains a synthetic operon under control of the pBAD promoter with genes in the following order: galE, pglB, pglA, wbnJ, wbnK.
For use in expressing fucosylated blood group H-antigen, the E. coli strain LPS1 (Yavuz E, Maffioli C, Ilg K, Aebi M, Priem B: Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12. Glycoconjugate journal 2011, 28(1):39-47.) was used to promote accumulation of GDP-fucose (GDP-Fuc). E. coli encodes a native pathway for synthesis of GDP-Fuc however this sugar nucleotide is then normally incorporated into the fucose-containing exopolysaccharide colanic acid. To prevent usage of GDP-Fuc in this competing pathway a mutation is present in the gene wcaJ (ECK2041) encoding a putative UDP-glucose lipid carrier transferase. To further promote glycosylation in this strain, a mutation in the waaL gene was introduced. The waaL (rfaL) gene has been previously mutated as part of the Keio collection and the resulting strain rfaL734(del)::kan (JW3597-1) (Baba et al.) was obtained from the Yale Coli Genetic Stock Center (CGSC). P1 vir phage was used to transduce the waaL mutation into the LPS1 recipient to make strain LPS1 ΔwaaL::kan.
To confirm the glycan structure produced by the glycosyltransferases encoded by pDisJK-07, the plasmid was used to transform strain LPS1ΔwaaL::kan for analysis of the lipid-released oligosaccharides. A 250 mL culture of the resulting strain was grown at 30° C. and induced when the optical density reached an ABS600 around ˜2.0. Cells were harvested after ˜20 hours for isolation of lipid-linked oligosaccharides by the method of Gao and Lehrman. Briefly, pellet was resuspended in 10 mL methanol and lysed by sonication. Material was dried at 60° C. and subsequently resuspended in 1 mL 2:1 chloroform:methanol (v/v, CM) via sonication and material was washed two times in CM. The pellet was then washed in water then lipids were extracted with 10:10:3 chloroform:methanol:water (v/v/v, CMW) followed by methanol. The CMW and methanol extracts were combined and loaded onto a DEAE cellulose column. CMW was used to wash the column and lipid-linked oligosaccharides were eluted with 300 mM NH4OAc in CMW. The lipid-linked oligosaccharides were extracted with chloroform and dried.
To release the glycans from the lipids, the material was resuspended in 1.5 mL 0.1N HCl in 1:1 isopropanol:water (v/v). The solution was heated at 50° C. for 2 hours and then dried at 75° C. Residue was suspended in water saturated butanol and the aqueous phase containing the glycans was dried, resuspended in water, and purified with AG50W-H8 (hydrogen atom) cation exchange resin followed by Ag1-X8 (formate form) anion exchange resin.
Purified oligosaccharides solubilized in water were subjected to incubation with α1,2 fucosidase (NEB) treatment) or a buffer only control and analyzed on an AB SCIEX TOF/TOF mass spectrometer using dihydroxybenzoic acid (DHB) as the matrix (
In order to improve conversion from the T antigen glycan to the fucosylated product, a system was devised in order to allow for expression of additional copies of the biosynthetic machinery for GDP-Fucose, UDP-Gal, and UDP-GalNAc. To accomplish this, the following genes were cloned as a synthetic operon under control of the pBAD promoter in pMQ70: galE (C. jejuni), galE, gmd, fcl, gmm, cpsB, cpsG (E. coli K12) to make plasmid pGNF-70 using homolgous recombination in yeast. The sequences of the E. coli genes cloned in pGNF-70 are provided as SEQ ID NOs: 21-26.
Strain LPS1 ΔwaaL::kan was transformed with plasmids pJK-07 and pGNF-70. The resulting strain was cultured in 250 mL LB medium under ampicillin and chloramphenicol selection and expression of both plasmids was induced at an optical density of approximately 2.0 and induction continued at 30° C. for approximately 16 hours. Pellets were harvested and LLOs were purified as previously described by the method of Gao and Lehrman.
Purified oligosaccharides were analyzed by Mass Spectrometry as described above (
Following analysis of the fucosylated glycan, it is necessary to confirm that the glycan is amenable to use in the glycosylation reaction. The TNFα Fab was selected as an initial target for glycosylation. A codon optimized version of the Fab including signal peptide sequences for each chain was obtained from DNA 2.0 and cloned into pTRCY using homologous recombination in S. cerevisiae to append a 4×GlycTag and hexahistidine tag to the heavy chain. The resulting plasmid is designated pTnfaFab4×-Y. The sequence of the modified TNFα Fab light and heavy chains are supplied as SEQ ID NOs: 27 and 28.
pTnfaFab4×-Y was used to transform strain LPS1 bearing glycosylation plasmid pJK-07 or empty vector pMW07 and the resulting strains were used to inoculate a 50 mL culture of LB and grown under selection of ampicillin and chloramphenicol. At an optical density of ABS600 of 1.5, expression of both plasmids was induced with the addition of 0.2% arabinose and 0.1 mM IPTG and cultures were maintained at 30° C. for approximately 16 hours. Protein was purified using nickel affinity chromatography was subjected to SDS PAGE followed by Western blot with anti Histidine antibody. A mobility shift was apparent for the Fab heavy chain grown in the presence of glycosylation plasmid pJK-07 but not vector pMW07 consistent with glycosylation (
Experiments described above indicated the potential for increasing the relative amount of fucosylated product as determined by Mass spectrometry through expression of additional copies of the GDP-Fucose biosynthetic pathway. A plasmid pMG1×-Y encoding the glycosylation acceptor peptide is modified using yeast homologous recombination to also include the following genes: galE (C. jejuni), galE (E. coli), gmd, fcl, gmm, cpsB, and cpsG to make plasmid pMG1×-GNF-Y. A similar plasmid was cloned in the same manner with the following genes in addition to the glucagon construct: wbnK, galE (E. coli), gmd, fcl, gmm, cpsB, and cpsG termed pMG1×-KGF-Y.
In preparation for glycosylation, strain LPS1 is transformed with plasmid pDisJK-07. To this, plasmids encoding the glycosylation acceptor protein (pMG1×-Y) or the acceptor protein with the GDP-Fucose biosynthetic machinery were added (pMG1×-GNF-Y, pMG1×-KGF-Y). Resulting strains were grown at 30° C. in 50 mL cultures in LB medium with ampicillin and chloramphenicol. Both plasmids were induced with the addition of 0.2% arabinose and 0.1 mM IPTG when the culture reached an approximate optical density of ABS600 1.5. After 16 hours, pellets were harvested and proteins purified by nickel affinity chromatography. Eluate was exchanged into 50 mM Tris, 100 mM NaCl and 30 μl of the concentrated protein was treated with TEV protease for 3 hours to release the glycopeptide.
Glycopeptide was analyzed on an AB SCIEX TOF/TOF mass spectrometer using dihydroxybenzoic acid (DHB) as the matrix (
Glycopeptide prepared from strain LPS1 pJK-07 pMG1×KGF-Y was divided and subjected to treatment with α1,2 fucosidase (NEB) or a buffer control for 8 hours at 37 degrees prior to analysis on an AB SCIEX TOF/TOF mass spectrometer using DHB as the matrix (
In order to examine glycosylation of a recombinant human protein, human growth hormone placental variant (GH2) was adapted for expression in this E. coli platform. Homolgous recombination in yeast was used to fuse the malE gene sequence with a 3′ TEV protease cleavage site to the gene encoding GH2 bearing a c-terminal hexahistidine motif in pTrcY. In addition, the sequence surrounding the native glycosylation site of this protein was modified to encode a DQNAT. The genes found to improve generation of the H antigen glycan (galE Cj, galE Ec, gmd, fcl, gmm, cpsB, cpsG) were inserted after the 3′ end of sequence encoding the GH2 fusion protein to make plasmid pG4-His-GNF-Y. The DNA sequence for the GH2 fusion protein is provided as SEQ ID NO 29.
pG4-HisGNF-Y was used to transform E. coli strain LPS1 for optimal fucosylation with the H antigen glycan. The resulting strain was made electrocompetent and transformed with a second plasmid containing the genes for expression of the glycosyltransferases required to produce the H antigen glycan and oligosaccharyltransferase PglB (pJK-07). To express GH2 and GH2-H antigen, one liter of LB was inoculated with 50 mL of overnight culture and grown at 30° C. under selection with 100 μg/mL ampicillin or 100 ug/mL ampicillin and 25 μg/mL chloramphenicol respectively. Cultures were incubated on a shaking platform at 30° C. until an approximate ABS600 of 3.0 was reached. The culture containing pG4-His-GNF-Y plasmid alone was induced with 0.1 mM IPTG while the culture containing G4-His-GNF-Y and pJK-07 plasmids was induced with 0.1 mM IPTG and 0.2% v/v arabinose for 16 hr at 30° C.
Purification and Determination of Glycoyslation
The cells were pelleted and then resuspended in Ni-NTA column binding buffer with lysozyme at 1 mg/mL. Cells were incubated on ice for 30 minutes then disrupted by sonication with five 10 second pulses. After sonication, the clarified lysate was then filtered through a 5 μm filter and then a 0.45 μm filter and Ni-affinity purified using 5 mL HisTrap FF column (GE Healthcare). The protein was buffer exchanged into DEAE loading buffer (20 mM Tris, pH 6.8) and purified using a 5 mL DEAE HiTrap FF column (GE Healthcare) and eluted with a NaCl gradient (0-500 mM) in 20 mM Tris pH 6.8. Eluted protein was pooled, concentrated, and exchanged into Ni-NTA column binding buffer containing 17 mM 0 Mercaptoethanol (BME) then treated with ˜1000U TEV protease and incubated overnight at room temperature. The success of cleavage from MBP was assessed by coomassie-stained SDS-PAGE. Once the cleavage was completed, the protein was then brought up in 20 mL of Ni-NTA binding buffer and purified by Ni-NTA resin as described above. Fractions were concentrated down to 1 mL and loaded onto a GST/Amylose mixed resin gravity flow column to remove MPB and TEV. hGH was found in the column wash buffer (50 mMTris, 1 mM EDTA, 200 mM NaCl, pH 7.4). Each protein was then concentrated to ˜1 mg/mL and stored at −80° C.
The purified GH2-H antigen was analyzed to assess glycosylation. Purified protein was separated by SDS PAGE and transferred to PVDF for Western blot with detection by with αhGH antibody (Abcam AB9821) (
To determine if glycosylation benefits protein stability, resistance to agitation was compared for glycosylated and aglycosylated forms of GH2. A 0.5 mg/mL solution of each GH2 form was vortexed for various lengths of time over a range of 0-10 minutes. The ABS 405 nm was immediately recorded to determine the turbidity of the solution. Additionally, the ABS280 nm was also monitored to determine if the increase in turbidity was due to aggregation, where loss of soluble protein would result in a decrease in the ABS280 nm reading. Each dataset was plotted and the rate of denaturation was determined (
To determine if glycosylation of GH2 affects receptor binding, both the aglycosylated and glycosylated forms of GH2 were subjected to an ELISA-based receptor binding assay. MaxiSorp ELISA plates were incubated with 2 μg/mL of the ectodomain of the hGH receptor fused to IgG (hGHR) (R and D systems) for 2 hours at room temperature. The plates were subsequently blocked for 1 hour with blocking buffer (5% BSA w/v, 0.1% Tween-20 v/v in PBS) then washed with PBS. A concentration range of 0-500 nM of each GH2 form was incubated in the hGHR-coated ELISA plate for 1 hour at room temperature and subsequently washed with blocking buffer. Each well was incubated with either an anti-HisTag-HRP antibody or an anti-hGH antibody for 1 hour at room temperature and subsequently washed with blocking buffer. In the case of the anti-hGH antibody, a mouse HRP-conjugated 2° antibody was incubated in each well for 45 minutes at room temperature. The wells were then washed and developed with 1-Step Ultra TMB-ELISA (Thermo) and the reaction was stopped with 2 M HCL and read at 450 nm. Each Kd value was determined by plotting the values on GraphPad Prism software. Calculated dissociation constants (Kd) were 1.5+/−0.7×101 nM for the GH2 and 1.6=/−0.9×101 nM for the GH2-H antigen suggesting that the effect of glycosylation on receptor binding is negligible (
In Vivo Half-Life
To determine if glycosylation has any affect to the half-life of GH2, each form was studied in a rat model in vivo half-life assay. A single intravenous bolus dose of 300 μg of a 1 mg/mL GH2 or GH2-H antigen solution was administered to a group of 4 Sprague Dawley male rats. Blood samples were drawn at various time points over a 24 hr period and the serum was separated by centrifugation. An anti-hGH antibody (Abcam) was diluted into a carbonate buffer pH 9.6 to a concentration of 2 ug/mL and used to coat MaxiSorp ELISA plates for 2 hours at room temperature. The plates were then blocked, washed and incubated with the serum of each time point for 30 min at room temperature. An anti-HisTag-HRP conjugate antibody was added to the wells for 30 min at room temperature and subsequently developed with 1-Step Ultra TMB-ELISA (Thermo). The reaction was stopped by adding 2 M HCl and absorbance recorded at 450 nm. Detection of the GH2-H antigen protein was more robust than that of the aglycosylated version, most notably in the last three time points suggesting glycosylation with the H antigen glycan enhanced serum persistence (
An important consideration in the design of strategies for N-glycoprotein production in E. coli is the efficiency of the glycosylation reaction which is likely to be determined by a number of factors. One such factor is any selectivity the OST may display for particular glycan structures. The glycoforms outlined in the preceding examples are all elaborations of an efficiently transferred “base” glycan consisting of a GalNAcα1,3 GlcNAc glycan from which structures including the T antigen, (2,3) sialyl T antigen, H antigen, and (2,6) sialylated T antigen were made. These examples have served in part as tools to illustrate the ability to generate E. coli glycoproteins containing galactosylated, sialylated, and fucosylated glycans, however are not meant to be an exclusive representation of the glycan structures that can be built from the basic structure.
The Lewis×glycan (Galβ1,4[Fucα1-3]GlcNAc) for example in addition to related structures, could be built from the T antigen glycan. To do this, genes encoding glycosyltransferases such as those from Haemophilus influenzae with β1,3 GlcNAc transferase (LsgE) and β1,4 Gal transferase (LsgD) activities, along with an α1,3 fucosyltransferase such as Helicobacter pylori FucT [23] would be inserted into the pdisJ-07 plasmid. This plasmid, when coexpressed with pGNF-70 or pMG1×-GNF-Y to allow sufficient accumulation of required sugar nucleotides, would be expected to result in production of a glycan with the structure Galβ1,4[Fucα1-3]GlcNAcβ1,3Galβ1,3GlcNAc. The sequences of the LsgE, LsgD, and FucT proteins are includes as SEQ ID NOs: 30-32.
Similarly, the H antigen glycan discussed in Examples 15-19 could be further built upon to generate additional related structures. The human blood group determinants AB and O are interrelated structures based on the blood group O glycan (H antigen). E. coli O86 naturally makes an oligosaccharide similar to the human blood group B glycan and thus is a potential source of the galactosyltransferase activity required to extend the H antigen glycan. By inserting the gene encoding the α1,3 galactosyltransferase WbnI[24] into the existing pJK-07 plasmid and expressing it under similar conditions used to generate the H antigen glycan, is expected to result in production of a glycan with the structure Galα1,3[Fuc α1,2] Galβ1,3 GalNAc α1,3GlcNAc. Similarly, an α1,3 GalNAc transferase such as BgtA from Helicobacter mutelae [25] could be used to generate a glycan containing the A antigen with the structure GalNAcα1,3[Fuc α1,2] Galβ1,3 GalNAc α1,3GlcNAc. The amino acid sequences of WbnI and BgtA are included as SEQ ID NOs: 33-34.
It is further expected that the oligosaccharides described herein could be assembled on an alternate UndPP-linked sugar. Alternatives may include GalNAc which can be attached to UndP by GNE from E. coli 0157 [26] (SEQ ID NO: 35) or Bacillosamine through the activity of C. jejuni glycosyltransferase PglC (SEQ ID NO: 36) and sugar nucleotide synthesis proteins PglFED[27] (SEQ ID NOs: 37-39).
N. meningitidis
P. damsela
Campylobacter jejuni
H. influenzae
E. coli K1
N. meningitidis
E. coli O86
E. coli K12
E. coli K12
E. coli K12
E. coli K12
E. coli K12
E. coli K12
Haemophilus influenzae
Haemophilus influenzae
Helicobacter pylori
E. coli 086
Helicobacter mustelae
E. coli
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
E. coli
This application is related to U.S. Provisional Application No. 61/801,948, filed Mar. 15, 2013, which is herein incorporated by reference, in its entirety, for all purposes.
This invention was made with government support under grant numbers 1R43GM093483-01, 5R43AI091336-01 and 5R43AI091336-02 by the National Institutes of Health. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/029897 | 3/15/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61801948 | Mar 2013 | US |
