Patent Grant
10597664
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 11, 2015, is named 8164.014.PCT_SL.txt and is 14,566 bytes in size.
This application is a continuation-in-part of U.S. application No. 15/h filed Jul. 27, 2016, which issued as U.S. Pat. No. 10,093,704 on Oct. 9, 2018, which is a National Stage Application, under 35 U.S.C. § 371, of International Application No. PCT/US2015/14130 filed Feb. 2, 2015, which claims priority to U.S. Provisional Application No. 61/934,377 filed Jan. 31, 2014, the entirety of each of which is specifically incorporated by reference.
The present invention relates to the field of recombinant protein production in bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of soluble recombinant CRM197 protein from E. coli. The invention also relates to purification and characterization methods for CRM197 as well as uses of the CRM197 produced by the method.
Diphtheria toxin (DT) is a proteinaceous exotoxin synthesized and secreted by pathogenic strains of Corynebacterium diphtheriae. These pathogenic strains contain a bacteriophage lysogen that carries the toxin gene. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified histidine 715 (diphthamide) of the elongation factor EF-2. This post-translational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. The A fragment of DT (also named C domain) carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication. The R domain, carried on the B fragment, mediates binding to receptors on the host cell surface and the T domain, also carried on the B fragment, promotes the pH-dependent transfer of fragment A to the cytoplasm. An Arginine-rich disulfide-linked loop connects fragment A to fragment B (or domain C to domains TR). This inter-chain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186. The isolation of various non-toxic and partially toxic immunologically cross-reacting forms of diphtheria toxins (CRMs or cross reacting materials) resulted in discovery of CRM197 (Uchida et al., Journal of Biological Chemistry 248, 3845-3850, 1973; see also Giannini et al. Nucleic Acids Res. 1984 May 25; 12(10):4063-9). Preferably, CRMs can be of any size and composition that contain all or a portion of DT.
CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution G52E. This mutation causes intrinsic flexibility of the active-site loop in front of the NAD-binding site and reduces the ability of CRM197 to bind NAD and eliminates toxic properties of DT (Malito et al., Proc Natl Acad. Sci. USA 109(14):5229-342012) Like DT, CRM197 has two disulfide bonds. One disulfide joins Cys186 to Cys201, linking fragment A to fragment B. A second disulfide bridge joins Cys461 to Cys471 within fragment B. Both DT and CRM197 have fragment A-associated nuclease activity (Bruce et al., Proc. Natl. Acad. Sci. USA 87, 2995-8, 1990).
CRM197 is commonly used as the carrier protein for protein-carbohydrate and hapten-protein conjugates. As a carrier protein, CRM197 has a number of advantages over diptheria toxoid as well as other toxoid proteins, many of which have been documented (Shinefield Vaccine, 28:4335, 2010, Broker et al, Biologicals, 39:195 2011). For example, since CRM197 is genetically detoxified, it retains a larger complement of lysines, which are used for conjugation but are blocked by chemical toxoiding. CRM197 has proven to be an effective carrier protein for Streptococcus pneumonia capsular polysaccharides, as evidenced by the success of PREVNAR™ (Pfizer), a vaccine consisting of up to 13 capsular polysaccharides chemically linked to CRM197. There is also evidence suggesting that compared with tetanus toxoid, there is less carrier-induced suppression of the immune response, especially when there are many individual polysaccharides linked to the same carrier protein.
CRM197 and native DT have a similar affinity for the diphtheria toxin receptor (DTR), which has an identical amino acid sequence to the HB-EGF precursor pro-HB-EGF (Mitamura et al., J. Biol. Chem. 272(43):27084-90, 1997). CRM197 binds to the soluble form of HB-EGF, as well as to the membrane form pro-HB-EGF, and inhibits HB-EGF mitotic action by preventing its binding to EGF receptor. Thus CRM197 may also have a future role in cancer therapy (Miyamoto et al., Anticancer Res. November-December 27(6A):3713-21, 2007).
CRM197 has been produced in the original host Corynebacterium, but yields are low, typically <50 mg/L and, in addition, Corynebacterium growth is relatively slow as compared with, for example, E. coli. There are proprietary strains of Corynebacterium that have been engineered to produce CRM197 at higher levels (U.S. Pat. No. 5,614,382). CRM197 has also been expressed in a proprietary strain of Psuedomonas fluorescens and expressed at high levels. Production of CRM197 in E. coli would be advantageous since E. coli is a BL1 level organism that is inexpensive to culture and propagate. Production of CRM197 in E. coli has mainly resulted in insoluble inclusion bodies (generally insoluble), which then requires a difficult refolding process, resulting in low yields. A method for the overexpression of soluble tag free CRM197 in E. coli suitable for the large quantity protein production, has not been reported. Thus, there is a need for better methods to produce CRM197 in an efficient and cost-effective manner.
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provide new compositions and methods for producing CRM.
One embodiment of the invention is directed to methods of producing all or a portion of a CRM protein, such as preferably CRM197, comprising; providing a recombinant cell that contains an expression vector, wherein the recombinant cell has been modified to shift the redox status of the cytoplasm to a more oxidative state as compared to an unmodified recombinant cell and the expression vector contains an inducible promoter functionally linked to a CRM coding sequence, a spacer sequence between a ribosome binding site and an ATG codon, an expression enhancer region upstream of the CRM coding sequence; inducing the expression vector to produce CRM protein; and isolating the CRM protein expressed. The recombinant cell may be a eukaryotic cell or a prokaryotic cell. Preferably the recombinant cell is a prokaryotic cells such as, for example, an E. coli cell or a derivative or strain of E. coli. Preferably, the recombinant cell modification comprises a reduced activity of one or more disulfide reductase enzymes such as, for example, one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, a protein reductase or a glutathione reductase. Preferably the reduced activity of the one or more disulfide reductase enzymes shifts the redox state of the cytoplasm of the recombinant cell to an oxidative state as compared with a non-recombinant cell. Preferably the CRM coding sequence encodes one or more CRM epitopes, CRM peptide sequences, CRM domains, or combinations thereof. Preferably the spacer comprises more or less than 9 nucleotides such as, for example, between 5 and 20 nucleotides. Preferably the expression enhancer comprises a ribosome binding site upstream of the CRM coding sequence and an ATG codon. Preferably the CRM protein expressed by the cell is soluble and is intracellular, periplasmic or secreted. Preferably the recombinant cell is propagated at a temperature from about 15° C. to about 32° C.
Preferably, the CRM protein is isolated from the cell by chromatography comprising, as a preferable chromatography medium, a dextran sulfate resin, a gel resin, an active sulfated resin, a phosphate resin, a heparin resin or a heparin-like resin.
Another embodiment of the invention is directed to CRM protein isolated by the methods of the invention. Preferably, the isolated CRM protein is conjugated and the conjugated CRM protein is formulated as a vaccine.
Another embodiment of the invention is directed to methods of producing all or a portion of a CRM protein such as for example a protein or peptide produced from a CRM coding sequence that encodes one or more CRM epitopes, CRM peptide sequences, CRM domains, or combinations thereof, and preferably CRM197, comprising providing a recombinant cell that contains an expression vector, wherein the expression vector contains a promoter functionally linked to a CRM coding sequence; expressing CRM protein from the CRM coding sequence; and isolating the CRM protein expressed. Preferably the recombinant cell is a prokaryotic or a eukaryotic cell and preferably the prokaryotic cell is an E. coli cell or a derivative or strain of E. coli. Preferably the promoter is constitutive or inducible. Preferably the recombinant cell has been modified to shift the redox status of the cytoplasm to a more oxidative state as compared to an unmodified recombinant cell. Preferably the modified recombinant cell has reduced activity of one or more disulfide reductase enzymes such as, for example, one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, a protein reductase or a glutathione reductase. Preferably the expression vector contains a spacer sequence between a ribosome binding site and an ATG codon such as, for example, wherein the spacer comprises more or less than 9 nucleotides and/or is between 5 and 20 nucleotides. Preferably the expression vector contains an expression enhancer such as, for example, a ribosome binding site upstream of the CRM coding sequence and an ATG codon.
Another embodiment of the invention is directed to methods for isolating and/or purifying CRM protein comprising: loading the CRM protein onto a chromatography column containing a resin with a loading buffer wherein the resin is preferably a dextran sulfate resin, a gel resin, an active sulfated resin, a phosphate resin, a heparin resin or a heparin-like resin; washing the resin with one or more washing buffers; and eluting CRM protein from the resin with an elution buffer. Preferably the loading buffer and the washing buffer are or contain the same components and at the same or in similar amounts. Preferably the loading buffer and the one or more washing buffers are low conductivity buffers such as, for example, a conductivity of about 10 mS/cm or less. Preferably the elution buffer is a high conductivity buffer such as, for example, a conductivity of about 10 mS/cm or more.
Another embodiment of the invention is directed to methods of characterizing folding of diphtheria toxin or CRM protein comprising: contacting diphtheria toxin or CRM protein to HB-EGF; determining the amount of binding of diphtheria toxin or CRM protein to HB-EGF; and determining the folding of diphtheria toxin or CRM protein by the amount of binding determined, wherein binding indicates correct folding. Preferably the diphtheria toxin or CRM contains a receptor binding domain. Preferably the CRM protein comprises CRM197. Also preferably the at least one of the diphtheria toxin or CRM protein and/or the HB-EGF is bound to a solid support. Preferably the amount of binding of diphtheria toxin or CRM protein to HB-EGF is determined by an ELISA and the CRM protein that binds to HB-EGF is soluable in PBS.
Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention.
Soluble, intact recombinant CRM197 was first produced in protease-deficient E. coli (Bishai et. al 1987). However, the amount of protein production was very low. Subsequently, CRM197 was produced in E. coli cells as inclusion bodies (Stefan A, et al. J Biotechnol. December 20; 156(4):245-52, 2010; International Application Publication No. WO 2011/126811, Chinese Patent Application No. 200610042194) or as soluble protein directed to the periplasm by signal peptide (International Application Publication No. WO 2011/042516). The periplasm of E. coli is an oxidizing environment that allows the formation of disulfide bonds. CRM197 has two disulfide bonds that are probably important for the correct folding and function, and for protein solubility.
It has been surprisingly discovered that a single, uncleaved chain of soluble recombinant CRM protein can be rapidly produced intracellularly and in commercial quantities from microorganisms and thereafter isolated and/or purified in large quantities and remain soluble. CRM is soluble in phosphate buffered saline (PBS, pH 7.5) and other similar buffers. Preferred CRM proteins produced are full length or partial regions such as, for example, peptides, single or multiple domains or epitopes, and any specific region expressed from native CRM coding sequences including CRM sequences that have been modified with one or more deletions, substitution and/or additions (e.g. conservative or non-conservative), and CRM sequences that have been modified with additional sequences (e.g., one or more promoters, start codons, and translation factor, ribosome or polymerase binding sites) that promote expression in a host organism. A preferred CRM protein is CRM197. Preferred is expression of CRM protein that is soluble and not otherwise bound as insoluble inclusion bodies of the cell. Preferred expression systems for the expression and production of CRM proteins include microorganisms with an intracellular oxidative state. Preferred expression systems may be recombinant or native eukaryotic or prokaryotic cells wherein recombinant cells include cells that contain a non-native CRM coding sequence. Preferred prokaryotic cells are strains of E. coli or another bacterial strain that contains one or more genetic alterations (e.g., one or more deletions or mutations). Preferably the one or more genetic alterations shift the redox state of the cytoplasm of the cell to a more oxidative state, as compared to wild-type, for example as disclosed in U.S. Pat. No. 7,410,788 (which is incorporated by reference). Alterations preferably reduce the activity of one or more disulfide reductase genes and/or other genes that reduce the oxidative state of the cytoplasm. Preferably, reduced activity is due to non-expression or reduced expression of one, two or multiple disulfide reductase or other genes, or one or more mutations that reduce activity of one or more expressed disulfide reductase proteins or other proteins. Preferred strains of microbial cells (e.g., recombinant, engineered or native eukaryotic or prokaryotic cells) have increased abilities to produce natively folded proteins containing disulfide bonds yet remain as functional proteins. The method of the invention produces quantities of CRM proteins containing full, truncated or modified CRM amino acid sequences. Quantities of CRM protein produced according to the invention are surprising such as, for example, 600 mg or more of CRM protein per liter of bacterial cell culture.
One embodiment of the invention is directed to methods for the production of large quantities of CRM protein, and preferably CRM197. Production quantities are typically quantified as mg/L of bacterial cell culture. CRM protein production, according to the methods of the invention, is 200 mg/L or more, 300 mg/L or more, 400 mg/L or more, 500 mg/L or more, 600 mg/L or more, 700 mg/L or more, 800 mg/L or more, 900 mg/L or more, 1,000 mg/L or more, 1,500 mg/L or more, or 2,000 mg/L or more. Preferred quantities CRM197 of the invention includes related proteins containing full length and truncated CRM protein, as well as modified amino acid sequences of CRM protein. Modifications include one or more of conservative amino acid deletions, substitution and/or additions. A conservative modification is one that maintains the functional activity and/or immunogenicity of the molecule, although the activity and/or immunogenicity may be increased or decreased. Examples of conservative modifications of CRM include, but are not limited to amino acid modifications (e.g., single, double and otherwise short amino acid additions, deletions and/or substitutions), modifications outside of the 39 ε-amino groups of lysine (primary amine groups of lysine) residues that are accessible for conjugation in forming a vaccine, modifications due to serotype variations of DT, modifications that increase immunogenicity or increase conjugation efficiency, modification that do not substantially alter binding to heparin, modifications that maintain proper folding or three dimensional structure, and/or modifications that do not significantly alter immunogenicity of the protein or the portions of the protein that provide protective immunity to DT.
Recombinant cells that are used in the method of the invention are preferably E. coli bacteria and, preferably, E. coli that are genetically engineered to shift the redox state of the cytoplasm to a more oxidative state such as, for example, by mutation of one or more disulfide reductase genes such as, for example, an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, a glutamate cysteine lyase, a disulfide reductase, a protein reductase, and/or a glutathione reductase. Preferably one or more disulfide reductase genes are mutated and rendered non-functional or marginally functional such that the redox state of the cytoplasm of the cell is shifted to a more oxidative state as compared to wild type. Oxidative protein folding involves the formation and isomerization of disulfide bridges and plays a key role in the stability and solubility of many proteins including CRM 197. Formation and the breakage of disulfide bridges is generally catalyzed by thiol-disulfide oxidoreductases. These enzymes are characterized by one or more Trx folds that consist of a four-stranded β-sheet surrounded by three α-helices, with a CXXC redox active-site motif. The assembly of various Trx modules has been used to build the different thiol oxidoreductases found in prokaryotic and in eukaryotic organisms. In the bacterial periplasm, the proteins are kept in the appropriate oxidation state by a combined action of the couples DsbB-DsbA and DsbD-DsbC/DsbE/DsbG (Inaba 2009, Gruber et al, 2006). Many protein expression systems are well known in the art and commercially available.
Especially preferred microbes include E. coli expression strains, for example, chemically competent E. coli K12 cells engineered to form disulfide bonded proteins in the cytoplasm (e.g., ORIGAMI™ (EMD Millipore) and SHUFFLE™ (New England Biolabs)). Other strains and types of cells and other E. coli strains with enhanced oxidative redox state also may be used. For example, ORIGAMI™ 2 host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in the E. coli cytoplasm. These strains are kanamycin sensitive; like the original Origami strains, the gor mutation is still selected for by tetracycline. To reduce the possibility of disulfide bond formation between molecules, strains containing mutations in trxB and gor are recommended only for the expression of proteins that require disulfide bond formation for proper folding. SHUFFLE™ cells are chemically competent E. coli K12 cells engineered to form proteins containing disulfide bonds in the cytoplasm. Preferably these cells contain mutations in trxB and gor and cytoplasmic chaperon disulfide bond isomerase DsbC (fhuA2 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC (SpecR, lacIq) ΔItrxB sulA11 R(mcr-73::miniTn10-TetS)2 [dcm] R(zgb-210::Tn10-TetS) endA1 Δgor A(mcrC-mrr)114::IS10). Also preferably, cells are suitable for T7 promoter driven protein expression and of the genotype F′ lac, pro, lacIQ/Δ(ara-leu)7697 araD139 fhuA2 lacZ::T7 gene1 Δ(phoA)PvuII phoR ahpC* galE (or U) galK λatt::pNEB3-r1-cDsbC (SpecR, lacIq) ΔtrxB rpsL150(StrR) Δgor Δ(malF)3. SHUFFLE™ strains expresses constitutively a chromosomal copy of the disufide bond isomerase DsbC. DsbC promotes the correction of mis-oxidized proteins into their correct form. Cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds.
Bacterial cultures are preferably cultured at temperatures such that solubility of the expressed protein increases (e.g., CRM or CRM197) as compared to solubility at higher temperatures (e.g., 37° C.). Preferred culture temperatures are 30° C. or lower, preferably 25° C. or lower, preferably 20° C. or lower, preferably 18° C. or lower, and preferably between 15° C. and 32° C.
Another embodiment of the invention comprises recombinant cells such as, for example, bacterial, mammalian or insect cells containing expressible CRM sequences and, preferably sequences of CRM197. Preferred host cells include, but are not limited to, cells genetically engineered to shift the redox state of the cytoplasm to a more oxidative state. Preferred cells include prokaryotic or eukaryotic cells such as, for example, E. coli cell expression systems, Baculovirus Expression System and other bacterial and/or eukaryotic cellular expression systems. Preferably the cells contain a protein expression system for expressing foreign or non-native sequences such as CRM peptides. Also preferable, the sequences to be expressed are comprised of an expression vector which contains one or more of an inducible promoter (e.g., auto-inducible preferably with specific media), a start codon (e.g., ATG), a ribosome binding site, and/or a modified sequence between ribosome binding site and ATG starting codon, or between start codon and the sequence to be expressed. Preferred modified sequences or spacer sequences include, for example, a number of nucleotides more or less than 9 (e.g., between 7 and 12 nucleotides), and preferably not 9 nucleotides. Specific examples of spacer nucleotides that can be utilized in an expression system include but are not limited to GATATAC (SEQ ID NO 3), GATATACCA (SEQ ID NO 4), and GATATACCATAT (SEQ ID NO 5). Accordingly, another embodiment of the invention comprises an expression construction of CRM, nucleotide and amino acids sequences, with or without defined spacer sequences and with and without a host cell.
Another embodiment of the invention is directed to recombinant CRM197 protein and the expression of recombinant CRM in E. coli or another host cell using an expression vector with an inducible promoter and/or a modified sequence between ribosome binding site and ATG starting codon. Preferably, the expression vector includes the lactose/IPTG inducible promoter, preferably a tac promoter, and the sequence between ribosome binding site and ATG starting codon. Preferably the expression system contains a spacer between the start codon and the expression sequence which is comprised of a number of nucleotides more or less than 9 (e.g., between 7 and 12 nucleotides), and preferably not 9 nucleotides. Specific examples of spacer nucleotides that can be utilized in an expression system include but is not limited to those identified herein. It was surprisingly discovered that the use of spacers of length seven or twelve resulted in dramatically increased levels of CRM197 expression when compared to spacers of nine nucleotides.
Another embodiment of the invention comprises an expression construction of CRM, nucleotide and amino acids sequences, with or without defined spacer sequences, as disclosed herein, and with or without an enhancer region. Enhancers regions promote protein expression by adding one or more sequences that promote nucleic acid recognition for increased expression (e.g., start codon, enzyme binding site, translation or transcription factor binding site). Preferably, an enhancer of the invention contains a ribosome binding site with a start codon upstream of and with a coding sequence that differs from the coding sequence of the CRM protein.
Another embodiment of the invention is directed to recombinant CRM, and in particular CRM197, purified according to the methods of invention. Purification preferably comprises heparin or heparin-like affinity chromatography. It was surprisingly discovered that CRM197 contains the sequence-based motif of typical heparin binding sites XBBXBX (SEQ. ID NO 6) where B is a lysine or arginine and X a hydropathic residue (Cardin A D, Weintraub H J., 1989: Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis 9: 21-32). This motif is located in the CRM197 receptor-binding domain and comprises of the following amino acids: GRKIRMRCR (SEQ ID NO 7), where G (Glycine), I (Isoleucine), M (Methionine) and C (Cysteine) are hydropathic residues. Presence of heparin binding site allows the use of heparin or heparin-like resins in the purification. Heparin-like resins include resins containing functional sulfate groups, such as dextran sulfate, e.g. Dextran sulfate (Sterogene), Capto Devirs (GE) or sulfate esters, e.g. Cellufine Sulfate (Asahi Kasei Bioprocess).
In a first step, crude E. coli extract may be clarified, for example, preferably by centrifugation or depth filtration. Optionally cleared lysate may be fractionated further, preferably by adding salts that have effect on protein solubility and salting out CRM197. In the second step, clarified lysate or re-solubilized salted out fraction containing CRM197 may be applied, for example, to anion exchange resin under conditions when CRM197 is in flow through. In the third step, the flow through fraction containing CRM197 may be applied to a column. Preferred column resins include, but are not limited to dextran sulfate resins, CELLUFINE™ resins (Chisso Corporation; chromatography gel), active sulfated resins, phosphate resins, or heparin or heparin-like resins. Preferably binding of CRM to resin is performed in a low salt buffer and eluted in higher salt buffer, yielding highly purified CRM197. Preferred binding buffers contain, for example, one or more ionic reagents and/or reagents that increase conductivity, one or more chaotropic agents, NaCl, KCl, glycerol, isopropyl alcohol, ethanol, arginine, acetate, guanidine, urea, ATP, one or more mono-, di-, tri-, and/or poly-phosphates, sulfates or pyrophosphates, and combinations thereof. Preferred elution buffers contain, for example, higher concentration of one or more components of the binding buffer.
Other preferred purification methods include any one or combination of an anion exchange chromatography, hydrophobic interaction chromatography and/or Cibacron-Blue resin (CN 101265288A, U.S. Pat. No. 8,383,783). Purification method of the invention produce recombinant CRM protein (e.g., CRM197) at high yields, as discussed herein, and with a purity level of greater than 80%, preferably greater than 85%, preferably greater than 90%, preferably greater than 95%, preferably greater than 99%, and preferably with an even greater purity.
Another embodiment of the invention is directed to methods to characterize recombinant DT and CRM proteins (e.g., binding activity) and, in particular CRM197, which contain a receptor binding domain (see SEQ ID NO 2). These methods comprise determination of the binding activity of proteins containing native or modified sequence of receptor binding domain of DT. Such modifications preferably preserve the ability of CRM to bind to HB-EGF (heparin binding epidermal growth factor). The method is applicable to both crude and purified CRM197. Binding activity represents binding to the soluble form of diphtheria toxin receptor HB-EGF (DTR). Soluble forms are believed to be properly folded. These methods comprise, preferably, determining the binding CRM197 to DTR and detection of with molecules (e.g., antibodies, antibody fragments, antigens) specific to the properly folded structure, the complex, binding, and/or the binding sites, and preferably in an ELISA format. Assays to determine and quantitate binding allow for the rapid determination that CRM197 is correctly folded, as only properly folded CRM197 binds to the receptor. Thus, the method monitors correct folding of manufactured CRM197 and related proteins during the development, production and purification process. In addition, this characterization method can be used to identify and track CRM protein after conjugation with another molecule such as in vaccine production. Using the detection method of the invention, properly folded and configured conjugated CRM protein can be monitored during the development of a vaccine for the treatment and/or prevention of diseases and disorders in patients.
Another embodiment of the invention is directed to CRM and proteins and peptides related to CRM, as well as portions and domains thereof, that can be manufactured according to the method of the invention. Proteins and peptides related to CRM comprise, but are not limited to, for example, those proteins and peptides that can be cytoplasmically expressed without leader or tag sequences and at commercially significant levels according to the methods disclosed and described herein. Preferably, these proteins and peptides show proper folding upon expression in recombinant cells of the invention. Recombinant cells of the invention preferably show reduced activity of one or more disulfide reductase enzymes, preferable reduced activity of less than five disulfide reductase enzymes, preferable reduced activity of less than four disulfide reductase enzymes, and also preferable reduced activity of less than three disulfide reductase enzymes. Preferably expression of the proteins and peptides is increased in recombinant cells of the invention, but may be not reduced or not significantly reduced compared with expression in recombinant cell that does not have reduced activity of one or more disulfide reductase enzymes. Proteins and peptides that can be expressed in the methods disclosed herein include, but are not limited to, for example, tetanus toxin, tetanus toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertusis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, Hemophilus influenzae protein D, Flagellin Fli C, Horseshoe crab Haemocyanin, and fragments, derivatives, and modifications thereof.
Another embodiment of the invention is directed to CRM and proteins and peptides related to CRM, as well as portions and domains thereof, fused genetically or by chemical modification or conjugation (e.g., carbodiimide, 1-cyanodimethylaminopyridinium tetrafluoroborate (CDAP)) with another molecule. Preferred other molecules are molecules such as, but not limited to, other proteins, peptides, lipids, fatty acids, saccharides and/or polysaccharides, including molecules that extend half-life (e.g., PEG, antibody fragments such as Fc fragments), stimulate and/or increase immunogenicity, or reduce or eliminate immunogenicity. CRM contains an N-terminal serine which useful for conjugation. Typical conjugation partner molecules include, but are not limited to polymers such as, for example, bacterial polysaccharides, polysaccharides derived from yeast, parasite and/or other microorganisms, polyethylene glycol (PEG) and PEG derivatives and modifications, dextrans, and derivatives, modified, fragments and derivatives of dextrans. One example of a conjugation compound is the polymer PEGASYS® (peginterferon alfa-2a). Other polymers, such as dextran, also increase the half-life of proteins and reduce immunogenicity of the conjugate partner. Polymers may be linked randomly or directed through site specific conjugation such as, for example, by modification of N-terminal serines and/or threonines. Also modifications may be used that selectively oxidize chemical groups for site specific conjugation.
Another embodiment of the invention is directed to methods of producing a peptide containing a domain, fragment and/or portion of a CRM or related protein comprising: expressing the peptide from a recombinant cell containing an expression vector that encodes the peptide, wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes and the expression vector contains a promoter functionally linked to a coding region of the peptide, wherein the one or more disulfide reductase enzymes comprises one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, or a glutathione reductase; and isolating the peptide expressed, wherein the peptide expressed is soluble. Preferably the domain comprises a CRM receptor binding domain, a CRM catalytic toxic domain, and/or cytoplasmic transfer domain. Preferably the CRM receptor binding domain comprises the sequence of SEQ ID NO. 2. Preferably the expression vector contains a ribosome binding site, an initiation codon, and, optionally, an expression enhancer region. Preferably the recombinant cell has a reduced activity of only one disulfide reductase enzyme, only two disulfide reductase enzymes, or two or more disulfide reductase enzymes. Preferably the reduced activity of the disulfide reductase enzymes results in a shift the redox status of the cytoplasm to a more oxidative state as compared to a recombinant cell that does not have reduced activity of one or more disulfide reductase enzymes. Preferably the recombinant cell is an E. coli cell or a derivative or strain of E. coli. Preferably the expression vector contains at least one spacer between the ribosome binding site and the initiation codon. Preferably the soluble peptide expressed comprises a natively folded domain of CRM. The promoter may be a constitutive or inducible promoter, whereby expression comprises inducing the inducible promoter with an inducing agent. Preferred inducing agents include, for example, lactose (PLac), isopropyl β-D-1-thiogalactopyranoside (IPTG), substrates and derivative of substrates. In one preferred embodiment, the recombinant cell contains a second expression vector that preferably contains a coding region for a peptidase that preferably acts upon and selectively cleaves the peptide or protein expressed from the first expression vector. Preferably the second expression vector contains a second promoter functionally linked to the coding region, and co-expressing comprises expressing the peptide and the peptidase. The two expression vectors may be induced together with the same inducing agent, or with different inducing agents, optionally at different times. Preferably the peptidase acts on and cleaves the peptide co-expressed with the peptidase. Preferably the peptide expressed is conjugated with a polymer such as, for example, dextran, a bacterial capsular polysaccharide, polyethylene glycol (PEG), or a fragment, derivative or modification thereof. Preferably the peptide expressed is coupled with a polymer which includes, for example, a polysaccharide, a peptide, an antibody or portion of an antibody, a lipid, a fatty acid, or a combination thereof.
Another embodiment of the invention comprises methods of treating cancer patients by exposing cancels ells of the patients to the peptide of the invention. Preferably exposing reduces proliferation of cancer cells and/or inhibits angiogenesis. Preferably the peptide expressed binds to a receptor of the cancer cells such as, for example, the receptor is a heparin-binding EGF-like growth factor. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF is implicated in cancer cell proliferation, malignancy, metastatic potential, and chemotherapy resistance. HB-EGF expression is elevated in many types of malignant tumors thus making HB-EGF a target for diagnosis and therapy of HB-EGF related cancers and related diseases. HB-EGF also serves as a receptor for diphtheria toxin (DT) and its mutant forms, including CRM197. Mutant form of DT and CRM prevent ectodomain shedding by binding to HB-EGF and represses mutagenic activity of HB-EGF. CRM induces only weak toxicity compare with DT and can involve an inflammatory response.
In another embodiment of the invention, it was surprisingly discovered that treatments with a domain of the invention conjugated or fused with a compound that increases half-life of the molecule increases effectiveness. The receptor binding domain of CRM197 (RBD CRM197, 381-535 aa) produced in recombinant cells according to this disclosure functions as an antitumor reagent and detection reagent in place of anti-HB-EGF monoclonal antibodies. Expressions of RBD CRM according to the disclosures herein (greater than 1 g/L) coupled with the purification disclosed herein makes RBD CRM197 an inexpensive reagent and tool for effective anticancer therapy. Binding of purified RBD CRM to human HB-EGF in plate based assay. Due to lack of A chain, RBD CRM197 does not produce toxicity as an anticancer drug. To increase the molecule blood permanence time and effectiveness, CRM receptor binding domain produced according to this disclosure is conjugated with dextran and utilized for antitumor drug delivery. Having serine as a first amino acid in the polypeptide chain allow for site specific conjugations which does not compromise HB-EGF binding. Conjugated to dextran to increase avidity and to HRP or dye for the detection RBD CRM is used to detect HG-EGF in IHC and plate based assays. The conjugate prevents HB-EGF binding to EGFR using ELISA format indicating that CRM according to this disclosure serves as a positive control and blocks EGFR phosphorylation and cell proliferation.
Another embodiment of the invention comprises conjugates of CRM and proteins related to CRM included fragments, domains, and portions thereof as disclosed and described herein.
Another embodiment of the invention comprises fusion molecules of CRM and proteins related to CRM included fragments, domains, and portions thereof as disclosed and described herein.
Another embodiment of the invention comprises a vaccine containing CRM and proteins related to CRM included fragments, domains, and portions thereof, as disclosed and described herein.
The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.
DNA encoded mature CRM197 was cloned into three vectors containing tac promoter and various nucleotide spacing between RBS and initial ATG codon, in particular 7, 9 or 12 nucleotides. The expression vectors named pTacCRM7, pTacCRM9 and pTacCRM12 were expressed in different E. coli strains, in particular ORIGAMI™ 2 cells, C41, High Control, Top 10. The SDS-PAGE analysis of the cells lysate shows that only pTacCRM7 and pTacCRM12 produce 58 kDa band corresponding to CRM197.
CRM197 was expressed insoluble at 37° C. When expression temperature was dropped below 37°, solubility of the protein expressed in ORIGAMI™ 2 cells and SHUFFLE™ cells, but not in the other tested E. coli strains, increases. CRM197 is mostly soluble when expressed in ORIGAMI™ 2 cells at 18° C.
The EES promotes transcription of CRM sequence in a CRM-containing vector and results in polycistronic mRNA that translates into two proteins; a short EES peptide and a CRM peptide. The coding sequence of the native CRM gene was analyzed for potential 3D structure formation and found to contain a number of potential hairpins, which could inhibit translation. A CRM sequence was created that would potentially result in an mRNA with no hairpins structures yet translate the same CRM amino acid sequence. This gene sequence is referred to an optimized CRM sequence and comprises SEQ ID NO 8.
The optimized CRM sequence expresses well in both E. coli (e.g., BL21) and in E. coli engineered to contain an oxidized cytoplasm (e.g., Shuffle). CRM peptide translated from polycistronic mRNA produces a full length protein and is believed to be more stable than the native CRM coding sequence. Unlike the native CRM sequence, the optimized CRM sequence expressed as full-length and as a soluble protein in Shuffle cells. In addition, compared to native CRM, higher expression of the optimized CRM sequence was observed with a lower cell density and with increased binding to chromatography resin resulting in greater production levels of CRM protein.
SHUFFLE™ cells expressing CRM197 were open using microfluidizer and 1M of sodium chloride was added to the cell lysate. To this was added enough ammonium sulfate to equal 1M followed by centrifugation for 30 minutes at 20,000× g, which removed mis-folded CRM197 and most of the bacterial proteins. Following clarification the ammonium sulfate concentration was further increased to 2.2M. The precipitate, which is mainly CRM197 was collected and re-solubilized in a low conductivity buffer.
Ammonium sulfate precipitated CRM197 was resolubilized in 20 mM Tris-HCl pH 8.0 to achieve conductivity 5 mS/cm and loaded on an column containing Heparin Sepharose CL-68 resin (GE). The purification was performed under the following conditions: flow rate was 5 ml/min, wash buffer A: 20 mM Tris-HCl pH8. Elution was done with a buffer B 0-100% gradient, buffer B: buffer A+1M NaCl in 20 CV. Eluted CRM197 was analyzed by SDS-PAGE in reduced and non-reduced conditions. The purity of eluted CRM197 was greater than 95%. The protein reduced with DTT appears as a single polypeptide confirming that the intact form of CRM197 is expressed in E. coli.
SHUFFLE™ cells expressing CRM197 were open using microfluidizer in 1×PBS, pH7.4, 1% sodium pyrophosphate. The lysate was clarified using depth filtration. Clarified lysate was loaded on a column containing Q Sepharose XL (GE) and flow through fraction was collected. To reduce volume and conductivity flow through fraction was subjected to tangential flow filtration using 10K cassette (Sartorius). Capto Devirs resin was equilibrated with 25 mM sodium phosphate buffer, pH8.0. CRM197 was bound to the column under the following conditions: conductivity was less than 10 mS/cm, in a binding buffer containing a chaotropic agent (e.g., in this case urea), wash buffer was 25 mM sodium phosphate, pH8.0. Elution was done with NaCl. Eluted CRM197 was analyzed by SDS-PAGE under reduced and non-reduced conditions. The purity of eluted CRM197 was greater than 95%. The protein reduced with DTT appears as a single polypeptide confirming that CRM197 remains intact during purification process.
The recombinant soluble diphtheria toxin receptor HB-EGF (DTR) (Sigma) was bound to the ELISA plate. Blocking solution of 5% dry non-fat milk was used to prevent high background. Recombinant CRM197 diluted in 1×PBS, pH7.4, 0.1% Twin 20 was incubated on the plate for 1 hour at 37° C. CRM197 bound to HB-EGF was detected by rabbit polyclonal anti-CRM197 antibody and goat anti-rabbit antibody conjugated to soybean peroxidase (Fina BioSolutions; Rockville, Md.). Denatured recombinant CRM197 did not bind to the receptor.
ELISA plates were coated with soluble HB-EGF and blocked with 5% dry non-fat milk. CRM197 was bound to the receptor and detected with rabbit anti-CRM197 polyclonal antibody and goat anti-rabbit polyclonal conjugated with SBP. CRM197 expressed in E. coli showed the same affinity to HB-EGF as CRM produced in Corynebacterium and Pseudomonas.
Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, containing and the like are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.
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| Number | Date | Country | |
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| 20190032063 A1 | Jan 2019 | US |
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| Number | Date | Country | |
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| Parent | 15114642 | US | |
| Child | 16154020 | US |
