COMPOSITIONS AND METHODS FOR GLYCOSYLATION OF PROTEINS

专利摘要:
The present invention relates to oligosaccharyl transferases for N-glycosylating proteins of interest in vitro and in cells. hosts. Also provided are methods of using these oligosaccharyl transferases, nucleic acids encoding these oligosaccharyl transferases, and host cells comprising these oligosaccharyl transferases. Furthermore, the invention relates to glycoconjugates generated by means of these oligosaccharyl transferases.
公开号:BE1022998B1
申请号:E2015/5846
申请日:2015-12-24
公开日:2016-10-28
发明作者:Amirreza Faridmoayer；Gerd Martin Lipowsky
申请人:Glycovaxyn Ag；
IPC主号:

专利说明:

COMPOSITIONS AND METHODS FOR GLYCOSYLATION OF PROTEINS 1. Introduction
The present invention relates to oligosaccharyl transferases for N-glycosylating proteins of interest in vitro and in host cells. The invention also relates to methods of using these oligosaccharyl transferases, nucleic acids encoding these oligosaccharyl transferases and host cells comprising these oligosaccharyl transferases. In addition, the invention relates to glycoconjugates generated using these oligosaccharyl transferases. 2. Background of the invention
Glycoconjugate vaccines are widely known for their ability to prevent many life-threatening bacterial infections. Glycoconjugate vaccines are generally considered safe and effective and have been used in humans for over 30 years. Conventional production of glycovaccins often involves chemical modification of immunogenic carrier proteins with polysaccharide antigens of pathogenic bacteria. However, more recently, biotechnological processes for the production of glycoconjugate vaccines have emerged, which is expected to reduce production costs and further increase the homogeneity and possible therapeutic activity and safety of vaccine preparations. glycoconjugate.
In eukaryotic cells, glycosylation of nitrogen is a crucial mechanism for post-translational protein modification involving multiple enzymes. In prokaryotic cells, glycosylation of nitrogen is catalyzed by certain bacterial N-oligosaccharyl transferases (N-OST). The glycosylation gene cluster of Campylobacter jejuni proteins (C. jejuni) includes the pglB gene, which encodes a membrane-bound N-OST (PglBcj). PglBcj can be expressed in conventional bacterial hosts, such as Escherichia coli (E. coli), and can glycosylate co-expressed periplasmic proteins carrying at least one D / EYNXS / T (Y, X Φ P) glycosylation pattern exposed surface. PglBcj can transfer bacterial polysaccharide antigens to C. jejuni proteins, as well as to immunogenic proteins supporting other organisms containing genetically modified glycosylation sites. PglBcj can transfer C. jejuni oligosaccharides and, to some extent, lipopolysaccharide structures of Gram-negative bacteria antigens and antigenic capsular polysaccharides of Gram-positive bacteria.
The present disclosure provides recombinant N-OSTs whose substrate specificities are modified and methods of using recombinant N-OSTs to produce glycoconjugate vaccines. These recombinant N-OSTs can be advantageously used in the N-glycosylation of proteins. 3. Summary
In one aspect, there is provided herein a recombinant modified N-oligosaccharyl transferase (N-OST), wherein the recombinant modified N-OST is Campylobacter jejuni PglB (PglBcj) comprising an amino acid substitution at the positions amino acid N311, K482, D483 and A669.
In certain embodiments, the recombinant modified N-OST comprises a substitution of the amino acid N311 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I).
In some embodiments, the recombinant modified N-OST comprises an N311V substitution.
In some embodiments, the recombinant modified N-OST comprises a substitution of the amino acid K482 with a basic amino acid selected from lysine (K) or arginine (R).
In some embodiments, the recombinant modified N-OST comprises a K482R substitution.
In some embodiments, the recombinant modified N-OST comprises a D483H substitution.
In some embodiments, the recombinant modified N-OST comprises a substitution of the amino acid A669 by an aliphatic amino acid selected from the group consisting of glycine (G), valine (V), leucine (L) or isoleucine (I).
In some embodiments, the recombinant modified N-OST comprises an A669V substitution.
In some embodiments, the recombinant modified N-OST comprises an N311V substitution, a K482R substitution, a D483H substitution, and an A669V substitution.
In some embodiments, the recombinant modified N-OST comprises an additional substitution at amino acid position N534.
In some embodiments, the recombinant modified N-OST comprises an N534Q substitution.
In some embodiments, the recombinant modified N-OST comprises an N311V substitution, a K482R substitution, a D483H substitution, and an A669V substitution.
A recombinant modified N-OST, wherein the recombinant modified N-OST is Campylobacter lari PglB (PglBci) comprising three amino acid substitutions at amino acid positions N314, K488 and D489, and another acid substitution amine in a region comprising amino acid positions P667 to 1672 of PglBci (PYAQFI). The recombinant modified N-OST according to claim 6, wherein the recombinant modified N-OST comprises an amino acid substitution at amino acid positions N314, K488, D489 and K668.
In another aspect, there is provided herein a recombinant modified N-OST, wherein the recombinant modified N-OST is Campylobacter lari PglB (PglBci) comprising an amino acid substitution at amino acid positions N314 , K488, D489 and K668. In some embodiments, the recombinant modified N-OST comprises a substitution of amino acid N314 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I).
In some embodiments, the recombinant modified N-OST comprises an N314V substitution.
In some embodiments, the recombinant modified N-OST comprises a substitution of the amino acid K488 with a basic amino acid selected from lysine (K) or arginine (R).
In some embodiments, the recombinant modified N-OST comprises a K488R substitution.
In some embodiments, the recombinant modified N-OST comprises a D483H substitution.
In some embodiments, the recombinant modified N-OST comprises a substitution of the amino acid K668 with an aliphatic amino acid selected from the group consisting of glycine (G), valine (V), leucine (L), or isoleucine (I).
In some embodiments, the recombinant modified N-OST comprises a K668V substitution.
In some embodiments, the recombinant modified N-OST comprises an N314V substitution, a K488R substitution, a D489H substitution and a K668V substitution.
In some embodiments, the recombinant modified N-OST comprises an amino acid substitution at amino acid positions N314, K488, D489, N535 and K668.
In some embodiments, the recombinant modified N-OST comprises an N535Q substitution.
In some embodiments, the recombinant modified N-OST comprises an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution, and a K668V substitution.
In some embodiments, the recombinant modified N-OST can detectably bind an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end to a carrier protein to produce an N-glycosylated carrier protein.
In some embodiments, the carrier protein is selected from the group consisting of P. aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus , agglutinating factor A, agglutinating factor B, FimH of E. E. coli, FimHC. coli, the thermolabile enterotoxin of E. coli. coli, the detoxified variants of E. thermolabile enterotoxin coli, cholera toxin subunit B (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli, the transient domain of E sat protein. coli, C. jejuni's AcrA and the natural glycoproteins of C. jejuni.
In some embodiments, the oligosaccharide or polysaccharide lacking the N-acetylated sugar at the reducing end comprises an antigen.
In some embodiments, the antigen comprises an E. coli antigen. coli, a Salmonella sp antigen, a Pseudomonas sp. , a Klebsiella sp. antigen, an Acinetobacter antigen, a Chlamydia trachomatis antigen, a Vibrio cholera antigen, a Listeria sp. antigen, a Legionella pneumophila serotype 1 to 15 antigen, a Bordetella parapertussis antigen, a Burkholderia mallei or pseudomallei antigen, a Francisella tularensis antigen, a Campylobacter sp. ; a Clostridium difficile antigen, a Streptococcus pyrogenes antigen, a Streptococcus agalacticae antigen, a Neisseria meningitidis antigen, a Candida albicans antigen, a Haemophilus influenza antigen, a Enterococcus faecalis antigen, a Borrelia burgdorferi antigen, a Neisseria meningitidis antigen, a Haemophilus influenza antigen, a Leishmania major antigen, or a Shigella sonnei antigen, or a Streptococcus pneumoniae antigen.
In some embodiments, the carrier protein and the oligosaccharide or polysaccharide originate from an organism other than C. jejuni or C. lari.
In some embodiments, the carrier protein and the oligosaccharide or polysaccharide are from different organisms.
In some embodiments, the carrier protein is from C. jejuni or C. lari.
In some embodiments, the carrier protein is from an organism other than C. jejuni or C. lari.
In some embodiments, the N-glycosylated carrier protein comprises the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) and the detoxified hemolysin A (H1A) of S. aureus.
In some embodiments, the N-glycosylated carrier protein comprises Staphylococcus aureus CP8 capsular polysaccharide (CPS 8) and E (ClfA) agglutinating factor A (ClfA). coli.
In some embodiments, the N-glycosylated carrier protein comprises the capsular polysaccharide CPI of Streptococcus pneumoniae (CPS 1) and exotoxin A of P. aeruginosa (EPA).
In some embodiments, the oligosaccharide or polysaccharide lacking an N-acetylated sugar at the reducing end has a galactose monosaccharide at its reducing end.
In some embodiments, the recombinant modified N-OST can produce a yield of the N-glycosylated carrier protein that is detectable at levels of more than 2-fold, more than 3-fold, more than 4-fold, and more. 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times the baseline level in a test the N-glycosylated carrier protein.
In some embodiments, the recombinant modified N-OST can increase the production yield of the N-glycosylated carrier protein by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3 times, more than 4 times, more than 5 times, more 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold compared to the yield obtained using a wild type N-OST or a recombinant modified N-OST having less amino acid substitution (eg, an N-OS Recombinant modified T presenting only a single, double or triple amino acid substitution, such as for example PglBcj (N534QV) or PglBci (N535Q)) as the recombinant modified N-OST proposed herein.
In some embodiments, the recombinant modified N-OST can increase the production rate of the N-glycosylated carrier protein by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3 times, more than 4 times, more than 5 times, more 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times compared to the speed of production obtained using an N-OST of wild type or a recombinant modified N-OST with less amino acid substitution (eg mple, a recombinant modified N-OST having only a single, double or triple amino acid substitution, such as PglBcj (N534Q) or PglBci (N535Q)) as the recombinant modified N-OST provided herein.
In certain embodiments, the recombinant modified N-OST can give an in vivo or in vitro glycosylation level of the support with the oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least at least 70%.
In another aspect, there is provided herein a recombinant modified N-OST, wherein the recombinant modified N-OST is PglBcj · comprising N311V substitution, K482R substitution, D483H substitution and A699V substitution.
In another aspect, there is provided herein a recombinant modified N-OST, wherein the recombinant modified N-OST is PglBcj comprising N311V substitution, K482R substitution, D483H substitution, N534Q substitution and A699V substitution. .
In another aspect, there is provided herein a recombinant modified N-OST, wherein the recombinant modified N-OST is PglBci comprising N314V substitution, K488R substitution, D489H substitution and K698.V substitution.
In another aspect, there is provided herein a recombinant modified N-OST, wherein the recombinant modified N-OST is PglBci comprising N314V substitution, K488R substitution, D489H substitution, N535Q substitution and K698V substitution. .
In another aspect, there is provided herein a nucleic acid encoding a recombinant modified N-OST provided herein.
In another aspect, there is provided herein a host cell comprising a recombinant modified N-OST provided herein.
In some embodiments, the host cell further comprises a recombinant glycosyltransferase.
In another aspect, there is provided herein a host cell comprising a nucleic acid provided herein.
In some embodiments, the host cell is a prokaryotic cell.
In some embodiments, the host cell is a cell of E. coli.
In another aspect, there is provided herein a method for producing a bioconjugate comprising culturing a host cell according to any one of the preceding claims in a cell culture medium.
In some embodiments, the host cell comprises a carrier protein and a recombinant modified PglBcj or a recombinant modified PglBci.
In some embodiments, the host cell further comprises a recombinant glycosyltransferase.
In some embodiments, the carrier protein is selected from P. aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, S. aureus detoxified hemolysin A, and agglutinating factor. A, the agglutinating factor B, FimH of E. E. coli, FimHC. coli, the thermolabile enterotoxin of E. coli. coli, the detoxified variants of E. thermolabile enterotoxin coli, cholera toxin subunit B (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli, the transient domain of E. sat protein. coli, C. jejuni's AcrA and the natural glycoproteins of C. jejuni.
In some embodiments, the bioconjugate is an N-glycosylated carrier protein.
In some embodiments, the bioconjugate is a natural N-glycosylated carrier protein of C. jejuni.
In some embodiments, the bioconjugate is a heterologous N-glycosylated carrier protein of C. jejuni.
In some embodiments, the N-glycosylated carrier protein does not have N-acetylated sugar at the reducing end of its oligosaccharide or polysaccharide component.
In some embodiments, the N-glycosylated carrier protein has a galactose at the reducing end of its oligosaccharide or polysaccharide component.
In some embodiments, the bioconjugate comprises the CPI capsular polysaccharide of Streptococcus pneumoniae (CPS 1) and the exotoxin A of P. aeruginosa (EPA).
In some embodiments, the bioconjugate comprises the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) and the detoxified hemolysin A (H1A) of S. aureus.
In some embodiments, the bioconjugate comprises Staphylococcus aureus CP8 capsular polysaccharide (CPS 8) and E (Clf A) agglutinating factor A (Clf A). coli.
In some embodiments, the bioconjugate comprises 0 Plesiomonas shigelloides 017 (017) antigen and EPA. .
In some embodiments, the recombinant modified N-OST can increase the production rate of the bioconjugate.
In some embodiments, the recombinant modified N-OST can produce a yield of the bioconjugate that is detectable at levels of more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold the baseline level in an assay detecting N-carrier protein. glycosylated.
In some embodiments, the recombinant modified N-OST can increase the production yield of the bioconjugate by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold the yield obtained using wild-type N-OST or recombinant modified N-OST presenting less than. amino acid substitution (for example, a recombinant modified N-OST having only a single, double or triple amino acid substitution, such as PglBcj (N534Q) or PglBci (N535Q)) as the modified N-OST recombinant proposed in this document.
In some embodiments, the recombinant modified N-OST can increase the production rate of the bioconjugate by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold the speed obtained using wild-type N-OST or modified N-OST recombinant having less amino acid substitution than the proposed recombinant modified N-OST d years of this document.
In some embodiments, at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of the carrier protein in a host cell are N-glycosylated to form the bioconjugate.
In some embodiments, the method further comprises purifying the bioconjugate from the culture of the host cell. 4. Brief description of the drawings
Figure 1 provides examples of results of screening for enhanced production of PglB-catalyzed CP5-Hla bioconjugate using Campylobacter jejuni-modified N-NOST PglB (PglBcj).
FIG. 2 shows examples of results of a screening of a production of the CP8-ClfA bioconjugate, using modified Campylobacter jejuni N-NOST PglB (PglBcj).
Figure 3 provides examples of results of screening for improved production of PglB-catalyzed CP1-EPA bioconjugate using Campylobacter jejuni-modified N-NOST PglB (PglBcj).
Figure 4 shows examples of results
screening for improved production of PglB-catalyzed Plesiomonas shigelloides (shigeiia sonnei) -EPA bioconjugate 017 using Campylobacter jejuni-modified N-NOST PglB (PglBcj).
Figure 5 illustrates a crystal structure of the Campylobacter jejuni PglB developed by Phyre homology modeling, a web-based modeling software, using information on the crystalline structure of C. lari PglB. The position of the amino acids used for the mutation is highlighted in red.
Figure 6 shows bacterial homologues of PglB (A) in the EL5 region, including the C. jejuni 287QLKFYxxR294 motif and the C. jejuni N311, and (B) in the vicinity of the C Y77 / S80 residues. jejuni and K482 / D843 of C. jejuni. The C. jejuni PGLB was used as a search pattern for Protein BLAST and the non-redundant sequences were aligned with the MegAlignTM program using the ClustalW algorithm (DNASTAR, Madison, WI, USA). The residues of PglBcj conserved in the sequences of other species are grayed out. The C. jejuni residues of interest are shown above and the corresponding amino acids in the homologous sequences of N-OST are boxed. 5. Abbreviations The abbreviation "cuo" as used in this document means "optimized codon usage". The abbreviation "CP" as used herein means "capsular polysaccharide". The abbreviation "EL" as used in this document means "outer loop". The abbreviation "N-OST" as used herein means N-oligosaccharyl transferase. The abbreviation "PglBcj" as used herein refers to the N-OST PglB of Campylobacter jejuni (C. jejuni). The abbreviation "PglBci" as used herein refers to N-OST PglB from Campylobacter lari (C. lari). 6. Detailed description
The present document proposes a recombinant modified N-oligosaccharyl transferase (N-OST) comprising four amino acid substitutions. In some embodiments, the recombinant modified N-OST comprises five or more amino acid substitutions (e.g., six, seven, eight, nine, or ten amino acid substitutions).
In one aspect, there is provided herein a recombinant modified Campylobacter jejuni N-OST PglB (PglBcj) comprising four amino acid substitutions. In some embodiments, the recombinant modified PglBcj proposed herein includes amino acid substitutions at amino acid positions N311, K482, D483 and A669. In some embodiments, the recombinant modified PglBcj proposed herein includes N311V substitution, K482R substitution, D483H substitution, and A669V substitution.
In some embodiments, the recombinant modified PglBcj proposed herein includes five amino acid substitutions. In some embodiments, the recombinant modified PglBcy provided herein includes amino acid substitutions at amino acid positions N311, K482, D483, N534, and A669. In some embodiments, the recombinant modified PglBcj proposed herein includes N311V substitution, K482R substitution, D483H substitution, N534Q substitution, and A669V substitution.
In another aspect, there is provided herein a recombinant modified Campylobacter lari N-OST PglB (PglBci) comprising four amino acid substitutions. In some embodiments, the recombinant modified PglBci provided herein comprises three amino acid substitutions at amino acid positions N314, K488 and D489, and another amino acid substitution in a region comprising the amino acid positions. amino acid P667 to 1672 from PglBci (PYAQFI). In some embodiments, the recombinant modified PglBci provided herein includes amino acid substitutions at amino acid positions N314, K488, D489 and K668. In some embodiments, the recombinant modified PglBcj comprises an N314V substitution, a K488R substitution, a D489H substitution, and a K669V substitution.
In some embodiments, the recombinant modified PglBci provided herein comprises five amino acid substitutions. In some embodiments, the recombinant modified PglBci provided herein comprises four amino acid substitutions at amino acid positions N314, K488, D489 and N535, and another amino acid substitution in a region comprising the positions. amino acid P667 to 1672 of PglBci (PYAQFI). In some embodiments, the recombinant modified PglBci provided herein includes amino acid substitutions at amino acid positions N314, K488, D489, N535 and K668. In some embodiments, the recombinant modified PglBcj comprises an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution, and a K669V substitution.
In some embodiments, the recombinant modified N-OST provided herein (e.g., PglBc1 (N311V-K482R-D483H-A669V) or PglBci (N314V-K488R-D489H-K668V)) is capable of using an oligosaccharide or a polysaccharide described herein (Section 6.2) as a substrate for the N-glycosylation of a carrier protein described herein (Section 6.3) at a consensus N-glycosylation sequence that can not be used (or which can not be used at detectable levels) by the wild-type form of N-OST (eg, PglBcj (wild-type) or PglBci (wild-type)).
In some embodiments, the recombinant modified N-OST provided herein may utilize such an oligosaccharide or polysaccharide to produce detectable levels of a N-glycosylated carrier protein, for example in vivo or in vitro.
Levels of the glycosylated carrier protein can be determined by methods known in the art such as, but not limited to, ELISA, HPLC, LC-MS and the like; see, for example, Section 6.8, Section 6.10 and Examples 2-4). In some embodiments, production of the N-glycosylated carrier protein is detected by ELISA. In some embodiments, production of the N-glycosylated carrier protein is detected by Western Blot.
In some embodiments, the level of the glycosylated carrier protein is detectable if the glycosylated carrier protein can be detected in an assay with a signal indicating the glycosylated carrier protein at levels of more than two or three standard deviations ( > 2o or> 3o) above the average or median baseline of the test, or more than twice, more than 3 times, more than 4 times, more than 5 times, more than 7 times times, more than 8 times, more than 9 times, or more than 10 times above the base signal of the test.
In some embodiments, the basic signal of the assay is the average or median test signal of negative control experiments performed in the absence of an N-OST. In some embodiments, the basic signal of the assay is the average or median test signal of negative control experiments performed in the presence of a wild-type N-OST.
In some embodiments, the glycosylated carrier protein can be detected at a level that is more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold the background level in a bioconjugate assay (for example, in an ELISA test, by HPLC, LC-MS, see also Section 6.8 and Section 6.10).
An alteration in an N-OST provided herein may be located at a specified distance from the monosaccharide moiety at the reducing end of the oligo- or polysaccharide component of a glycosylated carrier protein that is N-OST bound. To confirm that such a modification results in a modified substrate specificity, any routine test for glycosylation of the proteins can be used. Such modified N-OSTs can be used to generate bioconjugates in the prokaryotic host cells described herein. Compositions comprising the resulting bioconjugates are also described herein. In a specific embodiment, such a modified N-OST is capable of using as substrate an oligo- or polysaccharide which lacks an N-acetyl substituted sugar at the reducing end to produce detectable levels of a glycosylated carrier protein.
In some embodiments, recombinant modified N-oligo-saccharyl can increase the production yield of the bioconjugate to a level of more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times , more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold higher than baseline in a bioconjugate assay.
The baseline level in a bioconjugate assay may be, for example, the average or median signal obtained in control experiments that are performed in the absence of N-OST or that are performed using an N-OST of the type wild.
In some embodiments, the recombinant modified N-OST provided herein may produce a bioconjugate at higher yields and / or higher levels than a recombinant modified N-OST provided herein with less than 10 amino acid substitution (for example, a recombinant modified N-OST having only a single, double or triple amino acid substitution, such as, for example
PglBcj (N534Q) or PglBci (N535QV)) as the recombinant modified N-OST proposed herein. 6.1. N-Oligosaccharyl Transferases (Section 6.1)
In one aspect, there is provided herein a recombinant modified N-OST (Campylobacter jejuni PglB) N-oligosaccharyl transferase (PglBcj) comprising four amino acid substitutions. In some embodiments, the recombinant modified PglBc1 comprises amino acid substitution at amino acid positions N311, K482, D483 and A669.
In some embodiments, the recombinant modified BglC1 comprises five amino acid substitutions. In some embodiments, the recombinant modified BglBc comprises amino acid substitutions at amino acid positions N311, K482, D483 and A669.
In some embodiments, the recombinant modified PglBcj comprises a substitution of the amino acid N311 with an aliphatic amino acid, such as, for example, glycine (G), alanine (A), valine (V), leucine ( L) or isoleucine (I). In some embodiments, the recombinant modified PglBcj comprises an N311V substitution.
In some embodiments, the recombinant modified PglBc1 comprises a substitution of the amino acid K482 with a basic amino acid, such as, for example, lysine (K) or arginine (R). In some embodiments, the recombinant modified PglBcj comprises a K482R substitution.
In some embodiments, the recombinant modified PglBcj comprises a D483H substitution.
In certain embodiments, the recombinant modified BglBc comprises a substitution of the amino acid A669 by an aliphatic amino acid, such as, for example, glycine (G), valine (V), leucine (L) or isoleucine (I). In some embodiments, the recombinant modified PglBcj comprises an A669V substitution.
In some embodiments, the recombinant modified PglBcj comprises an N311V substitution, a K482R substitution, a D483H substitution, and an A669V substitution.
In some embodiments, the recombinant modified PglBcj comprises an N534Q substitution.
In some embodiments, the recombinant modified PglBcj comprises an N311V substitution, a K482R substitution, a D483H substitution, an N534Q substitution, and an A669V substitution.
In some embodiments, the recombinant modified BglBc can detectably bind an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end to a carrier protein to produce an N-glycosylated carrier protein.
In another aspect, there is provided herein a recombinant Campylobacter lari (PglBci) recombinant modified N-oligosaccharyl transferase (N-OST) comprising four amino acid substitutions.
In some embodiments, the recombinant modified PglBci provided herein comprises three amino acid substitutions at amino acid positions N314, K488 and D489, and another amino acid substitution in a region comprising the amino acid positions. amino acid P667 to 1672 from PglBci (PYAQFI).
In some embodiments, the recombinant modified PglBci provided herein includes amino acid substitutions at amino acid positions N314, K488, D489 and K668.
In certain embodiments, the recombinant modified PglBci comprises a substitution of the amino acid N314 with an aliphatic amino acid, for example glycine (G), alanine (A), valine (V), leucine ( L) or isoleucine (I). In certain embodiments, the recombinant modified PglBci comprises an N314V substitution.
In some embodiments, the recombinant modified PglBc1 comprises a substitution of the amino acid K488 with a basic amino acid, such as, for example, lysine (K) or arginine (R). In some embodiments, the recombinant modified PglBci comprises a K488R substitution.
In some embodiments, the recombinant modified PglBci comprises a D489H substitution.
In certain embodiments, the recombinant modified PglBci comprises a substitution of the amino acid K668 with an aliphatic amino acid, such as, for example, glycine (G), valine (V), leucine (L) or isoleucine ( I). In some embodiments, the recombinant modified PglBci comprises a K668V substitution.
In some embodiments, the recombinant modified PglBci comprises an N314V substitution, a K488R substitution, a D489H substitution, and a K668V substitution.
In some embodiments, the recombinant modified PglBci comprises five amino acid substitutions.
In some embodiments, the recombinant modified PglBci comprises an N535Q substitution.
In some embodiments, the recombinant modified PglBci comprises an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution, and a K668V substitution.
In some embodiments, the recombinant modified PglBci can detectably bind an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end to a carrier protein to produce an N-glycosylated carrier protein. The oligosaccharide and the polysaccharide may comprise any of the oligosaccharides or polysaccharides described herein. See, for example, Section 6.2. In some embodiments, the oligosaccharide and the polysaccharide comprise a capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5), a capsular polysaccharide CP8 of Staphylococcus aureus, a capsular polysaccharide CPI of Streptococcus pneumoniae (CPS 1) or the antigen 0 of Plesiomonas shigelloides 017 (017). See, for example, Figures 1 to 4.
The carrier protein may comprise any of the carrier proteins described herein. See, for example, Section 6.3. In some embodiments, the carrier protein is an α-toxin (hemolysin α, Hla), an agglutinating factor CP5 (Clfa5) or a P. aeruginosa exotoxin A (EPA). See, for example, Figures 1 to 4.
In some embodiments, the N-glycosylated carrier protein is a CP5-Hla bioconjugate, a CP8-ClfA bioconjugate, a CP1-EPA bioconjugate or a 017-EPA bioconjugate. See, for example, Figures 1 to 4.
Assays for confirming and quantifying the activity of recombinant modified N-OSTs provided herein are well known to those skilled in the art (e.g., ELISA, Western Blot, SDS-PAGE) and include tests described in Sections 6.8 and 6.10.
In some embodiments, the recombinant modified N-OST provided herein may increase the level of glycosylation in vivo or in vitro of a carrier protein with an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end between about 2-fold and about 100-fold, about 5-fold to about 80-fold, about 10-fold to about 60-fold, about 10-fold to about 20-fold, or about 20-fold to about 40-fold, ratio to a wild-type form of the recombinant modified N-OST provided herein, or to a recombinant modified N-OST having less amino acid substitution (e.g., having one, two or three amino acid substitutions) as the recombinant modified N-OST proposed herein.
In some embodiments, the recombinant modified N-OST provided herein may increase the level of glycosylation in vivo or in vitro of a carrier protein with an oligosaccharide or polysaccharide lacking an N-acetylated sugar at the same time. reducing end of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% , more than twice, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times , more than 90 times or more than 100 times compared to the rate of a form of the recombinant modified N-OST proposed herein, or of a recombinant modified N-OST having less amino acid substitution (e.g. having one, two or three amino acid substitutions) than the recombinant modified N-OST proposed herein.
In some embodiments, the glycosylation levels of the recombinant modified N-OST provided herein and the wild type form of recombinant N-OST can be compared by comparing the glycosylation levels of N-OST. The recombinant modification proposed herein and wild-type N-OST of a carrier protein with a polysaccharide or oligosaccharide lacking N-acetylated sugar at the reducing end.
In some embodiments, the glycosylation rate of the recombinant modified N-OST of a carrier protein with a polysaccharide or oligosaccharide lacking N-acetylated sugar at the reducing end is compared to the glycosylation rate of a wild-type N-OST of a carrier protein with a polysaccharide or oligosaccharide having an N-acetylated sugar at the reducing end. In some embodiments, the wild-type N-OST glycosylation rate of a polysaccharide or oligosaccharide carrier protein having N-acetylated sugar at the reducing end is defined as a relative level of 100%. . In some embodiments, the level of glycosylation of recombinant NOSTs of a carrier protein with a polysaccharide or oligosaccharide lacking an N-acetylated sugar at the reducing end is at least 10%, at least 15%, at less than 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least less than 70%, at least 75% or at least 80% of the relative rate of a wild-type N-OST.
In some embodiments, the recombinant modified N-OST provided herein may increase the in vivo or in vitro glycosylation yield of a carrier protein with an oligosaccharide or polysaccharide lacking an N-acetylated sugar at the same time. the reducing end between about 2 times and about 100 times, between about 5 times and about 80 times, between about 10 times and about 60 times, between about 10 times and about 20 times or about 20 times and about 40 times with respect to yield obtained with a wild-type form of the recombinant modified N-OST provided herein, or with respect to a recombinant modified N-OST having less amino acid substitution (e.g., one, two or three substitutions of amino acid) than the recombinant modified N-OST proposed herein.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a single substitution, such as a PglBcj (N534Q). recombinant modified. See, for example, Figure 1.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a double substitution, such as a PglBcj (N311V Recombinant modified PglBcy (K482R-N534Q), a recombinant modified PglBc1 (D483H-N534Q), a recombinant modified PglBcj (N311V-N534Q) or a recombinant modified PglBcj (N534Q-A669V). See, for example, Figure 1.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a triple substitution, such as a PglBcj (K482R- D483H-N534Q) recombinant modified. See, for example, Figure 3.
In some embodiments, the recombinant modified N-OST provided herein may increase the in vivo or in vitro glycosylation yield of a carrier protein with an oligosaccharide or polysaccharide lacking an N-acetylated sugar at the same time. reducing end of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% , more than twice, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times , more than 90 times or more than 100 times compared to the performance o obtained with a wild-type form of the recombinant modified N-OST provided herein, or with respect to a recombinant modified N-OST having less amino acid substitution (e.g., a recombinant modified N-OST presenting only a single, double or triple amino acid substitution, such as PglBcj (N534Q) or PglBci (N535Q)) as the recombinant modified N-OST proposed herein.
In some embodiments, the recombinant modified N-OST provided herein may increase the in vivo or in vitro glycosylation yield of a carrier protein with an oligosaccharide or polysaccharide lacking an N-acetylated sugar at the same time. reducing end at a level which is detectable more than twice, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70-fold, more than 80-fold, more than 90-fold, or more than 100-fold above basal level (for example, a mean or median signal of control experiments performed in the absence of N- OST ) in an assay detecting N-glycosylation of a carrier protein (e.g., ELISA, HPLC, LC-MS; see also Section 5.8 and Section 5.10).
In some embodiments, the recombinant modified N-OST provided herein may provide an in vivo glycosylation level or an in vitro glycosylation level of the carrier protein between about 1% and about 70%, between about 3% and about 65%, from about 5% to about 60%, from about 5% to about 55%, from about 10% to about 50%, from about 15% to about 45%, from about 20% to about 40%, or between about 25% and about 35%.
In some embodiments, the modified, recombinant N-OST provided herein may provide an in vivo glycosylation level or an in vitro glycosylation level of the carrier protein of at least 1%, at least 3 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%.
In some embodiments, the carrier protein comprises two or more N-glycosylation consensus sequences. In some embodiments, the recombinant modified N-OST provided herein may glycosylate in vitro or in vivo all consensus N-glycosylation sequences of the carrier protein. In some embodiments, the recombinant modified N-OST provided herein may glycosylate at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, minus 70%, at least 80% or at least 90% of all consensus N-glycosylation sequences of the carrier protein. In some embodiments, the recombinant modified N-OST provided herein may glycosylate in vitro or in vivo between about 10% and about 70%, between 20% and about 60%, or between about 30% and about 50% of all consensus N-glycosylation sequences of a carrier protein.
In some embodiments, the carrier protein comprises one or more N-glycosylation consensus sequences. In some embodiments, the carrier protein is a population of carrier proteins. In some embodiments, the recombinant modified NOST provided herein may glycosylate in vitro or in vivo at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% % of all N-glycosylation consensus sequences of the supporting proteins of a population of carrier proteins. In some embodiments, the recombinant modified N-OST provided herein may glycosylate in vitro or in vivo between about 10% and about 70%, between 20% and about 60%, or between about 30% and about 50% of all the N-glycosylation consensus sequences of the carrier proteins of a carrier protein population.
In some embodiments, the recombinant modified N-OST provided herein may increase the homogeneity of in vivo or in vitro glycosylation of a carrier protein with an oligosaccharide or polysaccharide lacking N-acetylated sugar. at the reducing end relative to the in vivo or in vitro glycosylation homogeneity obtained with a recombinant modified N-OST proposed herein with less amino acid substitution (eg, a recombinant modified N-OST having only a single, double or triple amino acid substitution, such as PglBcj (N534Q) or PglBci (N535Q)) as the recombinant modified N-OST proposed herein. The homogeneity of the in vivo or in vitro glycosylation of a carrier protein can be determined, for example, by determining the glycosylation levels of different carrier proteins of a population of glycosylated carrier proteins (e.g. percentage (%) of glycosylation sites that are glycosylated in different carrier proteins of a population of glycosylated carrier proteins or expressed as a standard deviation of glycosylation levels of glycosylated carrier proteins of a population of glycosylated carrier proteins). In some embodiments, the recombinant N-OST can decrease the standard deviation of glycosylation levels of carrier proteins in a population of glycosylated carrier proteins of at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, based on the standard deviation of the carrier protein glycosylation levels obtained with a recombinant modified N-OST with less acid substitution amino acid (e.g., having one, two or three amino acid substitutions) as the recombinant modified N-OST provided herein.
In some embodiments, the oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end comprises the CPI capsular polysaccharide of Streptococcus pneumoniae (CPS 1) and the carrier protein comprises P exotoxin A. aeruginosa (EPA). In some embodiments, the recombinant modified N-OST provided herein may increase the in vitro or in vivo glycosylation rate or yield of EPA by at least 2-fold, at least at least 4-fold or at least 5-fold, relative to the in vitro or in vivo glycosylation rate or yield obtained with a recombinant modified N-OST having less amino acid substitution (e.g. two or three amino acid substitutions) as the recombinant modified N-OST proposed herein. In some embodiments, the recombinant modified N-OST provided herein may increase the rate or yield of in vitro or in vivo glycosylation of EPA with Streptococcus pneumoniae (CPS 1) CPI capsular polysaccharide. at least 3-fold, at least 4-fold or at least 5-fold, relative to the in vitro or in vivo glycosylation rate or yield obtained with a recombinant modified N-OST with less than or equal to amino acid substitution (e.g., having one, two or three amino acid substitutions) as the recombinant modified N-OST provided herein.
In some embodiments, the oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end comprises the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) and the carrier protein comprises detoxified hemolysin A (H1A ) of S. aureus. In some embodiments, the recombinant modified N-OST provided herein may increase the rate or yield of in vitro or in vivo H1A glycosylation by at least 2-fold, at least 3-fold, from less than 4-fold or at least 5-fold, relative to the rate or yield of in vitro or in vivo glycosylation obtained with a recombinant modified N-OST with less amino acid substitution (eg, having one, two or three amino acid substitutions) as the recombinant modified N-OST proposed herein. In certain embodiments, the recombinant modified N-OST provided herein may increase the rate or yield of in vitro or in vivo H1A glycosylation with Staphylococcus aureus CP5 capsular polysaccharide at least 2-fold. at least 3-fold, at least 4-fold or at least 5-fold, relative to the in vitro or in vivo glycosylation rate or yield obtained with a recombinant modified N-OST with less amino acid substitution ( for example, having one, two or three amino acid substitutions) as the recombinant modified N-OST provided herein.
In some embodiments, the oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end comprises Staphylococcus aureus capsular polysaccharide CP8 (CPS 8) and the carrier protein comprises agglutinating factor A (ClfA). of. coli. In some embodiments, the recombinant modified N-OST provided herein may increase the in vitro or in vivo glycosylation rate or yield of ClfA by at least 2-fold, at least 3-fold, from less than 4-fold or at least 5-fold, relative to the rate or yield of in vitro or in vivo glycosylation obtained with a recombinant modified N-OST with less amino acid substitution (eg, having one, two or three amino acid substitutions) as the recombinant modified N-OST proposed herein. In some embodiments, the recombinant modified N-OST provided herein may increase the rate or yield of in vitro or in vivo glycosylation of ClfA with Staphylococcus aureus CP8 capsular polysaccharide (CPS 8) of at least 2. at least 3-fold, at least 4-fold or at least 5-fold, relative to the rate or yield of in vitro or in vivo glycosylation obtained with a recombinant modified N-OST with less amino acid (e.g., having one, two or three amino acid substitutions) as the recombinant modified N-OST provided herein.
In some embodiments, the oligosaccharide or polysaccharide lacking an N-acetylated sugar at the reducing end comprises Plesiomonas shigelloides O antigen 017 (017) and the carrier protein comprises an EPA. In some embodiments, the recombinant modified N-OST provided herein may increase the in vitro or in vivo glycosylation rate or yield of EPA by at least 2-fold, at least at least 4-fold or at least 5-fold, relative to the in vitro or in vivo glycosylation rate or yield obtained with a recombinant modified N-OST having less amino acid substitution (e.g. two or three amino acid substitutions) as the recombinant modified N-OST proposed herein. In some embodiments, the recombinant modified N-OST provided herein may increase the in vitro or in vivo glycosylation rate or yield of EPA with Plesiomonas shigelloides 017 (017) antigen 0. at least 3-fold, at least 4-fold or at least 5-fold, relative to the in vitro or in vivo glycosylation rate or yield obtained with a recombinant modified N-OST with less than or equal to amino acid substitution (e.g., having one, two or three amino acid substitutions) as the recombinant modified N-OST provided herein.
In some embodiments, the recombinant modified N-OST provided herein is derived from a procaryotic organism of the genus Campylobacter. In some embodiments, the recombinant N-OST is derived from Campylobacter jejuni or Campylobacter lari (e.g., the pglB product of the C. jejuni PglB gene, PglBcj, or C. lari, PglBci).
In some embodiments, the recombinant modified N-OST provided herein is a recombinant modified PglBcy, a homologue of
Recombinant modified pglBcj or a recombinant modified version of a naturally occurring PglBcj variant. Homologs of PglBcj may include homologues of naturally occurring PglBcj, for example, as exemplified in Figure 6, and unnatural counterparts of PglBcj. The homologs of PglBcj may comprise proteins having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with a PglBcj of SEQ ID NO: 1.
In some embodiments, the recombinant modified N-OST provided herein is a recombinant PglBci, a recombinant modified PglBci homologue or a recombinant modified version of a naturally occurring PglBci variant. Homologs of PglBci may include naturally occurring PglBci homologs, for example, as exemplified in Figure 6, and unnatural counterparts of PglBci. The homologs of PglBci may comprise proteins having sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with a PglBci of SEQ ID NO: 2.
In some embodiments, the recombinant modified N-OST provided herein includes a PglB fragment, for example a PglBc1 fragment or a PglBci fragment. In some embodiments, the PglB fragment comprises at least 350, at least 400, at least 450, at least 500, at least 550, at least 600 or at least 650 consecutive amino acids of a full length PglB. (a) Modifications of PglBçj
In some embodiments, the recombinant modified N-OST provided herein is wild-type, recombinantly modified N-OST, for example wild-type PglBcj. In some embodiments, the wild-type PglBcy is a wild-type PglBcy of SEQ ID NO: 1, or a naturally occurring variant thereof:
MLKKEYLKNP YLVLFAMIIL AYVFSVFCRF YWVWWASEFN EYFFNNQLMI ISNDGYAFAE GARDMIAGFH QPNDLSYYGS SLSALTYWLY KITPFSFESI ILYMSTFLSS LVVIPTILLA NEYKRPLMGF VAALLASIAN SYYNRTMSGY YDTDMLVIVL PMFILFFMVR MILKKDFFSL IALPLFIGIY LWWYPSSYTL NVALIGLFLI YTLIFHRKEK IFYIAVILSS LTLSNIAWFY QSAIIVILFA LFALEQKRLN FMIIGILGSA TLIFLILSGG VDPILYQLKF YIFRSDESAN LTQGFMYFNV NQTIQEVENV DLSEFMRRIS GSEIVFLFSL FGFVWLLRKH KSMIMALPIL VLGFLALKGG LRFTIYSVPV MALGFGFLLS EFKAIMVKKY SQLTSNVCIV FATILTLAPV FIHIYNYKAP TVFSQNEASL LNQLKNIANR EDYWTWWDY GYPVRYYSDV KTLVDGGKHL GKDNFFPSFA LSKDEQAAAN MARLSVEYTE KSFYAPQNDI LKTDILQAMM KDYNQSNVDL FLASLSKPDF KIDTPKTRDI YLYMPARMSL IFSTVASFSF INLDTGVLDK PFTFSTAYPL DVKNGEIYLS NGVVLSDDFR SFKIGDNVVS VNSIVEINSI KQGEYKITPI DDKAQFYIFY LKDSAIPYAQ FILMDKTMFN SAYVQMFFLG NYDKNLFDLV INSRDAKVFK LKIYPYDVPD YA (b) changes PglBci
In some embodiments, the modified N-OSTs described herein are recombinant modified wild-type NOSTs, for example wild-type PglBci (Campylobacter lari PglB). In some embodiments, the wild-type PglBcy is a wild-type PglBci of SEQ ID NO: 2, or a naturally occurring variant thereof:
MKLQQNFTDN NSIKYTCILI LIAFAFSVLC RLYWVAWASE FYEFFFNDQL
MITTNDGYAF AEGARDMIAG FHQPNDLSYF GSSLSTLTYW LYSILPFSFE
SIILYMSAFF ASLIVVPIIL IAREYKLTTY GFIAALLGSI ANSYYNRTMS
GYYDTDMLVL VLPMLILLTF IRLTINKDIF TLLLSPVFIM IYLWWYPSSY
SLNFAMIGLF GLYTLVFHRK EKIFYLTIAL MIIALSMLAW QYKLALIVLL
FAIFAFKEEK INFYMIWALI FISILILHLS GGLDPVLYQL KFYVFKASDV
QNLKDAAFMY FNVNETIMEV NTIDPEVFMQ RISSSVLVFI LSFIGFILLC
KDHKSMLLAL PMLALGFMAL RAGLRFTIYA VPVMALGFGY FLYAFFNFLE
KKQIKLSLRN KNILLILIAF FSISPALMHI YYYKSSTVFT SYEASILNDL
KNKAQREDYV VAWWDYGYPI RYYSDVKTLI DGGKHLGKDN FFSSFVLSKE
QIPAANMARL SVEYTEKSFK ENYPDVLKAM VKDYNKTSAK DFLESLNDKD
FKFDTNKTRD VYIYMPYRML RIMPVVAQFA NTNPDNGEQE KSLFFSQANA
IAQDKTTGSV MLDNGVEIIN DFRALKVEGA SIPLKAFVDI ESITNGKFYY
NEIDSKAQIY LLFLREYKSF VILDESLYNS SYIQMFLLNQ YDQDLFEQIT
NDTRAKIYRL KR
Oligosaccharides and polysaccharides (Section 6.2)
Oligosaccharides that may be bound to a carrier protein by a recombinant modified N-OST provided herein may contain between 2 and 100 monosaccharide units, for example, 2, 4, 6, 8, 10, 15, 20, 25 , 30, 40, 50, 60, 70, 80, 90 or 100 monosaccharide units. Polysaccharides that may be bound to a carrier protein by a recombinant modified N-OST provided herein may contain more than 100 monosaccharide units, for example, 101, 110, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 monosaccharide units or more.
The carrier proteins or N-OST may comprise any of the N-OSTs or any of the carrier proteins described herein. See, for example, Sections 6.1 and 6.3.
In some embodiments, the reducing end sugar of the oligosaccharide or polysaccharide is pentose, hexose or heptose. In some embodiments, the reducing end sugar of the oligosaccharide or polysaccharide is aldopentose or ketopentose. In some embodiments, the pentose is a D-arabinose, a D-lyxose, a D-ribose, a D-xylose, a D-ribulose or a D-xylulose. In some embodiments, the reducing end sugar of the oligosaccharide or polysaccharide is aldohexose or ketohexose. In some embodiments, hexose is, for example, D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-Talose, D-psicose, D-fructose, D-sorbose or D-tagatose. In some embodiments, the reducing end sugar of the oligosaccharide or polysaccharide is a deoxy or di-deoxy sugar, such as, for example, rhamnose, fucose or abequose. In some embodiments, the reducing end sugar of the oligosaccharide or polysaccharide is aldoheptose or ketoheptose. In some embodiments, the heptose is a mannoheptulose.
Oligosaccharides and polysaccharides that may be bound to a carrier protein by the recombinant N-OSTs provided herein may be from any organism, for example a prokaryotic organism or a eukaryotic organism. In some embodiments, the oligosaccharide or polysaccharide is from a pathogenic organism, for example a human pathogen or an animal pathogen (e.g., a farmed animal or a pet). In some embodiments, the oligosaccharide or polysaccharide is from a bacterial organism. In some embodiments, the oligosaccharide or polysaccharide may be from E. coli, Salmonella sp (for example, S. enterica subsp Enterica, S. enterica subsp Salamae, S. enterica subsp, arizonae, S. enterica subsp Diarizonae, S. enterica subsp Houtenae, S. bongori, and S Enterica subsp., Pseudomonas sp (P. aeruginosa), Klebsiella sp. (for example, K. pneumonia), Acinetobacter, Chlamydia trachomatis,
Vibrio cholera, Listeria sp., For example, L. monocytogenes, Legionella pneumophila, Bordetella parapertussis, Burkholderia mallei and Pseudomallei, Francisella tularensis, Campylobacter sp. (C. jejuni); Clostridium difficile, Staphylococcus aureus,
Streptococcus pyrogenes, E. coli, Streptococcus agalacticae, Neisseria meningitidis, Candida albicans, Haemophilus influenza, Enterococcus faecalis, Borrelia burgdorferi, Neisseria meningitidis, Haemophilus influenza, Leishmania major.
In some embodiments, the oligosaccharide or polysaccharide comprises an antigen, for example, an epitope that is immunogenic in a human or animal (e.g., a farmed animal or pet). In some embodiments, the oligosaccharide or polysaccharide comprises an E. coli O antigen (e.g., 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, 013 , 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 028, 029, 030, 032, 033, 034, 035, 036, 037, 038, 039 , 040, 041, 042, 043, 044, 045, 046, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065 , 066, 068, 069, 070, 071, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092 , 093, 095, 096, 097, 098, 099, 0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, 0110, YES, 0112, 0113, 0114, 0115, 0116, 0117, 0118 , 0119, 0120, 0121, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0138, 0139, 0140, 0141, 0142, 0143, 0144 , 0145, 0146, 0147, 0148, 0149, 0150, 0151, 0152, 0153, 0154, 0155, 0156, 0157, 0158, 0159, 0160, 0161, 0162, 0163, 0164, 0165, 0166, 0167, 0168, 0169 , 01 70, 0171, 0172, 0173, 0174, 0175, 0176, 0177, 0178, 0179, 0180, 0181, 0182, 0183, 0184, 0185, 0186, 0187), antigens of Salmonella sp (S. enterica subsp. Enterica, S. enterica subsp. Salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae, S. bongori or S. enterica subsp. indica and types 0-1 to 67, as described in Grimont P, Weill F: Antigenic Formula of the
Salmonella servoras. In., 9th edn edition. Geneva: WHO Collaborating Center for Reference and Research on Salmonella; 2007, Pseudomonas sp. (P. aeruginosa 0 serotypes 1 to 20 [Rocchetta HL, Burrows LL, Lam JS: Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa, Microbiology and Molecular Biology Reviews: MMBR 1999, 63 (3): 523-553]), Klebsiella sp. (for example, K. pneumonia serotypes 01, 02 (and sub serotypes), 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, [Trautmann M, Held TK, Cross AS: 0 Serumepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections, Vaccine 2004, 22 (7): 818-821]), Acinetobacter 0 antigens (eg, A. baumannii antigens identified in Pantophlet R). , Nemec A, Brade L, Brade H, Dijkshoorn L: 0-antigen diversity among Acinetobacter baumannii strains from the Czech Republic and Northwestern Europe, as determined by lipopolysaccharide-specific monoclonal antibodies Journal of Clinical Microbiology 2001, 39 (7): 25762580 ), 0 antigens of Chlamydia trachomatis (serotypes A, B, C, D, E, F, G, H, IJ, K, L1, L2, L3), Vibrio 0 antigens selected from 01 to 155, Listeria sp. , in particular L. monocytogenes type 1, 2, 3, 4 and their sub serotypes, Legionella pneumophila serotype 1 to 15 antigens, antigens Bordetella parapertussis 0, 0 Burkholderia mallei and Pseudomallei antigens, Francisella tularensis, Campylobacter sp. (C. jejuni); capsular polysaccharides of Clostridium difficile (serotypes A, G, H, K, SI, S4, D, Cd-5, K Toma et al., 1988, and C. perfringens serotypes A, B, C, D and E), Staphylococcus aureus type 5 and 8, Streptococcus pyrogenes (serotype group B streptococcal capsular polysaccharides), E. coli, Streptococcus agalacticae (group A streptococcal capsular polysaccharides), Neisseria meningitidis (serotypes A, B, C, W, Y, X ), Candida albicans, Haemophilus influenza, capsular polysaccharides of Enterococcus faecalis type I to V; and other surface polysaccharide structures, for example the glycolipids of Borrelia burgdorferi (Hossain H,
Wellensiek HJ, Geyer R, Lochnit G: Structural analysis of glycolipids from Borrelia burgdorferi. Biochemistry 2001, 83 (7): 683-692), the glycan 0 from the pilin of
Neisseria meningitidis [Borud B, Aas FE, Vik A, Winther-Larsen HC, Egge-Jacobsen W, Koomey M: Genetic, structural, and antigenic analyzes of glycan diversity in the O-linked protein glycosylation Systems of human Neisseria species. Journal of Bacteriology 2010, 192 (11): 2816-2829; Borud B, Viburiene R, Hartley MD,
Paulsen BS, Egge-Jacobsen W, Imperiali B, Koomey M:
Genetic and molecular analyzes reveal an evolutionary trajectory for glycan synthesis in a bacterial protein glycosylation system. Proceedings of the National Academy of Sciences of the United States of America 2011, 108 (23): 9643-9648] and a lipooligosaccharide (LOS), a Haemophilus influenzae LOS, a lipophosphoglycan of Leishmania major [McConville MJ,
Bacic A, Mitchell GF, Handman E: Lipophosphoglycan of Leishmania major that vaccine against cutaneous leishmaniasis contains an alkylglycerophosphoinositol lipid anchor. Proceedings of the National Academy of Sciences of the United States of America 1987, 84 (24): 8941-8945; McConville MJ, Ferguson MA: The structure, biosynthesis and function of glycosylated phosphatidylinositols in parasitic protozoa and higher eukaryotes. The Biochemical Journal 1993, 294 (Pt 2): 305-324]), tumor-associated carbohydrate antigens (malaria glycosyl phosphatidylinositol, mycobacterium tuberculosis arabinomannan [Astronomo RD, Burton DR: Carbohydrate vaccines: developing sweet solutions To sticky situations Nature Reviews Drug Discovery 2010, 9 (4): 308-324].
In some embodiments, the oligosaccharide or polysaccharide is a polysaccharide of Staphylococcus aureus (S. aureus) or Salmonella enterica sv. (S. enterica sv.). In some embodiments, the polysaccharide is a S. aureus CP5 polysaccharide or an S. enterica sv LT polysaccharide. Typhimurium.
In some embodiments, the oligosaccharide or polysaccharide comprises an N-acetylated sugar at the reducing end. In some embodiments, the oligosaccharide or polysaccharide comprising the N-acetylated sugar at the reducing end may comprise, for example, an E. coli O antigen. coli (e.g., 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, 013, 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 028, 029, 030, 032, 033, 034, 035, 036, 037, 038, 039, 040, 041, 042, 043, 044, 045, 046, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065, 066, 068, 069, 070, 071, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 095, 096, 097, 098, 099, 0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, 0110, YES, 0112, 0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120, 0121, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0138, 0139, 0140, 0141, 0142, 0143, 0144, 0145, 0146, 0147, 0148, 0149, 0150, 0151, 0152, 0153, 0154, 0155, 0156, 0157, 0158, 0159, 0160, 0161, 0162, 0163, 0164, 0165, 0166, 0167, 0168, 0169, 0170, 0171, 0172, 0173, 0174, 0175, 0176, 0177, 0178, 0179, 0180, 0181, 0182, 0183, 0184, 0185, 0186, 0187), a capsular polysaccharide of Staphylococcus aureus (S. aureus) (e.g. CP5 or CP8), a capsular polysaccharide of Francisella tularensis Schu4, a capsular polysaccharide of S. pneumoniae capsules (e.g., CPI, 4, 5, 12, 25, 38, 44, 45 or 46), glycan 0 of Neisseria meningitidis pilin [Borud B, Aas FE, Vik A, Winther-Larsen HC, Egge-Jacobsen W, Koomey M: Genetic, structural, and antigenic analyzes of glycan diversity in the 0-linked protein glycosylation Systems of human Neis species. Journal of Bacteriology 2010, 192 (11): 2816-2829; Borud B, Viburiene R, Hartley MD, Paulsen BS, Egge-Jacobsen W, Imperiali B, Koomey M: Genetic and Molecular Analyzes Reveal an Evolutionary Pathway for Glycan Synthesis in a Bacterial Protein Glycosylation System. Proceedings of the National Academy of Sciences of the United States of America 2011, 108 (23): 9643-9648], Burkholderia mallei and Pseudomallei antigen 0, Bordetella parapertussis 0 antigen, Legionella pneumophila serotype 1 antigen 0 to 15, Listeria sp. Antigen 0, particularly L. monocytogenes type 1, 2, 3, 4 antigen 0, Pseudomonas sp. (P. aeruginosa 0 serotypes 1 to 20 [Rocchetta HL, Burrows LL, Lam JS: Genetics of 0-antigen biosynthesis in Pseudomonas aeruginosa, Microbiology and Molecular Biology Reviews: MMBR 1999, 63 (3): 523-553]), a O antigen from Klebsiella sp. (for example, K. pneumonia serotypes 01, 02 (and sub serotypes), 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, [Trautmann M, Held TK, Cross AS: 0 An antigen of Shigella sp., Vaccine 2004, 22 (7): 818-821]), an antigen of Shigella sp. (eg, S. dysenteriae, S. sonnei, S. flexneri, S. boydii), an Acinetobacter antigen 0 (e.g., A. baumannii antigens 0 identified in Pantophlet R, Nemec A, Brade L, Brade H, Dijkshoorn L: 0-antigen diversity among Acinetobacter baumannii strains from the Czech Republic and Northwestern Europe, as determined by lipopolysaccharide-specific monoclonal antibodies, Journal of Clinical Microbiology 2001, 39 (7): 2576-2580), or an antigen 0 of Listeria sp.
The N-acetylated sugars may comprise an amino-acetyl (N-acetyl) substituent on one or more carbon atoms of the sugar. For example, an N-acetylated sugar may comprise an N-acetyl substituent on the C2 atom of a monosaccharide unit, such as a glucose (N-acetylglucosamine) unit.
In some embodiments, the oligosaccharide or polysaccharide comprises a reducing end sugar that is not N-acetylated. In certain embodiments, the oligosaccharide or polysaccharide comprising the non-N-acetylated sugar at the reducing end may comprise, for example, 020 of E. coli, an antigen of Salmonella sp (for example, S. enterica subsp Enterica, S. enterica subsp Salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp.houtenae, S. bongori or S. enterica subsp. Indica or S. Typhi), a type 1 to 67 antigen 0, a capsular polysaccharide of group A streptococci (S. pyrogenes), group B streptococci, and S. pneumoniae CPS serotypes ( encoding wchA, wcjG or wcjH in their capsular gene cluster, i.e. all serotypes except CPI, 4, 5, 12, 25, 38, 44, 45, 46), or Salmonella enterica antigen 0 sv. {S. enterica sv. ).
In some embodiments, the oligo saccharide or polysaccharide comprises a S. aureus CP5 polysaccharide or an S. enterica sv LT polysaccharide. Typhimurium, an O antigen
Vibrio cholera (for example, 01 to 155), or a 0 antigen from Listeria sp. (for example, L. monocytogenes type 1, 2, 3, 4).
In some embodiments, the oligosaccharide or polysaccharide comprises a DN-acetylfucosamine residue (D-FucNAc) at its reducing end, such as, for example, S. aureus serotype 5,8 capsular polysaccharide or a P. aeruginosa antigen. serotype 02, 05, 011, 016.
In some embodiments, the oligosaccharide or polysaccharide comprises a 4-amino-dN-acetylfucosamine residue (D-FucNAc4N) at its reducing end, such as certain S. pneumoniae oligosaccharides or polysaccharides, such as serotype 1, a Shigella sonnei 0 or 017 antigen of Plesiomonas shigelloides.
In some embodiments, the oligosaccharide or polysaccharide comprises a D-N-acetylquinosamine residue (D-QuiNAc) at its reducing end, such as for example a P. aeruginosa serotype 06 01 antigen or Francisella tularensis serotype Schu4.
In some embodiments, the oligosaccharide or polysaccharide comprises a galactose residue at its reducing end, such as, for example, LT2 of S. enterica.
In some embodiments, the oligosaccharide or polysaccharide comprises a capsular polysaccharide of S. pneumoniae serotype 5, E. coli 01, 02, Cronobacter sakazakii 05, i.e. end-poly- and oligo-saccharide D-GlcNAc reducing agent bound in 1-4 to L-Rhamnose in beta configuration.
Support Proteins (Section 6.3)
Supporting proteins can be linked to oligosaccharides or polysaccharides by recombinant N-OSTs provided herein. See, for example, Section 6.1.
The carrier protein can be any natural carrier protein (from the same organism as that of N-OST) or any heterologous carrier protein (from a different organism than that of N-OST). OST). In some embodiments, the carrier protein is an immunogen. The carrier proteins may be full-length proteins or fragments thereof. Examples of carrier proteins include, but are not limited to, P. aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, S. aureus detoxified hemolysin A, the factor agglutinating A (ClfA), agglutinating factor B, FimH dΈ. E. coli, FimHC. coli, the thermolabile enterotoxin of E. coli. coli, the detoxified variants of E. thermolabile enterotoxin coli, cholera toxin subunit B (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli, the transient domain of E sat protein. coli, C. jejuni's AcrA and the natural glycoproteins of C. jejuni. In some embodiments, the carrier protein is P. aeruginosa exotoxin A (EPA).
In some embodiments, the recombinant N-OST-N-glycosylated carrier proteins described herein are modified, for example, modified such that the protein is less toxic and / or more sensitive to glycosylation, etc. In some embodiments, the carrier proteins are modified such that the number of glycosylation sites in the carrier proteins is maximized in order to administer the protein at lower concentrations, for example in an immunogenic composition, under its bioconjugate form. Therefore, in some embodiments, the carrier proteins described herein are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 glycosylation sites which would normally be associated with the carrier protein (e.g., relative to the number of glycosylation sites associated with the carrier protein in its native / natural state, e.g., its "wild-type" state). In some embodiments, the introduction of glycosylation sites is accomplished by the insertion of consensus glycosylation sequences (eg, (i) the consensus sequence Asn-X-Ser (Thr), wherein X is / are selected (s) independently from amino acids except Pro; or (ii) the consensus sequence D / EXNZS / T, wherein X and Z are independently selected from amino acids except Pro) anywhere in the primary structure of the protein . The introduction of these glycosylation sites can be achieved, for example, by adding new amino acids to the primary structure of the protein (the glycosylation sites are added, in whole or in part), or by modifying the existing amino acids in the protein to generate the glycosylation sites (no amino acid is added to the protein, but selected amino acids of the protein are mutated to form glycosylation sites). It is known to those skilled in the art that the amino acid sequence of a protein can be easily modified using known approaches of the state of the art, for example, recombination approaches that include modification of the sequence of nucleic acid encoding the protein. In specific embodiments, consensus glycosylation sequences are introduced into specific regions of the carrier protein, for example, surface structures of the protein, at the N- or C-terminus of the protein and / or in loops that are stabilized by disulfide bridges at the base of the protein. In some embodiments, the conventional 5-amino acid glycosylation consensus sequence may be prolonated by lysine residues to allow more efficient glycosylation and, therefore, the inserted consensus sequence may encode 5, 6 or 7 amino acids which must be inserted or replacing the accepting amino acids of the protein.
N-OSTs may include any of the N-OSTs described in this document. See, for example, Section 6.1.
In some embodiments, the carrier proteins comprise a "tag", an amino acid sequence that allows the isolation and / or identification of the carrier protein. For example, the addition of a label to a carrier protein described herein may be useful in the purification of this protein and, therefore, in the purification of conjugate vaccines comprising the carrier protein marked. Examples of labels that may be used herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine tag or 6XHis tag), FLAG-TAG tags, and HA tags. In some embodiments, the labels used herein can be removed, for example, removed by chemical agents or enzymatic means, as soon as they are no longer needed, for example after purification of the protein.
Nucleic acids (Section 6.4)
In another aspect, there is provided herein nucleic acids encoding recombinant modified N-OSTs provided herein (e.g., Section 6.1). . In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising amino acid substitution in amino acids N311, K482, D483 and A669.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising a substitution of the amino acid N311 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I).
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising an N311V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising a substitution of the amino acid K482 with a basic amino acid selected from lysine (K) or arginine (R).
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising a K482R substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising a D483H substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising a substitution of the amino acid A669 by an aliphatic amino acid selected from the group consisting of glycine (G), valine (V), leucine (L ) or isoleucine (I).
In some embodiments, the nucleic acids encode a modified recombinant PglBcy comprising an A669V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising an N311V substitution, a substitution
K482R, a D483H substitution and a substitution A669V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising an amino acid substitution in amino acids N311, K482, D483, N534 and A669.
In some embodiments, the nucleic acids encode a modified recombinant PglBjj comprising an N534Q substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising N311V substitution, K482R substitution, D483H substitution, N534Q substitution, and A669V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising an amino acid substitution in amino acids N314, K488, D489 and another amino acid substitution in a region comprising amino acid positions P667 at 1672 from PglBci (PYAQFI).
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising an amino acid substitution in amino acids N314, K488, D489 and K668.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a substitution of amino acid N314 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I).
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising N314V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a substitution of the amino acid K488 with a basic amino acid selected from lysine (K) or arginine (R).
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a K488R substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a D489H substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a substitution of the amino acid K668 by an aliphatic amino acid selected from the group consisting of glycine (G), valine (V), leucine (L ) or isoleucine (I).
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising a K668V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising N314V substitution, K488R substitution, D489H substitution, and K668V substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBci comprising amino acid substitution in amino acids N314, K488, D489, N535 and K668.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising an N535Q substitution.
In some embodiments, the nucleic acids encode a modified recombinant PglBcj comprising an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution, and a K668V substitution.
In some embodiments, nucleic acids encoding Campylobacter jejuni pglB or Campylobacter lari are provided herein, wherein the nucleic acid is a codon optimized for expression in a host cell. In specific embodiments, the host cell is E. coli. In even more specific embodiments, the optimized codon sequences are the sequences mentioned in the examples (Section 7).
Host cells (Section 6.5)
In another aspect, there is provided herein a host cell comprising a recombinant modified N-OST provided herein. In some embodiments, the host cell comprises two or more modified recombinant N-OSTs provided herein (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10). Recombinant N-OST).
In another aspect, there is provided herein a host cell comprising a nucleic acid provided herein (e.g., encoding a recombinant N-OST provided herein, e.g., Section 6.1). See, for example, Section 6.5. In some embodiments, the host cell comprises two or more nucleic acids provided herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 nucleic acids) .
In some embodiments, the host cell comprises one or more additional enzymes useful for bioconjugate production or N-glycosylation of a carrier protein (e.g., glycosyltransferase). In some embodiments, at least one of the additional enzymes useful for the production of a bioconjugate is a recombinant enzyme. In some embodiments, the host cell comprises two or more additional enzymes useful for the production of a bioconjugate (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional enzymes).
In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a cell of E. coli. In some embodiments, the host cell comprises a recombinant modified N-OST provided herein. See, for example, Section 6.1. In some embodiments, the host cell comprises a carrier protein and a recombinant modified N-OST provided herein. See, for example, Sections 6.1 and 6.3. In some embodiments, the host cell comprises a carrier protein, a recombinant modified N-OST provided herein, and a recombinant glycosyltransferase. In some embodiments, the recombinant modified N-OST provided herein is a recombinant PglBcy. See, for example, Section 6.1 (a). In some embodiments, the recombinant modified N-OST provided herein is a recombinant PglBci. See, for example, Section 6.1 (a).
In some embodiments, the host cells used to produce the bioconjugates described herein are genetically engineered to include heterologous nucleic acids, for example, heterologous nucleic acids encoding one or more carrier proteins and / or nucleic acids. heterologous encoding one or more proteins, for example genes encoding one or more proteins. In some embodiments, heterologous nucleic acids encoding proteins involved in the glycosylation pathways (e.g., prokaryotic and / or eukaryotic glycosylation pathways) are introduced into the host cells described herein. Such nucleic acids may encode proteins such as, but not limited to, oligosaccharyl transferases and / or glycosyltransferases. Heterologous nucleic acids (e.g., nucleic acids encoding carrier proteins and / or nucleic acids encoding other proteins, e.g., proteins involved in glycosylation) can be introduced into the host cells described herein. using any method known to those skilled in the art, for example electroporation, heat shock chemical transformation, natural transformation, phage transduction and conjugation. In some embodiments, heterologous nucleic acids are introduced into the host cells described herein using a plasmid, for example the heterologous nucleic acids are expressed in the host cells by a plasmid (e.g., an expression vector ). In some embodiments, heterologous nucleic acids are introduced into the host cells described herein using the insertion method described in International Patent Application Publication No. WO 2014/057109.
In some embodiments, additional modifications may be introduced (e.g., using recombinant techniques) into the host cells described herein. For example, nucleic acids of the host cell (eg, genes) encoding proteins that form part of any competing or interfering glycosylation pathways (for example, that compete or interfere with one or more several heterologous genes involved in glycosylation that are introduced recombinantly into the host cell) can be deleted or modified in the background ("background") of the host cell (genome) so as to render them inactive / dysfunctional ( that is, nucleic acids of the host cell that are deleted / modified do not encode a functional protein or encode any protein). In some embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced with a desirable sequence, for example a sequence that is useful for the production of a glycoprotein.
Examples of genes that can be deleted in host cells (and, in some cases, replaced by other desired nucleic acid sequences) include host cell genes involved in glycolipid biosynthesis, such as waaL (see, for example, , Feldman et al., 2005, PNAS USA 102: 30163021), the lipid A core biosynthesis group (waa), the galactose group (gal), the arabinose group (ara), the group for coionic acid (wc), group for island polysaccharides, undecaprenol-p biosynthesis genes (eg, uppS, uppP), und-P recycling genes, metabolic enzymes involved in biosynthesis nucleotide-activated sugars, the group for the common enterobacterial antigens and the groups of prophage 0 antigen modifications such as the gtrABS group.
The host cells described herein can produce the N-glycosylated carrier proteins described herein. In some embodiments, the N-glycosylated carrier proteins produced by the host cells described herein are antigens, e.g., viral or bacterial antigens, that can be used in vaccines. In some embodiments, the N-glycosylated carrier proteins produced by the host cells described herein may be any of the carrier proteins described herein, wherein the carrier proteins are modified by the host cells described. in this document to possess one or more beneficial characteristics, for example the carrier protein is N-glycosylated.
Some of the examples below describe the application of the methods described herein to Gram-negative E. coli host cells; however, any host cell known to those skilled in the art can be used to produce N-glycosylated carrier proteins, including archaea, prokaryotic host cells different from E. coli and eukaryotic host cells.
Examples of prokaryotic host cells that can be used according to the methods described herein include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species and Clostridium species.
In some embodiments, the host cells described herein comprise a genome into which one or more DNA sequences have been introduced, wherein the DNA sequences encoding a protein or comprising a group of operons / genes involved in the N-glycosylation of proteins. For example, in some embodiments, a host cell described herein comprises a genome into which one or more of the following DNAs have been inserted: DNA encoding N-OST, DNA encoding a glycosyltransferase, DNA encoding a carrier protein, a DNA comprising a group of rfb genes, a DNA comprising a gene cluster for capsular polysaccharides and / or a DNA encoding an epimerase.
The host cells may comprise recombinant N-OSTs provided herein or nucleic acids encoding the recombinant N-OSTs provided herein, whereby the recombinant N-OSTs may originate from any organism having N-OSTs. OST, especially a eukaryotic organism or a prokaryotic organism. In some embodiments, the N-OST protein or nucleic acid encoding an N-OST is from the genus Campylobacter (e.g., the pglB gene of C. jejuni or C. lari).
The host cells described herein may comprise a known glycosyltransferase of the state of the art or a nucleic acid sequence encoding a glycosyltransferase known from the state of the art. In some embodiments, the glycosyltransferase is a glycosyltransferase disclosed in International Patent Application Publication No. WO 2011/138361, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the glycosyltransferase is from a Gram-positive bacterium, for example the glycosyltransferase is from S. aureus. In some embodiments, the glycosyltransferase is the capsular polysaccharide of S. aureus. In some embodiments, the glycosyltransferase is capsular polysaccharide 8 of S. aureus. In some embodiments, the glycosyltransferase is from a Gram-negative bacterium, for example E. coli. In some embodiments, the glycosyltransferase is from a eukaryote.
The host cells described herein may comprise or produce a known carrier protein of the state of the art or comprise a nucleic acid sequence encoding a known carrier protein of the state of the art. The carrier proteins produced by the host cells described herein include at least one N-glycosylation consensus sequence, for example the consensus sequence (i) Asn-X-Ser (Thr), wherein X is independently selected from amino acids except Pro; or (ii) D / E-X-N-Z-S / T, wherein X and Z are independently selected from amino acids except Pro. Therefore, the host cell may comprise DNA sequences encoding a consensus N-glycosylation sequence. The host cell may comprise any known carrier protein of the state of the art, including the carrier proteins described in Section 6.3. In some embodiments, the carrier protein is a P. aeruginosa exotoxin A (EPA), including an EPA that has been modified to include at least one consensus N-glycosylation sequence. In some embodiments, the carrier protein is cholera toxin B. In some embodiments, the carrier protein is AcrA. In some embodiments, the carrier protein is H1A (hemolysin a). In some embodiments, the carrier protein is ClfA.
Bioconjugates (Section 6.6)
The bioconjugates described herein are conjugates between a protein (e.g., any of the carrier proteins described herein, e.g., Section 6.3) and an oligosaccharide or polysaccharide (e.g. any of the oligosaccharides or polysaccharides described herein, see, for example, Section 6.2) prepared in a host cell, the host cell machinery binding the oligosaccharide or polysaccharide to the protein (eg, linkages to nitrogens) ). In some embodiments, the oligosaccharide or polysaccharide is an antigen (e.g., any of the antigens described herein, see, for example, Section 6.2). The glycoconjugates may include bioconjugates, as well as carbohydrate antigen conjugates (e.g., oligo- and polysaccharides) -protein prepared by other means, for example by chemical bonding of the protein and carbohydrate antigen.
The recombinant modified N-OSTs provided herein (see, for example, Section 6.1) can be used to produce host cells that produce bioconjugates comprising an N-glycosylated carrier protein. In some embodiments, bioconjugates comprising an N-glycosylated carrier protein with an antigen (eg, oligosaccharide or polysaccharide) described herein are provided herein. In some embodiments, the carrier protein is EPA, ClfA or H1A. The bioconjugates described herein may include, for example and without limitation, any of the carrier proteins described herein. The bioconjugates described herein may include, for example and without limitation, any of the oligosaccharides or polysaccharides described herein.
In some embodiments, there is provided in this document a bioconjugate containing a carrier protein conjugated to one or more of 01, 02, 04, 06, 07, 08, 011, 015, 016, 017, 018, 020, 022, 025, 073, 075 and / or 083 of E. coli. In some embodiments, the carrier protein is EPA.
In some embodiments, there is provided herein a bioconjugate containing a carrier protein conjugated to one or more polysaccharides other than P. aeruginosa. In some embodiments, the carrier protein is EPA, ClfA, or HLA.
In some embodiments, there is provided herein a bioconjugate containing a carrier protein conjugated to one or more polysaccharides other than Streptococcus pneumoniae. In some embodiments, the carrier protein is EPA, ClfA, or HLA. In some embodiments, the bioconjugate comprises the CPI capsular polysaccharide of Streptococcus pneumoniae (CPS 1) and the exotoxin A of P. aeruginosa (EPA). See, for example, Figure 3.
In some embodiments, there is provided herein a bioconjugate comprising a carrier protein conjugated to one or more polysaccharides other than Staphylococcus aureus. In some embodiments, the carrier protein is EPA, ClfA, or H1A. In some embodiments, the bioconjugate comprises the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) detoxified hemolysin A (H1A) of S. aureus. See, for example, Fig. 1. In some embodiments, the bioconjugate comprises Staphylococcus aureus CP8 capsular polysaccharide (CPS 8) and E (ClfA) agglutinating factor A (ClfA). coli. See, for example, Figure 2.
In some embodiments, there is provided herein a bioconjugate comprising a carrier protein conjugated to one or more polysaccharides other than Plesiomonas shigelloides. In some embodiments, the carrier protein is EPA, ClfA, or H1A. In some embodiments, the bioconjugate comprises 0 Plesiomonas shigelloides 017 (017) antigen and EPA. See, for example, Figure 4.
In some embodiments, the bioconjugate is S. dysenteriae Pseudomonas aeruginosa exotoxin (EPA) -01 (EPA-01), S. aureus type 5 capsular EPA-polysaccharide (EPA-CP5), EPA-LT2 of Salmonella enterica (S. enterica) (EPA-LT2).
Methods of producing a bioconjugate (Section 6.7)
In some embodiments, the recombinant modified N-OSTs provided herein (see, for example, Section 6.1) may be used to produce a bioconjugate proposed herein (see, for example, Section 6.6), such as a glycoconjugate. In some embodiments, the recombinant modified N-OSTs provided herein may be used to produce conjugate vaccines, i.e., vaccines that contain an oligosaccharide or a polysaccharide (see, for example, 6.2) and a protein antigen of the pathogen against which the vaccine is designed.
In another aspect, there is provided herein a method for producing a bioconjugate, the method comprising culturing a host cell provided herein (see, for example, Section 6.5) in a culture medium. of cells. In some embodiments, the host cell comprises a nucleic acid encoding a recombinant modified N-OST provided herein (see, for example, Section 6.1 and Section 6.4). In some embodiments, the host cell comprises a nucleic acid encoding a carrier protein described herein (see, for example, Section 6.3 and Section 6.4). In some embodiments, the carrier protein contains one or more N-glycosylation consensus sequences. In some embodiments, the host cell comprises a nucleic acid encoding a glycosyl transferase (see, for example, Section 6.2 and Section 6.5).
In some embodiments, the bioconjugate is an N-glycosylated carrier protein. The N-glycosylated carrier protein may comprise an oligosaccharide or polysaccharide component including any of the oligosaccharides or polysaccharides described herein. See, for example, Section 6.2. The N-glycosylated carrier protein can comprise any of the carrier proteins described herein. See, for example, Section 6.3. In some embodiments, the bioconjugate is a naturally occurring N-glycosylated polypeptide of C. jejuni (including an oligosaccharide or polysaccharide component of C. jejuni and a C. jejuni carrier protein). In some embodiments, the bioconjugate is a heterologous C. jejuni glycosylated polypeptide (including a polysaccharide component and / or a carrier protein that does not originate from C. jejuni). In some embodiments, the glycosylated polypeptide does not contain N-acetylated sugar at its reducing end. In some embodiments, the glycosylated polypeptide has galactose at its reducing end.
In some embodiments, the bioconjugate is produced at a rate of between about 2-fold to about 100-fold, about 5-fold to about 80-fold, about 10-fold to about 60-fold, about 10-fold to about 20-fold or about 20-fold to about 40-fold higher when using a host cell comprising a recombinant modified N-OST provided herein than when using a host cell comprising a rescuer-like form of recombinant N-OST, or when a host cell comprising a recombinant modified N-OST having less amino acid substitution (e.g., having one, two or three amino acid substitutions) than the recombinant modified N-OST proposed herein is used.
In some embodiments, the bioconjugate is produced at a rate more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12. times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times higher when using a host cell comprising a recombinant modified N-OST proposed herein. document when using a host cell comprising a wild-type form of the recombinant modified N-OST, or when using a host cell comprising a recombinant modified N-OST having less amino acid substitution (e.g. , two or three amino acid substitutions) as the recombinant modified N-OST proposed herein.
In some embodiments, the bioconjugate is produced in a yield of between about 2-fold and about 100-fold, between about 5-fold and about 80-fold, between about 10-fold and about 60-fold, between about 10-fold and about 20-fold. or between about 20-fold and about 40-fold higher when using a host cell comprising a recombinant N-OST of the present disclosure than when using a host cell comprising a wild-type form of recombinant modified N-OST, or when a host cell comprising a recombinant modified N-OST having less amino acid substitution (e.g., having one, two or three amino acid substitutions) than the recombinant modified N-OST proposed herein is used.
In some embodiments, the bioconjugate is produced at a yield that is more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times more when using a host cell comprising a recombinant modified N-OST provided herein when using a host cell comprising a wild-type form of recombinant modified N-OST, or when using a host cell comprising an N Recombinant modified -OST having less amino acid substitution (e.g., having one, two or three amino acid substitutions) than the recombinant modified N-OST proposed herein.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a single substitution, such as PglBcj (N534Q). recombinant modified. See, for example, Figure 1.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a double substitution, such as a PglBcj (N311V- N534Q) recombinant modified, recombinant modified PglBcy (K482R-N534Q), recombinant modified PglBcy (D483H-N534Q), recombinant modified PglBcj (N311V-N534Q) or recombinant modified PglBcj (N534Q-A669V). See, for example, Figure 1.
In some embodiments, the recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST provided herein comprises a recombinant modified N-OST having a triple substitution, such as a PglBcj (K482R- D483H-N534Q) recombinant modified. See, for example, Figure 3.
In some embodiments, the bioconjugate is produced at a yield that is detectable more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times. more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold baseline (eg, a mean or median signal of control experiments performed in the absence of an N-OST) in an assay detecting N-glycosylation of a carrier protein (e.g., ELISA, HPLC, LC-MS, see also Section 6.8 and Section 6.10).
In some embodiments, from about 1% to about 70%, from about 3% to about 65%, from about 5% to about 60%, from about 10% to about 55%, from about 15% to about 50% between about 20% and about 45%, between about 20% and about 45%, between about 25% and about 40%, or between about 30% and about 35% of the carrier protein in the host cell are glycosylated to form the bioconjugate.
In some embodiments, at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of the carrier protein in the host cell is glycosylated to form the bioconjugate .
In some embodiments, the bioconjugate comprises the CPI capsular polysaccharide of
Streptococcus pneumoniae (CPS 1) and exotoxin A of P. aeruginosa (EPA).
In some embodiments, the bioconjugate comprises the CP5 capsular polysaccharide of
Staphylococcus aureus (SPC 5) and detoxified hemolysin A (H1A) of S. aureus.
In some embodiments, the bioconjugate comprises the CP8 capsular polysaccharide of
Staphylococcus aureus (CPS 8) and the agglutinating factor A (ClfA) of E. coli.
In some embodiments, the bioconjugate comprises 0 Plesiomonas shigelloides 017 (017) antigen and EPA.
In some embodiments, the methods further include purifying the bioconjugate from the host cell culture. Methods for purifying bioconjugates, such as N-glycosylated carrier proteins, from host cell cultures are known from the state of the art. See, for example, Jan-Christer Janson, Protein Purification: Principles, High Resolution Methods, and Applications. Wiley; 3rd edition (March 22, 2011).
Analytical procedures (Section 6.8)
Various methods can be used to analyze the structural compositions and carbohydrate chain lengths of the bioconjugates or N-glycosylated carrier proteins described herein.
In one embodiment, hydrazinolysis can be used to analyze the glycans. First, the polysaccharides are released from their protein carriers by incubation with hydrazine according to the manufacturer's instructions (Kit Ludger Liberate Hydrazinolysis Glycan Release, Oxfordshire, UK). The nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows the release of the attached glycans. The N-acetyl groups are lost during this treatment and must be reconstituted by a new N-acetylation. The free glycans are purified on carbon columns and then labeled at the reducing end with the fluorophore 2-aminobenzamide (Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB.) Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid Anal Biochem 20 Sept. 1995; 230 (2): 229-38). The labeled polysaccharides are separated on a GlycoSep-N (GL Sciences) column according to the HPLC protocol of Royle et al. (Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM.) An analytical and structural database provides a strategy for sequencing O-glycans from micrograms of glycoproteins Anal Biochem. May 1, 2002; 304 (1): 70-90). The resulting fluorescence chromatogram indicates the length of the polysaccharide and the number of repeating units. Structural information can be collected by collecting individual peaks and then performing MS / MS analysis. Thus, the monosaccharide composition and the repeating unit sequence can be confirmed and, furthermore, the homogeneity of the polysaccharide composition can be identified. Specific low molecular weight peaks can be analyzed by MALDI-MS / MS and the result is used to confirm the glycan sequence. Each peak corresponds to a polymer consisting of a number of repeating units and fragments thereof. The chromatogram thus makes it possible to measure the length distribution of the polymers. The elution time is an indication of the length of the polymer, the intensity of the fluorescence being correlated with the molar abundance for the respective polymer.
In another embodiment, SDS-PAGE or capillary gel electrophoresis may be used to analyze glycans and glycoconjugates. The length of the polymers for the antigenic glycans 0 which are synthesized here is defined by the number of repeating units which are linearly assembled. This means that the classic ladder-type pattern is a consequence of different numbers of repeating patterns that make up the glycan. Therefore, two bands close to one another in SDS PAGE or other size-separated techniques differ from only one repetitive pattern. These discrete differences are exploited in glycoprotein analysis for glycan size: the unglycosylated carrier protein and the glycoconjugate with different polymer chain lengths separate as a function of their electrophoretic mobility. The first number of detectable repetitive units (ni) and the average number of repeating units (nmoyen) present on a glycoconjugate are measured. These parameters can be used to demonstrate batch-to-batch uniformity or polysaccharide stability.
In another embodiment, high mass MS and size exclusion HPLC can be used to measure the size of the complete glycoconjugates.
In another embodiment, an anthronic acid-sulfuric acid assay can be used to measure polysaccharide yields (Leyva A, Quintana A, Sanchez M, Rodriguez EN, Cremata J, Sanchez JC.) Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation, Biologicals Mar. 2008; 36 (2): 134-41.
Epub 26 November 2007). (a) Change in use of glycosylation sites ·
To show that the use of sites in a specific protein is changed, the use of glycosylation sites can be quantified. Methods for doing this are presented below. LC-MS / MS on glycopeptide: the glycoconjugates are digested with one or more proteases, and the peptides are separated by a suitable chromatographic process (C18, HPLC HILIC with hydrophilic interaction, GlycoSepN columns, SE HPLC, AE HPLC), and the different peptides are identified by MS / MS. This method can be used with or without prior foreshortening of carbohydrate chains by chemical (smith degradation) or enzymatic methods. Quantification of the peaks of the glycopeptides by UV detection between 215 nm and 280 nm allows the relative determination of the use of the glycosylation sites. Size exclusion HPLC: Higher utilization of glycosylation sites is reflected by a faster elution time from a HPLC SE column. See also (a). (b) Homogeneity. The homogeneity of glycoconjugates (the homogeneity of bound carbohydrate residues) can be assessed by methods that measure glycan length and hydrodynamic radius. Benefits (Section 6.9).
The recombinant N-OSTs provided herein (see, for example, Section 5.1) and the methods provided herein (see, e.g., Section 6.7) for using the recombinant N-OST provided herein (see , for example, Section 6.1) are particularly important and commercially attractive because they allow rapid fermentation, with high yields, on a large scale and at a low cost of preparing highly homogenous bioconjugates (for example, a glycoconjugate preparation). or vaccine preparations based on conjugates). The recombinant N-OSTs provided herein enable economically viable production of bioconjugates of commercial and therapeutic value, such as conjugate vaccines.
The enzymatic processes using recombinant N-OSTs provided herein are expected to provide more homogeneous and reproducible bioconjugate preparations than commonly used chemical synthesis methods. It is expected that the reproducibility and robustness of bioconjugate biotechnology production processes using recombinant N-OSTs proposed herein will reduce production costs. In general, it is believed that homogeneity particularly of conjugated biotherapeutic vaccines affects the clinical safety of drug products.
Analytical procedures for testing benefits (Section 6.10)
Yield. Yield is measured by the amount of carbohydrate derived from one liter of a bacterial production culture made in a bioreactor under controlled and optimized conditions. After the purification of the glycoconjugate, the carbohydrate yield can be measured directly by the anthrone or ELISA assay using carbohydrate specific antisera. Indirect measurements are possible using the amount of protein (measured by the well known BCA, Lowry or bardford assays) and the length of the glycans and the structure to calculate a theoretical amount of carbohydrate per gram of protein. In addition, the yield can also be measured by drying the glycoprotein preparation from a volatile buffer and using a scale to measure the weight.
Homogeneity. Homogeneity means the variability of the length of the glycans and possibly the number of glycosylation sites. The methods mentioned above can be used for this purpose. SE-HPLC is used to measure the hydrodynamic radius. The higher the number of glycosylation sites in the support, the greater the variation of the hydrodynamic radius with respect to a support containing fewer glycosylation sites. However, when individual chains of glycan are analyzed, they can be more homogeneous because of the more regulated length. The length of the glycans is measured by hydrazinolysis, SDS PAGE and CGE. In addition, homogeneity may also mean that certain patterns of utilization of glycosylation sites change to a wider / narrower range. These factors can be measured by LC-MS / MS glycopeptides.
Examples (Sections 7) ·
Example 1: Conception and construction of mutants of
PGLB (Section 7.1)
This example shows that the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) is a substrate for the modified C. jejuni PglB oligosaccharyl transferases proposed herein.
C-terminal HA-tagged wild type (wt) PglB was amplified from C. jejuni genomic DNA and cloned into the Eco RI and Bam HI restriction sites of pEXT21 under the control of the pTacI promoter, thereby make its expression inducible by IPTG, and the plasmid pGVXN114 was obtained.
Based on the amino acid sequence of PglBwt, the use of codons has been optimized for expression in E. coli. Unprotected codon-optimized pglB (cuo) was then cloned into the Eco RI and Bam HI restriction sites of pEXT21 to obtain pGVXN970. A series of PglB mutants were produced containing one, two, three, four or five amino acid exchanges relative to the PglBcuo sequence present in pGVXN970.
The amino acid sequence of PglB in pGVXN970 was modified by site-directed mutagenesis, so that the asparagine residue (N) at position 534, which provides a potential N-glycosylation site, is changed to a glutamine residue (Q). ), which made it possible to obtain the plasmid pGVXN971 (N534Q).
In addition, the following amino acid exchanges were introduced into the PglB sequence by site-directed mutagenesis: • N311V-N534Q-A669V, to obtain plasmid pGVXN1219; • N311V-K482R-D483H-A669V, to obtain the plasmid pGVXN1221.
Using pGVXN1219, the following amino acid exchanges were introduced by site-directed mutagenesis: • N311V-K482R-D483H-N534Q-A669V, to obtain pGVXN1220. PGVXN971 was used to introduce the following mutations by site-directed mutagenesis: • K482R- D483H-N534Q, to obtain the plasmid pGVXN1222; • K482R-N534Q, to obtain the plasmid pGVXN1223; • D483H-N534Q, to obtain the plasmid pGVXN1224; • N311V-N534Q, to obtain the plasmid pGVXN1225; • N534Q-A669V, to obtain the plasmid pGVXN1226.
Example 2: targeting of PglB for enhanced production of the CP5-Hla bioconjugate using different PglB mutants (Section 7.2)
This example shows that the capsular polysaccharide CP5 of Staphylococcus aureus (CPS 5) is a substrate for examples of recombinant modified N-OST PglBcj provided herein.
Figure 1 presents the results of a screening for improved production of the PglB catalyzed CP5-Hla bioconjugate using examples of recombinant modified N-OST PglBcj.
E. StGVXN1717 (W3110 AwecA-wzzE, ArlmB-wecG :: Clm, AwaaL, AwbbL :: IS5) coli containing pGVXN393 (CPS5) were co-transformed by electroporation with plasmids pGVXN570 (Hlanisg) and one of the indicated PglB plasmids (pGVXNH4). pGVXN97 0, pGVXN971, pGVXN1219, pGVXN1221, pGVXN1223, pGVXN1224, pGVXN1225 or pGVXN1222, respectively) and the transformed bacteria were cultured overnight at 37 ° C on selective agar plates containing the three antibiotics tetracycline [20 μg / ml]. ml], ampicillin [100 μg / ml] and spectinomycin [40 μg / ml]. 50 ml of liquid pre-cultures in 100 ml Erlenmeyer flasks containing the antibiotics tetracycline [20 μg / ml], ampicillin [100 μg / ml] and spectinomycin [40 μg / ml] were inoculated the next day with the colonies that had grown on the selection agar plates and were incubated overnight at 37 ° C and 170 rpm. The precultures were then diluted with a liquid medium to a DC> 600nm of 0.1 and the expression of HlaHise and PglB was induced at a DC> 6oonm between 0.71 and 0.84 by the addition of 0.1% arabinose and 1mM isopropyl β-D1-thiogalactopyranoside (IPTG), respectively.
After incubating overnight at 37 ° C and 150 rpm, the cells were collected by centrifugation and 50 equivalents of OD were washed with 2 ml of 0.9% NaCl. To extract the soluble periplasmic content, the washed cell pellet was resuspended in 1 ml of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA pH 8.0, 20% sucrose, containing lysozyme [1 mg / ml final]), and incubated for 30 minutes on a rotating wheel at 4 ° C. The suspension was clarified by centrifugation at 14,000 rpm at 4 ° C for 20 minutes and the supernatant was stored as a periplasmic extract (PPE).
The labeled periplasmic proteins (His6) were enriched by immobilized metal ion affinity chromatography (IMAC) using the resin
IMAC Hypercel from PALL. In detail, 1 ml of the clarified PPE supernatant was supplemented with 0.25 ml of 5x binding buffer (150 mM Tris-HCl pH 8.0, 2.5 M NaCl, 50 mM imidazole pH 8.0 4 mM MgCl 2) and
incubated with 100 μl of IMAC resin which had been previously equilibrated with 1 × binding buffer (30 mM Tris-HCl pH 80, 500 mM NaCl, 10 mM imidazole pH 8.0) for 30 minutes at room temperature on a rotating wheel. The resin was pelletized by centrifugation and unbound proteins removed. The resin was transferred to a purification column (Costar, Spin-X centrifuge tube filter) and washed twice with 200 μl of wash buffer (1x PBS pH 7.0, 10 mM imidazole) and eluted with 100 μl of elution buffer (1x PBS pH 7.0, 500 mM imidazole). 45 μl of the eluted fraction were mixed with 15 μl of Laemmli buffer [4x] and boiled for 5 minutes at 98 ° C. before being loaded on an SDS-PAGE (4-12% of NuPAGE gel). 25 μΐ were used per lane on SDS-PAGE (4-12% NuPAGE gel, MOPS migration buffer, 80 minutes at 170 V). The proteins were then electroblotted onto a nitrocellulose membrane using the devices and methods. Thermo Fisher Scientific Inc. Inc. iBlot System Dry-Blotting System
His electroblot-tagged proteins were detected on the nitrocellulose membrane with an anti-His antibody (Qiagen No. 34660) and a secondary peroxidase-conjugated anti-mouse antibody (Sigma). A0412).
Anti-His Western Blot analysis was performed on proteins extracted from E. coli
StGVXN1717 (wbbL :: IS5, waaL ", ΔECA (wecA-wzzE), (rlmB-wecG) :: Clm) co-transformed with the plasmid pGVXN393 which directs the recombinant expression of CPS 5, with a plasmid for the recombinant expression of the Hlatus6 support protein containing a glycosylation site and a C-terminal hexahistidine (His6) tag (pGVXN570) and one of the variant plasmids conferring expression of the indicated pglB mutants.
The indicated plasmids (see Figure 1) were tested in the pGLB screen described and the formation of the corresponding CP5-Hla glycoconjugate is shown in lanes 2 to 10 of Figure 1.
After induction, the cells were collected and the proteins were extracted from the periplasm and enriched by immobilized metal affinity chromatography (IMAC) and eluted in PBS buffer containing imidazole. The purified samples were subjected to SDS-PAGE (4-12% gel) and electroblotting on a nitrocellulose membrane developed by anti-His.
Figure 1 shows the results for the following samples: • Corridor 1: scale of precolored proteins serving as a page rule;
Lane 2: Protein sample from St1717 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN970 (pg1bcu)] induced; • Corridor 3: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN971 (pgBcuO N534Q)] induced; • Corridor 4: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN1219 (pgbcu N311V-N534Q-A669V)] induced; • Corridor 5: Protein sample from
Stp177 [GVXN3 93 (cap5HIJK), pGVXN570 (Hla), pGVXN1221 (pgbcuo N311V-K482R-D483H-A669V)] induced; • Corridor 6: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN1223 (pgBcuo K482R-N534Q)] induced; • Corridor 7: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN1224 (pgBcuO D483H-N534Q)] induced; • Corridor 8: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN1225 (pgbcu N311V-N534Q)] induced; • Corridor 9: Protein sample from
Stp177 [GVXN3 93 (cap5HIJK), pGVXN570 (H1a), pGVXN1222 (pg160N534Q-A669V)] induced; • Corridor 10: Protein sample from
Stp177 [GVXN393 (cap5HIJK), pGVXN570 (Hla), pGVXN114 (pGLB-) -THj.
Example 3: Targeting PglB for enhanced production of the CP8-ClfA bioconjugate using different PglB mutants (Section 7.3)
This example shows that the capsular polysaccharide CP8 of Staphylococcus aureus is a substrate for examples of recombinant modified N-OST PglBcj provided herein.
Figure 2 provides examples of screening results for production of the CP8-Clfa bioconjugate using examples of recombinant modified N-OST PglBcj.
Anti-His Western Blot analysis was performed on proteins extracted from E. coli
StGVXN1795 (wbbL :: IS5, waaL-, ΔECA, (rlmB-wecG)) cotransformed with the plasmid pGVXNN564 which directs the recombinant expression of CP 8, with a plasmid for the recombinant expression of the ClfAHis6 support protein containing a glycosylation site and a C-terminal hexahistidine (His6) tag (pGVXNN1188) and one of the variant plasmids conferring expression of the indicated pGLB mutants.
The indicated plasmids (see Figure 2) were tested in the pGLB screen described and the corresponding CP8-ClfA glycoconjugate.
Figure 2 shows the results for the following samples: • Corridor 1: Protein Scale Protein as a Page Rule • Corridor 2: Protein Sample from
Stl795 [GVXN564, pGVXNH88 (ClfA), pGVXN970 (pGLB codon optimized coding (cuo)] induced • Lane 3: protein sample from stl7 95 [GVXN564, pGVXNH88 (ClfA), pGVXN971 (pgCBcuo K482R)] induced; • Lane 4: protein sample from stl795 [GVXN564, pGVXNH88 (ClfA), pGVXN1214 (pglBcuoD483H)] induced • Lane 5: protein sample from
Stp17 95 [GVXN5 64, pGVXN1188 (ClfA), pGVXN1215 (pglBcuoK482R-D483H)] induced; • Corridor 6: Protein sample from
Stp197 [GVXN564, pGVXN1188 (ClfA), pGVXN1217 (pg1cuNoN311V)] induced; • Lane 7: Protein sample from stl7 95 [GVXN564, pGVXN1188 (ClfA), pGVXN1218 (pglBcuo N311V-A669V)] induced; • Lane 8: Protein sample from stl7 95 [GVXN564, pGVXN1188 (ClfA), pGVXN1219 (pglBcuo N311V-K482R-D483H-N534Q-A669V)] induced; • Lane 9: Protein sample from stl795 [GVXN564, pGVXN1188 (ClfA), pGVXN1221 (pgBcuo N311V-K482R-D induced.
Example 4: Example of PglB targeting for improved production of the CP1-EPA bioconjugate using different PglB mutants (Section 7.4)
This example shows that the CPI capsular polysaccharide of Streptococcus pneumoniae (CPS 1) is a substrate for examples of recombinant modified N-OST PglBcj provided herein.
Figure 3 provides examples of screening results for enhanced production of the PglB catalyzed CP1-EPA bioconjugate using examples of recombinant modified N-OST PglBcj.
E. StGVXN3600 (W3110 ΔιvecA-wzzE to waaL Arfb016 :: rf bPs017AwbgW) coli containing pGVXN767 (CPS1) and pGVXN930 (RNAtrare) were cotransformed by electroporation with plasmids pGVXN1077 (EPA hIs6) and one of the indicated pglB plasmids (pGVXN114, pGVXN970, pGVXN971, pGVXN1219, pGVXN1220, pGVXN1221, pGVXN1222, pGVXN1223, pGVXN1224, pGVXN1225, or pGVXN1226, respectively) or pGVXN72 (control plasmid pEXT21 "empty") and the transformed bacteria were grown overnight at 37 ° C on plates selection agar containing the four antibiotics tetracycline [20 μg / ml], chloramphenicol [30 μg / ml], kanamycin [50 μg / ml] and spectinomycin [40 μg / ml]. 50 ml of liquid pre-cultures in 100 ml Erlenmeyer flasks containing antibiotics tetracycline [20 μg / ml], chloramphenicol [30 μg / ml], kanamycin [50 μg / ml] and spectinomycin [40 μg / ml] were inoculated the next day with the colonies that had grown on the selection agar plates and were incubated overnight at 37 ° C and 150 rpm. The pre-cultures were then diluted with a liquid medium to a DC> 6oonm of 0.1 and the expression of EPAhiss and the variant PglB was induced at a DOeoonm between 0.48 and 0.52 by the addition of 0.1% arabinose and 1 mM isopropyl β-D1-thiogalactopyranoside (IPTG), respectively.
After incubation overnight at 37 ° C and 150 rpm, 50 equivalents of OD were collected by centrifugation (4000 rpm, 15 min, 4 ° C) and washed with 4 ml of 0% NaCl. , 9%. To extract the soluble periplasmic content, the washed cell pellet was resuspended in 1 ml of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA pH 8.0, 20% sucrose, containing lysozyme [1 mg / ml final]), and incubated for 30 minutes on a rotating wheel at 4 ° C. The suspension was clarified by centrifugation at 14,000 rpm at 4 ° C for 15 minutes and the supernatant was stored as a periplasmic extract (PPE).
The labeled periplasmic proteins (His6) were enriched by immobilized metal ion affinity chromatography (IMAC) using IMAC Hypercel resin from PALL. In detail, 1 ml of the clarified PPE supernatant was supplemented with 0.25 ml of 5x binding buffer (150 mM Tris-HCl pH 8.0, 2.5 M NaCl, 50 mM imidazole pH 8.0 4 mM MgCl 2) and incubated with 100 μl of IMAC resin which had previously been equilibrated with 1 × binding buffer (30 mM Tris-HCl pH 80, 500 mM NaCl, 10 mM imidazole pH 8.0). for 30 minutes at room temperature on a rotating wheel. The resin was pelletized by centrifugation and unbound proteins removed. The resin was transferred to a spin column (Costar, Spin-X centrifuge tube filter) and washed twice with 200 μl of wash buffer (30 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole) and eluted with 100 μl of elution buffer (30 mM Tris-HCl pH 8.0, 50 mM NaCl, 500 mM imidazole).
The protein concentration in the eluted sample was determined by Nanodrop analysis.
The fraction eluted (100 μl) was mixed with 33.3 μl of Laemmli buffer [4x] and boiled for 5 minutes at 98 ° C. before being loaded onto an SDS-PAGE (4-12% of NuPAGE gel). ). 5 μg were used per lane on SDS-PAGE (4-12% NuPAGE Midi gel, MOPS migration buffer, 75 minutes at 200 V at 4 ° C). The proteins were then electroblotted onto a nitrocellulose membrane using Thermo Fisher Scientific Inc.'s iBlot system dry drying and dry-blotting devices.
Electroblot-tagged His-tagged proteins were detected on the nitrocellulose membrane with an anti-His antibody (Qiagen No. 34660) and a peroxidase-conjugated secondary anti-mouse antibody (Sigma, AO412).
Anti-His Western Blot analysis was performed on proteins extracted from E. coli StGVXN3600 (W3110 ΔwecA-wzzE ΔwaaL ArfbOl 6:: rfjbPs017AwbgW) co-transformed with plasmids pGVXN930 (providing rare codon tRNAs), pGVXN767 which directs the recombinant expression of CPS 1, with a plasmid for the recombinant expression of the EPAhis6 support protein containing two glycosylation sites and a C-terminal hexahistidine (His6) tag (pGVXN1077) and one of the indicated variant plasmids conferring the expression of the indicated pglB mutants or the empty plasmid backbone pEXT21 (pGVXN72) as witness.
The corresponding plasmids were tested in the pGLB screen described and the formation of the corresponding CP1-EPA glycoconjugate is shown in lanes 3 to 13 of Figure 3.
After induction, the cells were collected and the proteins were extracted from the periplasm and enriched by immobilized metal affinity chromatography (IMAC) and eluted with Tris buffer containing imidazole. The purified samples were subjected to SDS-PAGE (4-12% NuPAGE) and electroblotting on a nitrocellulose membrane developed by anti-His.
After induction, the cells were collected and the proteins were extracted from the periplasm and enriched by immobilized metal affinity chromatography (IMAC) and eluted with Tris buffer containing imidazole. The purified samples were subjected to SDS-PAGE (4-12% NuPAGE) and electroblotting on a nitrocellulose membrane developed by anti-His.
Figure 3 illustrates the results for the following samples: • Corridor 1: scale of precolored proteins serving as a page rule; • Corridor 2: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAh16), pGVXN767 (S.p.CPS1), pGVXN72 (pEXt21)] induced; • Corridor 3: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAmse), pGVXN767 (S.p.CPS1), pGVXN970 (pglBCUo)] induced; • Corridor 4: Protein sample from
St3600 [GVXN930, pGVXNl077 (ΕΡΑηιξ <ϊ), pGVXN767 (S.p.CPS1), pGVXN971 (pglBCUo N534Q)] induced; • Corridor 5: Protein sample from
St3600 [GVXN930, pGVXNl077 (EPAhiss), pGVXN767 (S.p.CPS1), pGVXN1219 (pglBC10 N311V-N534Q-A669V)] induced; • Corridor 6: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAmse), pGVXN767 (S.p.CPS1), pGVXN1220 (pglBCUoN311V-K482R-D483H-N534Q-A669V)] induced; • Corridor 7: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAhiss), pGVXN767 (S.p. CPS1), pGVXN1221 (pglBC10 N311V-K482R-D483H-A669V)] induced; • Corridor 8: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAniss), pGVXN767 (S.p.CPS1), pGVXN1222 (pglBCUo K482R-D483H-N534Q)] induced; • Corridor 9: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAhisI), pGVXN767 (S.p.CPS1), pGVXN1222 (pglBCUo K482R-N534Q)] induced; • Corridor 10: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAHise) pGVXN767 (S.p.CPS1), pGVXN1224 (pglBCUo D483H-N534Q)] induced; • Corridor 11: Protein sample from
St3600 [GVXN930, pGVXN1077 (EPAnise), pGVXN767 (S.p.CPS1), pGVXN1225 (pglBCUo N311V-N534Q)] induced; • Lane 12: Protein sample from St3600 [GVXN930, pGVXN1077 (EPA Mise), pGVXN767 (S.p.C PSI), pGVXN122 6 (pglBCUo N534Q-A669V)] induced; • Lane 13: Protein sample from St3600 [GVXN930, pGVXN1077 (EPAHiSe), pGVXN767 (S.p. CPS1), pGVXN114 (pglB "t-HA) induced • Lane 14: scale of precolored proteins serving as a page rule.
Example 5: Targeting PglB for Improved Production of the 017-EPA Bioconjugate Using Different PglB Mutants (Section 7.5)
This example shows that P. shigelloides 017 antigen 0 (017) is a substrate for examples of recombinant modified N-OST PglBcj provided herein.
Figure 4 provides examples of screening results for improved production of PglB-catalyzed P38B-based PUCB-based P17-based biosugonate shiga-GNA (Shigella sonnei) -PNA using examples of recombinant modified N-OST PglBcj provided herein.
An analysis by Western Blot anti-S. sonnei was performed on proteins extracted from E. coli StGVXN2174 (W3110 ΔwecA-wyze AwaaL ArfbO16 :: rfbPs017) co-transformed with plasmids pGVXN930 (providing rare codon tRNAs), with a plasmid for the recombinant expression of the EPAHis6 support protein containing two glycosylation sites and one C-terminal hexahistidine (His6) label (pGVXN150) and one of the indicated variant plasmids conferring the expression of the indicated pglB mutants or the empty plasmid backbone pEXT21 (pGVXN72) as a control.
The corresponding plasmids were tested in the pGLB screen described and the formation of the corresponding 017-EPA glycoconjugate is shown in lanes 3-7 of Figure 4.
After induction, the cells were collected and the proteins were extracted from the periplasm and enriched by immobilized metal affinity chromatography (IMAC) and eluted with Tris buffer containing imidazole. The purified samples were subjected to SDS-PAGE (4-12% NuPAGE) and electroblotting on a nitrocellulose membrane developed by anti-S. sonnei.
Figure 4 shows the results for the following samples: • Corridor 1: scale of precolored proteins used as a page rule • Corridor 2: protein sample from
St2174 [GVXN930, pGVXN150 (EPAaseff), pGVXN72 (pEXT21)] induced; • Corridor 3: Protein sample from
St2174 [GVXN930, pGVXN150 (EPAmse), pGVXN970 (pgIbcu)] induced; • Corridor 4: Protein sample from
St2174 [GVXN930, pGVXN150 (EPAmse), pGVXN1220 (pgBcu N311V-K482R-D483H-N534Q-A669V)] induced; • Corridor 5: Protein sample from
St2174 [GVXN930, pGVXN150 (mse EPA), pGVXN114 (pGLB "t-HA).
The scope of this disclosure should not be limited by the specific embodiments described in this document. Indeed, various modifications of the subject of the invention proposed herein, in addition to those described, will become apparent to those skilled in the art from reading the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications, patents and various patent applications are cited herein, their disclosure being incorporated herein by reference in their entirety. The invention is further described in the following paragraphs: 1. A recombinant modified N-oligosaccharyl transferase (N-OST), wherein the recombinant modified N-OST is Campylobacter jejuni PglB (PglBcj) comprising an acid substitution amino acid positions N311, K482, D483 and A669. 2. The recombinant modified N-OST of paragraph 1, wherein the recombinant modified N-OST comprises a substitution of the amino acid N311 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I). 3. The recombinant modified N-OST of paragraph 1 or 2, wherein the recombinant modified N-OST comprises an N311V substitution. 4. The recombinant modified N-OST according to any of paragraphs 1 to 3, wherein the recombinant modified N-OST comprises a substitution of the amino acid K482 with a basic amino acid selected from lysine (K) or arginine (R). 5. The recombinant modified N-OST according to any one of paragraphs 1 to 4, wherein the recombinant modified N-OST comprises a K482R substitution. 6. The recombinant modified N-OST according to any one of paragraphs 1 to 5, wherein the recombinant modified N-OST comprises a D483H substitution. 7. The recombinant modified PglBc according to any one of paragraphs 1 to 6, wherein the recombinant modified N-OST comprises a substitution of the amino acid A669 by an aliphatic amino acid selected from the group consisting of glycine (G) , valine (V), leucine (L) or isoleucine (I). 8. The recombinant modified N-OST according to any one of paragraphs 1 to 7, wherein the recombinant modified N-OST comprises a substitution • A66 9V.
9. The recombinant modified N-OST according to any one of paragraphs 1 to 8, wherein the recombinant modified N-OST comprises an N311V substitution, a K482R substitution, a D483H substitution and an A669V substitution. 10. The recombinant modified N-OST according to any one of paragraphs 1 to 9, wherein the recombinant modified N-OST comprises an additional substitution at the amino acid position N534. 11. The recombinant modified N-OST of paragraph 10, wherein the recombinant modified N-OST comprises an N534Q substitution. 12. The recombinant modified N-OST of paragraph 11, wherein the recombinant modified N-OST comprises an N311V substitution, a K482R substitution, a D483H substitution and an A669V substitution. 13. A recombinant modified N-OST, wherein the recombinant modified N-OST is the PglB of
Campylobacter lari (PglBci) comprising three amino acid substitutions at amino acid positions N314, K488 and D489, and another amino acid substitution in a region comprising amino acid positions P667 to 1672 'of PglBci (PYAQFI ). 14. The recombinant modified N-OST of paragraph 6, wherein the recombinant modified N-OST comprises an amino acid substitution at amino acid positions N314, K488, D489 and K668. 15. The recombinant modified N-OST of paragraph 13 or 14, wherein the recombinant modified N-OST comprises a substitution of the amino acid N314 with an aliphatic amino acid selected from the group consisting of glycine (G), alanine, valine (V), leucine (L) or isoleucine (I). 16. The recombinant modified N-OST of paragraph 6, wherein the recombinant modified N-OST comprises an N314V substitution. 17. The recombinant modified N-OST according to any one of paragraphs 13 to 16, wherein the recombinant modified N-OST comprises a substitution of the amino acid K488 by a basic amino acid selected from lysine (K) or arginine (R). 18. The recombinant modified N-OST according to any one of paragraphs 3 to 17, wherein the recombinant modified N-OST comprises a K488R substitution. 19. The recombinant modified N-OST according to any one of paragraphs 3 to 18, wherein the recombinant modified N-OST comprises a D483H substitution. 20. The recombinant modified N-OST according to any one of paragraphs 13 to 19, wherein the recombinant modified N-OST comprises a substitution of the amino acid K668 by an aliphatic amino acid selected from the group consisting of glycine ( G), valine (V), leucine (L) or isoleucine (I). 21. The recombinant modified N-OST according to any one of paragraphs 13 to 20, wherein the recombinant modified N-OST comprises a K668V substitution. 22. The recombinant modified N-OST according to any one of paragraphs 13 to 21, wherein the recombinant modified N-OST comprises an N314V substitution, a K488R substitution, a D489H substitution and a K668V substitution. 23. The recombinant modified N-OST according to any one of paragraphs 6 to 22, wherein the recombinant modified N-OST comprises an amino acid substitution at the amino acid positions N314, K488, D489, N535 and K668. 24. The recombinant modified N-OST of paragraph 23, wherein the recombinant modified N-OST comprises an N535Q substitution. 25. The recombinant modified N-OST of paragraph 24, wherein the recombinant modified N-OST comprises an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution and a K668V substitution. 26. The recombinant modified N-OST according to any one of paragraphs 1 to 25, wherein the recombinant modified N-OST can detectably bind an oligosaccharide or polysaccharide lacking an N-acetylated sugar at the end. reducing to a carrier protein to produce an N-glycosylated carrier protein. 27. The recombinant modified N-OST of paragraph 7, wherein the carrier protein is selected from the group consisting of P. aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, 1 detoxified hemolysin A of S. aureus, agglutinating factor A, agglutinating factor B, FimH of E. coli, E. coli, FimHC. coli, the thermolabile enterotoxin of E. coli. coli, the detoxified variants of E. thermolabile enterotoxin coli, cholera toxin subunit B (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli, the transient domain of E sat protein. coli, C. jejuni's AcrA and the natural glycoproteins of C. jejuni. 28. The recombinant modified N-OST of paragraph 7 or 27, wherein the oligosaccharide or polysaccharide lacking the N-acetylated sugar at the reducing end comprises an antigen. 29. The recombinant modified N-OST according to any one of paragraphs 7 to 28, wherein the antigen comprises an E. coli antigen. coli, Salmonella sp antigen, Pseudomonas sp. antigen, Klebsiella sp. antigen, Acinetobacter antigen 0, Chlamydia trachomatis antigen, Vibrio cholera antigen, Listeria sp. antigen, antigen Legionella pneumophila serotypes 1 to 15, Bordetella parapertussis antigen, Burkholderia mallei or pseudomallei antigen, Francisella tularensis antigen, Campylobacter sp. ; a Clostridium difficile antigen, a Streptococcus pyrogenes antigen, a Streptococcus agalacticae antigen, a Neisseria meningitidis antigen, a Candida albicans antigen, a Haemophilus influenza antigen, an Enterococcus faecalis antigen, a Borrelia burgdorferi antigen, a Neisseria meningitidis antigen, a Haemophilus influenza antigen, a Leishmania major antigen, or a Shigella sonnei antigen, or a Streptococcus pneumoniae antigen. 30. The recombinant modified N-OST according to any one of paragraphs 7 to 29, wherein the carrier protein and the oligosaccharide or polysaccharide originate from an organism other than C. jejuni or C. lari. The recombinant modified N-OST according to any one of the 7 to 30, wherein the carrier protein and the oligosaccharide or polysaccharide come from different organisms. 32. The recombinant modified N-OST according to any one of 7 to 31, wherein the carrier protein is from C. jejuni or C. lari. 33. The recombinant modified N-OST according to any one of 7 to 31, wherein the carrier protein is from an organism other than C. jejuni or C. lari. 34. The recombinant modified N-OST according to any one of the 7 to 33, wherein the N-glycosylated carrier protein comprises Staphylococcus aureus capsular polysaccharide CP5 (CPS5) and detoxified hemolysin A (H1A) of S. aureus. 35. The recombinant modified N-OST according to any one of paragraphs 7 to 33, wherein the N-glycosylated carrier protein comprises Staphylococcus aureus capsular polysaccharide CP8 (CPS 8) and agglutinating factor A (ClfA) of E. coli. 36. The recombinant modified N-OST according to any one of 7 to 33, wherein the N-glycosylated carrier protein comprises Streptococcus pneumoniae CPI (CPS 1) capsular polysaccharide and P. aeruginosa exotoxin A ( LFS). 37. The recombinant modified N-OST according to any one of 7 to 33, wherein the oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end has a galactose monosaccharide at its reducing end. 38. The recombinant modified N-OST according to any one of paragraphs 1 to 37, wherein the recombinant modified N-OST can produce a yield of the N-glycosylated carrier protein which is detectable at levels of more than 2 times , more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times above baseline in an assay detecting the N-glycosylated carrier protein. 39. The recombinant modified N-OST according to any one of paragraphs 1 to 37, wherein the recombinant modified N-OST can increase the production yield of the N-glycosylated carrier protein by more than 20%, by more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3% times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times time relative to the yield obtained using a wild-type N-OST or a recombinant modified N-OST presentan t less amino acid substitution than the recombinant modified N-OST according to any one of paragraphs 1 to 37. 40. The recombinant modified N-OST according to any one of paragraphs 1 to 37, wherein the N-OST is Recombinant modified OST can increase the production rate of N-glycosylated carrier protein by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times , more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 faiths s with respect to the production rate obtained using a wild type N-OST or a recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST according to any one of paragraphs 1 to 37 41. The recombinant modified N-OST according to any one of paragraphs 1 to 37, wherein the recombinant modified N-OST can give a level of glycosylation in vivo or in vitro of the support with the oligosaccharide or polysaccharide lacking the N-acetylated sugar at the reducing end of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20% at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%. 42. A recombinant modified N-OST, wherein the recombinant modified N-OST is PglBcj comprising N311V substitution, K482R substitution, D483H substitution and A699V substitution. 43. A recombinant modified N-OST, wherein the recombinant modified N-OST is PglBcj comprising N311V substitution, K482R substitution, D483H substitution, N534Q substitution and A699V substitution. 44. A recombinant modified N-OST, wherein the recombinant modified N-OST is PglBci comprising an N314V substitution, a K488R substitution, a D489H substitution and a K698V substitution. 45. A recombinant modified N-OST, wherein the recombinant modified N-OST is PglBci comprising an N314V substitution, a K488R substitution, a D489H substitution, an N535Q substitution and a K698V substitution. 46. A nucleic acid encoding a recombinant modified N-OST according to any one of paragraphs 1 to 45. 47. A host cell comprising a recombinant modified N-OST according to any one of paragraphs 1 to 45. 48. The cell host of 10, further comprising a recombinant glycosyltransferase. 49. A host cell comprising a nucleic acid of paragraph 46. 50. The host cell according to any one of the preceding paragraphs, wherein the host cell is a prokaryotic cell. 51. The host cell of paragraph 50, in which the host cell is a cell dΈ. coli. 52. A method of producing a bioconjugate comprising culturing a host cell according to any one of the preceding paragraphs in a cell culture medium. 53. The method of paragraph 14, wherein the host cell comprises a carrier protein and a recombinant modified PglB. 54. The method of paragraph 14 or 53, wherein the host cell further comprises a recombinant glycosyl transferase. 55. The process according to any one of paragraphs 14 to 54, the carrier protein is selected from the group consisting of P. aeruginosa exotoxin A (EPA), CRM 197, diphtheria toxoid, tetanus toxoid, Detoxified hemolysin A of S. aureus, agglutinating factor A, agglutinating factor B, FimH d. E. coli, FimHC. coli, the thermolabile enterotoxin of E. coli. coli, the detoxified variants of E. thermolabile enterotoxin coli, cholera toxin subunit B (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli, the transient domain of E sat protein. coli, C. jejuni's AcrA and the natural glycoproteins of C. jejuni. 56. The method of any one of paragraphs 14 to 55, wherein the bioconjugate is an N-glycosylated carrier protein. 57. The method of any of paragraphs 14 to 56, wherein the bioconjugate is a natural N-glycosyl carrier protein of C. jejuni. 58. The method of any of paragraphs 14 to 56, wherein the bioconjugate is a heterologous N-glycosylated carrier protein of C. jejuni. 59. The method of any one of 14 to 58, wherein the N-glycosylated carrier protein does not have N-acetylated sugar at the reducing end of its oligosaccharide or polysaccharide component. 60. The process of any one of 14 to 59, wherein the N-glycosylated carrier protein has a galactose at the reducing end of its oligosaccharide or polysaccharide component. 61. The method of any one of paragraphs 14 to 59, wherein the bioconjugate comprises the capsular polysaccharide CPI (CPS 1) of Streptococcus pneumoniae and the exotoxin A of P. aeruginosa (EPA). 62. The process of any one of 14 to 59, wherein the bioconjugate comprises the capsular polysaccharide CP5 (CPS 5) of Staphylococcus aureus and the detoxified hemolysin A (H1A) of S. aureus. 63. The process of any one of 14 to 59, wherein the bioconjugate comprises Staphylococcus aureus CP8 (CPS 8) capsular polysaccharide and E (ClfA) agglutinating factor A (ClfA). coli. 64. The method of any one of paragraphs 14 to 59, wherein the bioconjugate comprises 0 Plesiomonas shigelloid.es 017 (017) antigen and EPA. 65. The process according to any one of the preceding paragraphs, wherein the recombinant modified N-OST can increase the production rate of the bioconjugate. 66. The method according to any one of the preceding paragraphs, wherein the recombinant modified N-OST can produce a yield of the bioconjugate that is detectable at levels of more than 2 times, more than 3 times, more than 4 times , more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times the baseline level. an assay detecting the N-glycosylated carrier protein. 67. The process according to any one of the preceding paragraphs, wherein the recombinant modified N-OST can increase the production yield of the bioconjugate by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than twice, more than 3 times, more than 4 times, more than 5 times , more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45-fold, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold or more than 100-fold compared to the performance achieved using an N-OST of wild-type or a recombinant modified N-OST with less amino acid substitution The recombinant modified N-OST according to any one of the preceding paragraphs, wherein the recombinant modified N-OST can increase the production rate of the bioconjugate by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 2 times , more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100-fold with respect to the rate obtained using a wild-type N-OST or a A recombinant modified N-OST having less amino acid substitution than the recombinant modified N-OST according to any one of paragraphs 1 to 45. 69. The process according to any one of the preceding paragraphs, wherein at least 1% at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of the carrier protein in a host cell is N-glycosylated to form the bioconjugate. 70. The method according to any one of the preceding paragraphs, further comprising purifying the bioconjugate from the culture of the host cell.

权利要求:
Claims (9)
[1]
1. Recombinant modified N-oligosaccharyl transferase (N-OST), wherein the recombinant modified N-OST is Campylobacter jejuni PglB (PglBcj) comprising an amino acid substitution at amino acid positions N311, K482, D483 and A669 wherein the recombinant modified N-OST comprises a substitution of the amino acid A669 by an aliphatic amino acid selected from the group consisting of glycine (G), valine (V), leucine (L) or isoleucine (I). 2. The recombinant modified N-OST according to claim 1, wherein the recombinant modified N-OST comprises a substitution of the amino acid N311 with an aliphatic amino acid selected from the group consisting of glycine (G ), alanine, valine (V), leucine (L) or isoleucine (I). The recombinant modified N-OST according to claim 1 or 2, wherein the recombinant modified N-OST comprises a substitution of the amino acid K482 with arginine (R). The recombinant modified N-OST according to any one of claims 1 to 3, wherein the recombinant modified N-OST comprises a D483H substitution.
[5]
The recombinant modified PglBC1 according to any one of claims 1 to 4, wherein the recombinant modified N-OST comprises the substitution of the amino acid A669 with valine (V). 6. Recombinant modified N-OST, wherein the recombinant modified N-OST is Campylobacter lari PglB (PglBC1) comprising three amino acid substitutions at amino acid positions N314, K488 and D489, and a further substitution of amino acid to amino acid K668. A recombinant modified N-OST according to any one of claims 1 to 6, wherein the recombinant modified N-OST can detectably bind an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end to a carrier protein to produce an N-glycosylated carrier protein. A recombinant modified N-OST according to any one of claims 1 to 7, wherein the recombinant modified N-OST can detectably bind an oligosaccharide or polysaccharide lacking N-acetylated sugar at the reducing end to a carrier protein to produce an N-glycosylated carrier protein.
[9]
Nucleic acid encoding a recombinant modified N-OST according to any one of claims 1 to 8.
[10]
A host cell comprising a recombinant modified N-OST according to any one of claims 1 to 8.
[11]
The host cell of claim 10, further comprising a recombinant glycosyltransferase.
[12]
12. A host cell comprising a nucleic acid according to claim 9.
[13]
The host cell of any one of claims 10 to 12, wherein the host cell is an E. coli cell. coli.
[14]
A process for producing a bioconjugate comprising culturing the host cell of any one of claims 10 to 12 in a cell culture medium.
[15]
The method of claim 14, further comprising purifying the bioconjugate from the host cell culture.

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法律状态:
2018-10-18| FG| Patent granted|Effective date: 20161028 |

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2018-10-18| MM| Lapsed because of non-payment of the annual fee|Effective date: 20171231 |

优先权:

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申请号 | 申请日 | 专利标题

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US201462097975P| true| 2014-12-30|2014-12-30|

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US201462098071P| true| 2014-12-30|2014-12-30|

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