Polypeptide antigen conjugates with unnatural amino acids

Abstract

To provide methods and compositions for the production of immunogenic compositions containing a non-natural amino acid.SOLUTION: The non-natural amino acid can be a site for attachment of antigens, such as bacterial capsular polysaccharides, to make immunogenic conjugates. Bio-orthogonal attachment chemistry incorporated into the non-natural amino acids allows for more efficient and potent antigen presentation to the immune system, simplified purification, and more well-defined structure of these semi-synthetic immunogens.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 44,115, filed Dec. 30, 2016; U.S. Provisional Patent Application No. 62 / 530,803, filed Jul. 10, 2017; U.S. Provisional Patent Application No. 62 / 568,201, filed Oct. 4, 2017; and U.S. Provisional Patent Application No. 62 / 591,160, filed Nov. 27, 2017, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Isolated antigenic polymer - based vaccines (e.g., first - generation meningococcal, pneumococcal, and Haemophilus polysaccharide vaccines) have shown significant improvements over previous vaccine formulations centered on live attenuated or inactivated microbial vaccines.

[0003] Purified polymers are significantly easier to manufacture, have improved safety characteristics, and can generate a more productive specific immune response (e.g., they can target more conserved or antigens important for pathogenesis). Further, they provide a simplified template for vaccine production where the immune response can target a specific site or a specific microorganism simply by providing an appropriate immunogen. However, this approach has the disadvantageous fact that not every polymer generates a strong immune response. Many lipids, polysaccharides, and certain protein antigens (and most small molecules) elicit an immune response that is essentially weak, transient, and / or does not function properly in certain patient populations (including, for example, infants or the elderly). Such weak immune responses are thought to arise from antigen structures that do not primarily activate B cells or, alternatively, do not activate the T - cell - dependent pathways involved in immune memory and antibody maturation.

Summary of the Invention

[0004] This disclosure relates to methods, compositions, and techniques for producing immunogenic compositions containing non-natural amino acids. Bio-orthogonal adhesion chemistry incorporated into non-natural amino acids enables more efficient and potent antigen presentation to the immune system, simplified purification, and more clearly defined structures of these semi-synthetic immunogens.

[0005] In one embodiment, the disclosure provides a conjugate comprising a polypeptide and an antigen, wherein the polypeptide is a carrier protein comprising at least one T cell activation epitope and at least one non-native amino acid, i.e., “nnAA”, and the antigen is conjugated to the nnAA. In another embodiment, the carrier protein comprises at least one T cell activation epitope derived from a protein selected from the group consisting of Corynebacterium diphtheriae toxin, Tetanus tetanospasmin (also known as tetanus toxin), Haemophilus influenzae protein D (PD, HiD), Serogroup B meningococcus (OMPC) outer membrane protein complex, and CRM197. In another embodiment, the carrier protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs. In yet another embodiment, at least one nnAA is replaced with lysine in the native carrier protein. For example, the carrier protein comprises CRM197 in which at least two (e.g., at least three, at least four, at least five, or at least six) of the 39 lysine residues in the natural CRM197 are replaced by nnAAs. In another embodiment, at least one nnAA is replaced with phenylalanine in the natural carrier protein. In another embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine nnAAs are replaced with lysine in the natural carrier protein. In another embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine nnAAs are replaced with phenylalanine in the natural carrier protein. In another embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine nnAAs are replaced with lysine, phenylalanine, or both lysine and phenylalanine in the natural carrier protein.In another embodiment, nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. In another embodiment, the carrier protein has at least 80% sequence identity with a protein selected from the group consisting of diphtheria toxin (DT), tetanus toxin (TT), Haemophilus influenzae protein D (PD), and CRM197. In another embodiment, the carrier protein has at least 80% sequence identity with SEQ ID NO: 1. In another embodiment, at least one T cell activation epitope is derived from CRM197 according to SEQ ID NO: 1. In another embodiment, at least one nnAA is replaced with K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527 of SEQ ID NO: 1. In another embodiment, at least one nnAA is replaced with F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. In another embodiment, at least two nnAAs are replaced with K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, K527, F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. In another embodiment, at least one nnAA is replaced with K265 of SEQ ID NO: 1. In another embodiment, at least one nnAA is replaced with K386 of SEQ ID NO: 1. In another embodiment, at least one nnAA is replaced with K265 and K386 of SEQ ID NO: 1.In another embodiment, nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. In another embodiment, the antigen is conjugated to nnAA via a triazole linkage. In another embodiment, the antigen is a polysaccharide. In another embodiment, the antigen is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae (specifically type b, i.e., Hib), Streptococcus pyogenes, or Streptococcus agalactia. In yet another embodiment, the antigen is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F and any combination thereof. In yet another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).

[0006] In related embodiments, the conjugate comprises a polypeptide and an antigen, the polypeptide being a carrier protein comprising at least one T cell activation epitope and at least one, preferably at least two nnAAs, the antigen conjugating to at least one nnAA, the at least one nnAA being a 2,3-disubstituted propanoic acid having an amino substituent at position 2 and an azide-containing substituent, a 1,2,4,5-tetradinyl-containing substituent, or an ethynyl-containing substituent at position 3.

[0007] In another related embodiment, the conjugate comprises a polypeptide and an antigen, the polypeptide being a carrier protein comprising at least one T cell activation epitope and at least one, preferably at least two nnAA residues, the antigen conjugating with nnAA, and further, the nnAA residues corresponding to amino acids having the structure of formula XII. [ka] During the ceremony, Ar optionally comprises a 5-membered or 6-membered aromatic ring containing at least one heteroatom. W 5 C1~C 10 Selected from alkylene, -NH-, -O-, and -S-, Q1 is either zero or 1. W 6 It is selected from azide, 1,2,4,5-tetradinyl which is optionally substituted with a lower alkyl group, and ethynyl. As a result, the nnAA residue in the polypeptide has the structure of formula XIII, [ka] In the formula, R 3 R is an OH or an amino acid residue of the carrier protein. 4 This is an amino acid residue of H or a carrier protein.

[0008] In one embodiment, the disclosure provides a polypeptide comprising at least one nnAA that is exchanged with a naturally occurring amino acid in the natural polypeptide according to SEQ ID NO: 1, wherein at least one nnAA is exchanged with K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527 of SEQ ID NO: 1, and the nnAA comprises a linking moiety. In another embodiment, the disclosure provides a polypeptide comprising at least one nnAA that is exchanged with a naturally occurring amino acid in the natural polypeptide according to SEQ ID NO: 1, wherein at least one nnAA is exchanged with F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1, and the nnAA comprises a linking moiety. In another embodiment, the Disclosure provides a polypeptide comprising at least two nnAAs that are exchanged with naturally occurring amino acids within the natural polypeptide according to SEQ ID NO: 1, wherein at least one nnAA is exchanged with K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, K527, F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1, and the nnAA comprises a linking moiety. In another embodiment, nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. In another embodiment, K265 of SEQ ID NO: 1 is replaced. In another embodiment, K386 of SEQ ID NO: 1 is replaced. In another embodiment, K265 and K386 of SEQ ID NO: 1 are replaced.In another embodiment, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs. In another embodiment, the nnAAs are selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof.

[0009] In the relevant embodiments, at least one, and preferably at least two, nnAAs in the polypeptide are 2,3-disubstituted propanoic acids having an amino substituent at position 2 and an azide-containing substituent, a 1,2,4,5-tetradinyl-containing substituent, or an ethynyl-containing substituent.

[0010] In another related embodiment, at least one, and preferably at least two, nnAAs in the polypeptide have the structure of formula XII, [ka] During the ceremony, Ar optionally comprises a 5-membered or 6-membered aromatic ring containing at least one heteroatom. W 5 C1~C 10 Selected from alkylene, -NH-, -O-, and -S-, Q1 is either zero or 1. W 6 The compounds are selected from azides, 1,2,4,5-tetradinyl which is optionally substituted with a lower alkyl group, and ethynyl.

[0011] In one embodiment, the disclosure provides a composition comprising a polypeptide antigen conjugate, wherein the polypeptide is a carrier protein comprising at least one T cell-activating epitope and at least one nnAA, and the antigen is conjugated to the nnAA. In another embodiment, the polypeptide antigen conjugate is crosslinked through protein-antigen-protein linkages. In another embodiment, the composition comprises a plurality of carrier-protein antigen conjugates, each conjugate comprising a different antigen (e.g., capsular polysaccharides derived from different serotypes of Streptococcus pneumoniae). In another embodiment, the antigen is derived from different serotypes (e.g., for Streptococcus pneumoniae) or serogroups (e.g., for Neisseria meningitidis) of the same microorganism. In another embodiment, the antigen is a polysaccharide. In another embodiment, the antigen is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae (e.g., Hib), Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5 and / or K6). In yet another embodiment, the antigen is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F and any combination thereof. In another embodiment, the composition comprises protein-carrier-antigen conjugates as described herein, having at least 14, 20, 21, 24, or 25 different carrier protein-capsular polysaccharide conjugates, each conjugate containing a different capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. In another embodiment, the ratio of polysaccharides to carrier protein (w / w) is greater than 1. In another embodiment, the carrier protein contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs.In another embodiment, nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. In another embodiment, the carrier protein has at least 80% sequence identity with a protein selected from diphtheria toxin (DT), tetanus toxin (TT), Haemophilus protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), and CRM197. In another embodiment, the carrier protein has at least 80% sequence identity with CRM197. In another embodiment, the carrier protein has at least 80% sequence identity with SEQ ID NO: 1. In another embodiment, the carrier protein has at least 80% sequence identity with SEQ ID NO: 1, and furthermore, at least one nnAA replaces an amino acid that occurs naturally within it. In another embodiment, at least one nnAA replaces an amino acid selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527 of SEQ ID NO: 1. In another embodiment, at least one, and preferably at least two nnAAs replace an amino acid selected from the group consisting of F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. In another embodiment, at least one, and preferably at least two, nnAAs replace amino acids selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, K527, F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. In another embodiment, the antigen is conjugated to the nnAA via a linking portion.In another embodiment, the antigen is conjugated to the nnAA via a triazole linking moiety.

[0012] In related embodiments, the polypeptide antigen conjugate in the composition comprises, as a polypeptide, a carrier protein comprising at least one T cell activation epitope and at least one, and preferably at least two, nnAAs, the antigen is conjugated to the nnAA, and further, at least one nnAA is a 2,3-disubstituted propanoic acid having an amino substituent at the 2-position and an azide-containing substituent, a 1,2,4,5-tetrazinyl-containing substituent, or an ethynyl-containing substituent at the 3-position.

[0013] In another related embodiment, the polypeptide antigen conjugate in the composition comprises, as a polypeptide, a carrier protein comprising at least one T cell activation epitope and at least one, and preferably at least two, nnAAs, the antigen is conjugated to the nnAA, and further, at least one nnAA in the polypeptide has the structure of formula XII,

Chemical formula

[0014] In one embodiment, the present disclosure provides a method for generating a conjugate, comprising: (a) providing an activated antigen comprising a plurality of functional groups including a first chemical handle that can be conjugated to a second chemical handle of nnAA; (b) combining an activated antigen with a polypeptide comprising at least one of nnAA, under conditions that the first and second chemical handles react to form an antigen-polypeptide conjugate, wherein the polypeptide comprises at least one T cell-activating epitope; and (c) recovering a composition comprising the conjugate. In another embodiment, the antigen is a polysaccharide. In another embodiment, the antigen is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae (e.g., Hib), Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the antigen is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6). In another embodiment, the antigen is reacted with a first reagent selected from the group consisting of CDAP, CDI, or periodate in the production of an activated antigen. In another embodiment, the first reagent is a periodate less than 1 M. In another embodiment, the multiple functional groups include hydroxyl groups. In another embodiment, the functional groups include aldehyde groups. In another embodiment, the antigen is reacted with a second reagent containing a functional group selected from the group consisting of propargyl, DIFO, DBCO, and DBCO(PEG)n-NH2. In another embodiment, the antigen is reacted with a second reagent containing DBCO-NH2. In another embodiment, the first chemical handle includes an alkyne group. In another embodiment, the second chemical handle includes an azide group. In another embodiment, the ratio of antigen to polypeptide in the conjugate in the composition (w / w) is greater than 1.

[0015] In related embodiments, a method for generating a conjugate includes (a) activating an antigen to incorporate at least one first chemical handle internally, wherein the first chemical handle can be conjugated to a second chemical handle of an nnAA in the polypeptide; (b) combining an activated antigen with a polypeptide containing at least one of the nnAAs under conditions that the first and second chemical handles react to form an antigen-polypeptide conjugate, wherein the polypeptide contains at least one T cell-activating epitope; and (c) recovering a composition containing the conjugate. In one aspect of this embodiment, antigen activation includes incorporating at least one alkynyl group into the antigen as a first chemical handle.

[0016] In another related embodiment, a method is provided for generating a polypeptide antigen conjugate, comprising activating the antigen by reacting the antigen with a polypeptide comprising at least one nnAA, preferably at least two nnAAs, which incorporates at least one alkynyl group as a first chemical handle and has an azide group as a second chemical handle, thereby enabling a non-catalytic covalent bioconjugation reaction between the polypeptide and the antigen. In a preferred embodiment, the alkynyl group is constrained to increase reactivity, for example, in a ring structure such as diaryl-strained cyclooctin.

[0017] In one embodiment, the present disclosure provides a method for inducing an immunoprotective antibody response to an antigen in a subject, comprising administering a conjugate, as described herein, to the subject with an excipient suitable for parenteral administration.

[0018] In one embodiment, the present disclosure provides a method for inducing an immunoprotective antibody response to an antigen in a subject, comprising administering to the subject a composition such as those described herein, with an excipient suitable for parenteral administration.

[0019] In one embodiment, the disclosure provides a method for synthesizing a polypeptide comprising at least two non-natural amino acids (nnAAs) in a cell-free expression mixture maintained at a temperature of about 10°C to about 30°C, wherein the resulting polypeptide comprises both a soluble fraction and an insoluble fraction, with a ratio of at least 30% (w / w) of soluble to insoluble fractions. For example, for 100 g of total polypeptide, the insoluble fraction would be 70 g or less and the soluble fraction would be 30 g or more. In another embodiment, the temperature is above about 20°C. In another embodiment, the temperature is below about 20°C. In another embodiment, the temperature is between about 14°C and about 18°C. In another embodiment, the polypeptide is encoded by a nucleic acid containing a repressor codon. In another embodiment, the cell-free expression mixture comprises an orthogonal tRNA / aminoacyl-tRNA synthetase pair specific to nnAAs. In another embodiment, the tRNA concentration is at least 20 μM (i.e., the concentration of orthogonal tRNA). In another embodiment, the nnAA concentration is less than about 2 mM, and the aminoacyl-tRNA synthase concentration is less than about 5 μM (i.e., the concentration of orthogonal synthase). In another embodiment, the method includes conjugating a polypeptide to the active site. In another embodiment, the active site is selected from the group consisting of haptens, bacterial antigens, viral antigens, peptide toxins, macrolides, polyethers, and any combination thereof. In another embodiment, the expression mixture includes a cell extract of Escherichia coli, wheat germ, or rabbit reticulocytes. In another embodiment, the expression mixture includes at least 30% cell extract. In another embodiment, the polypeptide includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs.In another embodiment, nnAA is selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In another embodiment, the resulting polypeptide comprises both a soluble fraction and an insoluble fraction, with the ratio of the soluble fraction to the insoluble fraction being at least 60% (w / w). In another embodiment, the resulting polypeptide comprises both a soluble and an insoluble fraction, with a ratio of at least 80% (w / w) of soluble to insoluble fractions. For example, for 100 g of total polypeptide, the insoluble fraction may be 20 g or less, and the soluble fraction may be 80 g or more.

[0020] In one embodiment, the disclosure provides an improved method for producing a protein-conjugate vaccine, wherein an antigen is conjugated to a carrier protein that provides a T cell-dependent immune response, and the improvement comprises using a polypeptide comprising at least one non-natural amino acid as the carrier protein, wherein the non-natural amino acid comprises a bioorthogonal reactive moiety through which the antigen is conjugated to the polypeptide. In another embodiment, the antigen is a bacterial polysaccharide. In another embodiment, the polypeptide comprises at least two non-natural amino acids comprising a bioorthogonal reactive moiety through which the antigen is conjugated to the polypeptide. In another embodiment, the polypeptide comprises at least one T cell-activating epitope that does not contain a non-natural amino acid and comprises a bioorthogonal reactive moiety through which the antigen is conjugated to the polypeptide. In another embodiment, the T cell-activating epitope is derived from a protein selected from the group consisting of Corynebacterium diphtheriae toxin, Tetanus tetanus tetanospasmin, Haemophilus influenzae protein D (PD, HiD), Serogroup B meningococcus (OMPC) outer membrane protein complex, and CRM197. In another embodiment, the antigen is a bacterial polysaccharide, and the bacteria are selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae (e.g., Hib), Streptococcus pyogenes, and Streptococcus agalactia. In another embodiment, at least one of the non-natural amino acids is selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0021] In one embodiment, the present disclosure provides a method for generating a carrier protein that incorporates a plurality of non-natural amino acids into its structure, comprising: (a) providing a nucleic acid encoding the carrier protein and comprising a plurality of repression codons; (b) preparing a reaction mixture by combining the nucleic acid with a cell-free bacterial extract comprising non-natural amino acids, tRNA complementary to the repression codons, and aminoacyl-tRNA synthetase; and (c) culturing the reaction mixture from (b) under conditions sufficient to selectively incorporate the non-natural amino acids at sites corresponding to each repression codon in the carrier protein. In another embodiment, the non-natural amino acid is 4-azidomethylphenylalanine (pAMF). In another embodiment, step (c) comprises culturing the reaction mixture at a temperature below 20 degrees Celsius. In another embodiment, the method further comprises purifying the carrier protein immediately after (c). In another embodiment, the repression codon is selectively substituted with codons 25, 34, 38, 40, 213, 215, 228, 245, 265, 386, 523, or 527 of SEQ ID NO: 2. In another embodiment, the reaction mixture of (b) further comprises biocomponents necessary for protein synthesis. In another embodiment, the tRNA of (b) can be charged with pAMF. In another embodiment, the aminoacyl-tRNA synthetase of (b) preferentially aminoacylates the tRNA with pAMF compared to 20 native amino acids.

[0022] In another embodiment, the Disclosure provides a composition comprising at least 14, 20, 21, 24, or 25 distinct carrier protein-antigen conjugates, wherein the antigen is a capsular polysaccharide, (a) the capsular polysaccharide in each distinct carrier protein-antigen conjugate is derived from a different serotype of Streptococcus pneumoniae, (b) the carrier protein of the carrier protein-antigen conjugate comprises a polypeptide comprising at least one T cell-activating epitope and at least two non-natural amino acids (nnAAs), and (c) the capsular polysaccharide is conjugated with the nnAAs. In a preferred form of this embodiment, at least one T cell activating epitope is derived from CRM197 by SEQ ID NO: 1, the polypeptide has at least 80% or 95% sequence identity with SEQ ID NO: 1, (i) the polypeptide comprises 2 to 9 nnAAs, (ii) the polypeptide comprises 4 to 6 nnAAs, and / or (iii) at least one nnAA is (a) K25, K34, K38, K40, K213, K215, K228, K245, K265 of CRM197 (b) is substituted with an amino acid product selected from the group consisting of K386, K523, K527, (b) F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1, or (c) K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, K527, F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1.A preferred type of the earlier embodiment comprises a composition comprising at least 14, 20, 21, 24, or 25 distinct carrier protein-antigen conjugates, each distinct carrier protein-antigen conjugate comprising serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 2 Antigens individually selected from the capsular polysaccharides of Streptococcus pneumoniae serotypes selected from the group consisting of 4F, 31, and 33F, 24 of the distinct carrier protein-antigen conjugates, are used for Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and The present invention relates to a composition comprising at least 24 distinct carrier protein-antigen conjugates derived from 33F, a composition comprising at least 25 distinct carrier protein-antigen conjugates in which at least one capsular polysaccharide of the distinct carrier protein-antigen conjugates is derived from a Streptococcus pneumoniae serotype selected from the group consisting of 6C, 7C, 13, 15A, 15C, 16, 16F, 23A, 23B, 24F, 31, 34, 35B, 33F, 35F, 37, and 38, and a composition comprising at least 25 distinct carrier protein-antigen conjugates in which at least one capsular polysaccharide of the distinct carrier protein-antigen conjugates is derived from 15A and 35B, or alternatively, from a Streptococcus pneumoniae serotype selected from the group consisting of 20A, 20B, and 24B.

[0023] In one embodiment, the present disclosure is a composition comprising at least 14, 20, 21, 24, or 25 distinct carrier protein-antigen conjugates, wherein the antigen is a capsular polysaccharide of Streptococcus pneumoniae, (a) the capsular polysaccharide in each distinct carrier protein-antigen conjugate is derived from a different serotype of Streptococcus pneumoniae, (b) the carrier protein of the carrier protein-antigen conjugate is a polypeptide comprising at least one T cell-activating epitope and at least two non-natural amino acids (nnAAs), the capsular polysaccharide conjugates with the nnAAs, and (c) Streptococcus pneumoniae serotype 1 The present invention provides a composition comprising (d) a separate carrier protein-antigen conjugate containing a capsular polysaccharide for each of serotypes 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, and (d) at least one additional separate carrier protein-antigen conjugate containing a capsular polysaccharide derived from a serotype of Streptococcus pneumoniae selected from the group consisting of serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38. For example, the composition may comprise at least 20 or 21 distinct carrier protein-antigen conjugates, each containing a conjugate for each of the serotypes of Streptococcus pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F; or (ii) at least 24 distinct carrier protein-antigen conjugates, each containing a distinct carrier protein-antigen conjugate with a capsular polysaccharide for each of the serotypes of Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0024] In one embodiment, the disclosure provides a polypeptide antigen conjugate in which the polypeptide comprises three or more nnAA residues and the conjugate has a molecular weight of at least 500 kDa. The polypeptide may be CRM197 (for example, containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, as discussed in Section 5a below) containing three or more nnAA residues (e.g., 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6 nnAA residues). The antigen may be a bacterial polysaccharide such as pneumococcal capsular polysaccharide. The conjugate may have a molecular weight of at least 600 kDa, at least 800 kDa, at least 900 kDa, or at least 1 MDa, for example, 1 to 5 MDa. As further discussed herein, multiple preparations of such conjugates, each prepared from pneumococcal capsular polysaccharides derived from different serotypes of Streptococcus pneumoniae, can be incorporated into compositions of the present invention useful as polyvalent vaccines. Preferred selections of Streptococcus pneumoniae serotypes represented by such conjugates will also be discussed further herein.

[0025] In one embodiment, the disclosure provides a polypeptide antigen conjugate in which the polypeptide comprises four or more nnAA residues. The polypeptide may be CRM197 (for example, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, as discussed in Section 5a below) containing four or more nnAA residues (e.g., 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6 nnAA residues). The antigen may be a bacterial polysaccharide such as pneumococcal capsular polysaccharide. The conjugate may have a molecular weight of at least 500 kDa (e.g., at least 600 kDa, at least 800 kDa, at least 900 kDa, or at least 1 MDa, e.g., 1 to 5 MDa). As further discussed herein, multiple preparations of such conjugates, each prepared from pneumococcal capsular polysaccharides derived from different serotypes of Streptococcus pneumoniae, can be incorporated into compositions of the present invention useful as polyvalent vaccines. Preferred selections of Streptococcus pneumoniae serotypes represented by such conjugates will also be discussed further herein.

[0026] In one embodiment, the present disclosure provides a protein suitable for preparing immunogenic polysaccharide-protein conjugates, wherein the protein (i) comprises at least one nnAA and (ii) has a solubility of at least 50 mg / L in Tris buffer at pH 7.4 at 20°C. The polypeptide comprises at least one T cell activation epitope (as considered above), which may be CRM197 (for example, containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, as considered in section 5a below) containing two or more nnAA residues, e.g., 3-9, or 4-9, or 3-8, or 4-8, or 3-7, or 4-7, or 3-6, or 4-6 nnAA residues. The protein may conjugate with bacterial polysaccharides such as Streptococcus pneumoniae capsular polysaccharide to produce conjugates. The solubility may be at least 100 mg / L, at least 200 mg / L, or even at least 250 mg / L. As will be further discussed herein, multiple preparations of such conjugates, each prepared with pneumococcal capsular polysaccharides derived from different Streptococcus pneumoniae serotypes, can be incorporated into the composition of the present invention useful as a polyvalent vaccine. Preferred selections of Streptococcus pneumoniae serotypes represented by such conjugates will also be further discussed herein. [Brief explanation of the drawing]

[0027] The features and advantages of the embodiments described herein will be further described by reference to the following detailed description and accompanying drawings illustrating exemplary embodiments.

[0028] [Figure 1] The yields of six nnAA-containing eCRMs produced at 30, 25, or 20 degrees Celsius in a CFPS reaction, with optionally selected increases in tRNA (otRNA) or nnAA / aaRS synthase (nnAA), are shown. Each column shows two bars, representing both the total yield and the soluble yield. [Figure 2]Figure 2A shows Coomassie (2A) and fluorescent (2B) gel images demonstrating the relative yield of the synthesized protein (2A) and the ability of the pAMF incorporated into the eCRM to react with DBCO-fluorescein (2B) for the single-site eCRM produced in the cell-free protein synthesis (CFPS) reaction. In Figure 2A, the ladders are shown from top to bottom as 10, 15, 20, 25, 37, 50, 75, 100, 150, and 250 kDa. In Figure 2B, the fluorescent markers are 25 and 75 kDa. The lanes are as follows: L = ladder, W = wild type, C = C-terminal TAG. Then lanes 1-12 have TAGs for exchanging Lys at positions 11, 25, 34, 38, 40, 52, 60, 77, 83, 91, 96, and 103, respectively. [Figure 3] This graph shows the opsonization phagocytic activity (OPA) (GMT) of monovalent pneumococcal polysaccharide-eCRM conjugates in mice after administration. Serotypes are shown on the X-axis. White bars represent adjuvant-modified polysaccharides, and black bars represent adjuvant-modified conjugates. [Figure 4] This graph shows the IgG response (GMT) in mice after administration of monovalent pneumococcal polysaccharide-eCRM conjugates. Serotypes are shown on the X-axis. White bars represent adjuvanted but unconjugated polysaccharides, while black bars represent adjuvanted conjugates. [Figure 5] This graph shows the IgG response (GMT) in rabbits after administration of a polyvalent pneumococcal vaccine. Each serotype (X-axis) has data for the 24-valent conjugated vaccine of the present invention (left), Prevnar-13® (middle), and the 24-valent unconjugated vaccine (right). Data are expressed with a mean + / - 95% confidence interval. [Figure 6] Similar to Figure 5, but showing the OPA response (GMT). [Modes for carrying out the invention]

[0029] In protein-conjugate vaccines, the immune response to a “weak” antigen is amplified by attachment to a known “strong” protein antigen. In these semi-synthetic biomolecules, the proteins that purify a strong, long-lived T cell-dependent immune response (“T cell-dependent antigen”) typically attach to the “weak” antigen via nonspecific oxidation / reduction chemistry. The T cell activation features for these immunogenic proteins replenish helper T cells in B cells that recognize the attached weak antigen, thus enabling a differently strong, long-lived immune response to weakly immunogenic molecules.

[0030] Current methods and components used for protein-zygote vaccine production hinder the broader application of zygote vaccines for the treatment and prevention of disease. Firstly, relatively few strong protein antigens are chemically resistant, non-toxic, and sufficiently extensible for use as carriers in zygote vaccines. Secondly, the oxidation / reduction chemistry commonly used for zygote vaccine production makes it difficult to preserve epitopes for carriers and antigens required for maximum immunogenicity. Thirdly, the relatively low efficiency of these oxidation / reduction reactions complicates quality control and purification, particularly on a commercial scale.

[0031] While the generation of recombinant proteins allows for the optimization of the antigenicity and non-toxicity of carrier proteins, existing carrier proteins are difficult to generate in recombinant cells, and fully engineered proteins are difficult to generate in high yield. Looser conjugation reactions minimize denaturation / occlusion of carrier and antigen epitopes, but the lower efficiency of these reactions leads to lower antigen loadings on carrier proteins and more complex purification schemes. Importantly, relatively low antigen levels on carriers lead to a higher likelihood of immune "interference" through antibody reactions against the carrier protein itself or the recognized phenomenon of carrier-induced epitope suppression.

[0032] Therefore, the need for strategies and reagents that enable a combination of these techniques to produce more readily manufactured conjugate vaccines with higher immunogenicity has been identified. Accordingly, this specification describes, in particular, polypeptide antigen conjugates of (1) and (2) comprising (1) a polypeptide comprising an enhanced carrier protein containing a non-natural amino acid, (2) an antigen suitable for conjugation to the polypeptide comprising an enhanced carrier protein containing a non-natural amino acid, and (3) an antigen conjugated with an enhanced carrier protein containing a non-natural amino acid, (4) a vaccine composition comprising the foregoing, and (5) a method for preparing and using the foregoing.

[0033] 1.Definition The term "repression codon" refers to a nucleotide triplet introduced into a polynucleotide at a specific location, recognized by a specific tRNA capable of recognizing a stop codon (e.g., amber, ochre, or opal stop codons), allowing translation to read the codon and produce a protein, thereby repressing the stop codon.

[0034] "Non-natural amino acids" (nnAAs) refer to amino acids that are not one of the 20 common amino acids, pyrrolidines, or selenocysteines. Other terms used synonymously with "non-natural amino acids" include "non-naturally encoded amino acids," "non-natural amino acids," "non-naturally occurring amino acids," and their various hyphenated and unhyphenated forms. Non-natural amino acids with bioorthogonal reactive chemical side chains can be used as chemical "handles" to attach various payloads to discontinuous sites in proteins.

[0035] In the context of two or more nucleic acid or polypeptide sequences, the terms “sequence identity” or “percent identity” refer to two or more sequences that, when compared and aligned for the maximum correspondence across a comparison window, are identical or have a specified percentage of identical amino acid residues or nucleotides, as measured using a sequence comparison algorithm (e.g., BLASTP for amino acid sequences). For the purposes of this document, percent identity is determined across full-length sequences, such as the reference sequence described in Sequence ID No. 1. A method for calculating sequence identity as provided herein is the BLASTP program, with a word length (W) of 3, an expected value (E) of 10, and its initial value set in the BLOSUM62 scoring matrix (see, for example, Henikoff & Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). See, for example, the BLAST alignment tool available at WWW at blast.ncbi.nlm.nih.gov / Blast.cgi or elsewhere.

[0036] The term "antigen" refers to any molecule or linear molecular fragment that can be recognized by the adaptive immune system's highly variable antigen receptors (B cell receptors, T cell receptors, or both). Non-restrictive examples of antigens include polysaccharides or glycans (e.g., bacterial capsular polysaccharides), polynucleotides, polyamino acids, lipids, and small molecules (e.g., haptens, drugs of abuse).

[0037] The term "T cell activation epitope" refers to a structural unit of molecular structure that can induce T cell immunity. The function of carrier proteins containing T cell activation epitopes is well known and documented in zygotes. While we do not wish to be bound by theory, T cell activation epitopes in carrier proteins are thought to be covalent antigens that are processed by antigen-presenting cells and induce immunological memory against the antigen in CD4 +ve This allows it to be presented to T cells.

[0038] The term "B cell epitope" generally refers to these characteristics of macromolecular structures that can induce a B cell response. In contrast to T cell epitopes, B cell epitopes do not need to contain peptides because treatment by antigen-presenting cells and loading of MHC peptide bond clefts are not required for B cell activation.

[0039] As used herein, “carrier protein” refers to a non-toxic or detoxified polypeptide containing a T-cell activating epitope that can be attached to an antigen (e.g., a polysaccharide) to enhance a humoral response to the antigen conjugated in a subject. The term includes any of the bacterial proteins used as epitope carriers in FDA-approved vaccines. In some embodiments, the carrier protein is Corynebacterium diphtheriae toxin, Tetanus tetanus tetanospasmin, Haemophilus influenzae protein D (PD, HiD), Serogroup B meningococcus (OMPC) outer membrane protein complex, CRM197, or malaria orkinate-specific surface protein Pfs25. In another embodiment, the carrier protein is BB derived from the G protein of Streptococcus strain G148. A “natural carrier protein” has amino acids that occur only naturally. An “enhanced carrier protein” has at least one non-natural amino acid that is replaced with a naturally occurring amino acid in the carrier protein.

[0040] As used herein, the term “immunogenic polypeptide” means a polypeptide comprising at least one T cell-activating epitope, where the T cell epitope is derived from a protein capable of inducing immunological memory in animals.

[0041] The terms “eCRM” or “enhanced CRM” as used interconvertibly herein refer to a modified form of the G52E codon variant of diphtheria toxin, wherein at least one native amino acid residue is substituted with a non-native amino acid and the polypeptide retains at least one T cell activation epitope.

[0042] As used herein, the terms “modified,” “exchanged,” “enhanced,” and “substituted” are considered synonymous when used to describe polypeptide residues, and in all cases refer to the exchange of a non-natural amino acid for a naturally occurring amino acid within a polypeptide chain.

[0043] As used herein, the term “T-independent antigen” refers to an antigen that does not induce features of B cell-mediated immunity or processes associated with helper T cell-mediated immunity, such as isotype switching or immunological memory.

[0044] As used herein, the term “polysaccharide” is used in its ordinary sense, including, but not limited to, polysaccharides having 50 or more repeating units, and oligosaccharides having 50 or fewer repeating units, as well as saccharides having multiple repeating units. Typically, polysaccharides have about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about 2,000 or more repeating units, and optionally, about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1,000 repeating units to about 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, or 1,900 repeating units. Oligosaccharides typically have about 6, 7, 8, 9, or 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units.

[0045] As used herein, the term “glycan” refers to any linear or branched polymer consisting of monosaccharide (e.g., glucose) residues linked to one another by glycosidic linkages. Examples of glycans include glycogen, starch, hyaluronic acid, and cellulose. Other examples of “glycans” include bacterial capsular polysaccharides.

[0046] As used herein, the term “molecular weight” of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) combined with multi-angle laser scattering (MALS).

[0047] As used herein, unless otherwise specified, the term “lower alkyl” refers to a straight-chain saturated or branched hydrocarbon having 1 to 6 carbon atoms, i.e., C1 to C6 alkyl groups. In certain embodiments, the lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted moieties. See also US2014 / 0066598. The term “lower alkylene” refers to the alkylene group of a lower alkyl group.

[0048] Compounds of various embodiments disclosed herein, or pharmaceutically acceptable salts thereof, comprising one or more chiral centers, resulting in enantiomers, diastereomers, and other isomeric stereomorphisms defined in absolute stereochemistry as (R) or (S) relative to an amino acid, or as (D) or (L). This disclosure is intended to include all such isomers, as well as their racemic and optionally pure forms. nnAAs as used herein are generally α-amino acids having a chiral center at the α-carbon, and they are preferably (L) isomers.

[0049] The chemical naming protocols and structural diagrams used herein are modified forms of IUPAC nomenclature using the ACD / Name type 9.07 software program and / or the ChemDraw Ultra type 11.0.1 software naming program (CambridgeSoft). All bonds are identified in the chemical structural diagrams herein, except for all bonds on several carbon atoms that are assumed to be bonded with enough hydrogen atoms to complete their valence, as described below.

[0050] 2. General methods Unless otherwise specified, all technical and scientific terms used herein have their commonly understood meanings. For definitions and terms, practitioners are particularly advised to consult Green & Sambrook (eds.) Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), which are incorporated herein by reference; Ausubel, FM, et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012); and Plotkin, SA, Orenstein, WA, & Offit, PA, Vaccines, 6th ed, Elsevier, London (2013). Standard methods are also described in detail in Bindereif, Schon, & Westof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which is incorporated herein by reference. Appropriate molecular techniques for generating recombinant nucleic acids, and examples of instructions for many cloning movements, are incorporated herein by reference: Green & Sambrook (Id.), Ausubel, FM, et al. (Id.), Berger & Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology (Volume 152, Academic Press, Inc., San Diego, Calif. 1987), and PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, Calif. 1990). Appropriate bioorganic techniques for activating and derivatizing biomolecules by chemical handles, and examples of instructions for designing such rigidities, are found in Hermanson, GT, Bioconjugate Techniques, 2nd This information can be found in ed., Elsevier, London (2008). For examples of techniques and components necessary for parenteral administration of the biomolecules described herein, practitioners are directed to Remington, Essentials of Pharmaceutics, Pharmaceutical Press, London (2012). Methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization are described in Coligan et al. (2000), Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Methods for cell-free synthesis are described in Spirin & Swartz (2008), Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. Methods for incorporating non-natural amino acids into proteins using cell-free synthesis are also described in Shimizu et al. (2006) FEBS Journal, 273, 4133-4140, and Chong (2014) Curr Protoc Mol Biol. 108: 16.30.1-11.

[0051] PCR amplification methods are described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif., 1990 and Domingues (ed.), PCR: Methods and Protocols, ISBN 1493970593 (2017). The amplification reaction typically involves the DNA to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, and magnesium. Typically, the desired number of thermal cycles is 1–25. Methods for designing and optimizing primers for PCR conditions can be found in Ausubel et al., Short Protocols in Molecular Biology, 5th Edition, Wiley, 2002, and Innis et al., PCR Protocols, Academic Press, 1990. Computer programs are useful in designing primers with the required specificity and optimal amplification characteristics (e.g., Oligo type 5.0 (National Biosciences)). In some embodiments, the PCR primers additionally contain a recognition site for restriction endonucleases to facilitate the insertion of the amplified DNA fragment into a specific restriction enzyme site in the vector. If the restriction site is to be added to the 5' end of the PCR primer, it is preferable to include several (e.g., two or three) extra 5' bases to allow for more efficient cleavage by the enzyme. In some embodiments, the PCR primers also contain an RNA polymerase promoter site, such as T7 or SP6, to allow for subsequent in vitro transcription. Methods for in vitro transcription can be found in sources such as Van Gelder et al., Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990 and Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992.

[0052] The molecular weight of polysaccharides or carrier protein-polysaccharide conjugates is measured by size exclusion chromatography (SEC) combined with multi-angle laser scattering (MALS). The SEC MALS-UV-RI setup consists of an Agilent HPLC 1100 (including degassing agent, quaternary pump, temperature-controlled autosampler, temperature-controlled column compartment, and UV-VIS diode array detector) according to a DAWN-HELEOS multi-angle laser scattering detector and an Optilab T-rEX differential refractometer (Wyatt Technology, Santa Barbara, California, USA) for eluting species detection. The following series of columns are fitted to this system: TSKgel Guard PWXL 6.0 mm ID × 4.0 cm length, 12 μm particles, TSKgel 6000 PWXL 7.8 mm ID × 30 cm length, 13 μm particles, and TSKgel 3000 PWXL 7.8 mm ID × 30 cm length, 7 μm particles. The column compartment is set to 25°C, and the sample compartment to 4°C. A mobile phase consisting of 1×PBS filtered through 0.2 μm with 5% v / v acetonitrile is used at a flow rate of 0.5 mL / min. Samples are injected into polysaccharides in a concentration range of 0.2–1.5 mg / mL, and the injection volume is adjusted to produce a phase injection mass of 30–40 μg. Agilent Open Lab software is used to control the HPLC, and Wyatt Astra 7 software is used for data retrieval and analysis. This technique reveals the absolute molecular weight distribution of conjugates in the sample, and results for individual populations are expressed as average values.

[0053] In some embodiments, the capsular polysaccharide from which Streptococcus pneumoniae is isolated is obtained directly from the bacteria using isolation procedures known to those skilled in the art (see, for example, the methods disclosed in U.S. Patent Publications 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498, and WO2008 / 118752). In other embodiments, the capsular polysaccharide from which Streptococcus pneumoniae is isolated is obtained from a commercial source (e.g., ATCC).

[0054] 3. polypeptide Polypeptides comprising at least one nnAA residue are disclosed herein. Preferred polypeptides include any biologically active polypeptide. In some embodiments, the polypeptide is an immunogenic polypeptide. In some embodiments, the nnAA residue is substituted for a native residue of the specific polypeptide. In other embodiments, the nnAA residue is added before, after, or inserted into the sequence of the specific polypeptide. In further embodiments, the polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAA residues. In another embodiment, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues. In yet another embodiment, the polypeptide comprises 2 to 9 nnAA residues, and preferably 4 to 6 nnAA residues. In yet another embodiment, the polypeptide comprises two or more chemically distinct nnAA residues.

[0055] In one embodiment, the disclosure provides an immunogenic polypeptide comprising nnAA residues. In another embodiment, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAA residues. In yet another embodiment, the at least 2 non-natural amino acid residues comprise at least 2 different non-natural amino acids. In another embodiment, at least two different non-natural amino acids are selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In another embodiment, the polypeptide contains a T-cell activation epitope of the carrier protein. In another embodiment, the polypeptide is the carrier protein. In another embodiment, nnAA is not a T-cell activation epitope of the carrier protein. In another embodiment, nnAA is substituted with a lysine residue. In another embodiment, the polypeptide is conjugated with the antigen. In another embodiment, the antigen is conjugated with nnAA. In another embodiment, the antigen comprises a T cell-independent antigen selected from the group consisting of haptens, bacterial capsular polysaccharides, bacterial lipopolysaccharides, or tumor-derived glycans. In another embodiment, the antigen comprises an exopolysaccharide, such as a bacterial non-capsular polysaccharide, e.g., the exopolysaccharide of Staphylococcus aureus.

[0056] In one embodiment, the disclosure provides a carrier protein comprising nnAA residues. In another embodiment, the carrier protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAA residues. In another embodiment, the non-natural amino acid is selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In another embodiment, nnAA is substituted with a lysine residue. In another embodiment, the nnAA residue is located outside the T-cell activation epitope of the carrier protein. In another embodiment, the substitution is selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K265, K386, K523, and K527 of SEQ ID NO: 1, and any combination thereof. In another embodiment, the substitution includes the combination of K25, K213, K245, K265, K386, and K523 of SEQ ID NO: 1. In another embodiment, the carrier protein includes an antigen. In another embodiment, the antigen includes a T-independent antigen selected from the group consisting of haptens, bacterial capsular polysaccharides, bacterial lipopolysaccharides, or tumor-derived glycans. In another embodiment, the antigen is a polysaccharide. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the polypeptide can induce a T cell-dependent immune response.

[0057] In one embodiment, the disclosure provides a protein comprising an antigen conjugated to an amino acid residue of a carrier protein, wherein the antigen does not conjugate to a native amino acid residue of the carrier protein. In another embodiment, the antigen does not conjugate to a lysine residue of the carrier protein. In another embodiment, the amino acid is not present in the T cell activation epitope of the carrier protein. In another embodiment, the antigen comprises a T-independent antigen selected from the group consisting of haptens, bacterial capsular polysaccharides, bacterial lipopolysaccharides, or tumor-derived glycans. In another embodiment, the antigen is a polysaccharide. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof.

[0058] Ideally, the carrier protein should have a solubility of at least 50 mg / L (e.g., at least 100 mg / L, at least 150 mg / L, at least 200 mg / L, or at least 250 mg / L) when expressed in a cell-free protein synthesis system.

[0059] If the carrier contains more than one nnAA residue, it is preferable that it contains only one type of nnAA (for example, only the nnAA in the carrier is pAMF). This makes it possible to use the same conjugation chemistry for each nnAA simultaneously. If it is desired to attach two different antigens to a single carrier molecule, this can be achieved by using different nnAA species within a single carrier and conjugating each antigen to a different nnAA, but conjugation to a single type of nnAA in the carrier is preferred. Furthermore, if the composition contains multiple different conjugates (for example, different Streptococcus pneumoniae serotypes), it is preferable that each conjugate contains the same single type of nnAA. Furthermore, if the composition contains multiple different conjugates (for example, different Streptococcus pneumoniae serotypes), it is preferable that each conjugate contains the same carrier protein.

[0060] In another embodiment, the disclosure provides a polynucleotide encoding a polypeptide described herein. In another embodiment, the disclosure provides an expression vector comprising a polynucleotide encoding a polypeptide described herein. In another embodiment, the disclosure provides a host cell comprising an expression vector.

[0061] 4. Non-natural amino acids The nnAA residues optionally include any of the non-natural amino acids described in this application or in other applications identified as compatible with cell-based or cell-free protein synthesis (see, for example, Schultz et al. Annu Rev Biochem. 2010;79:413-44 particularly pp.418-420, and Chin et al. Annu Rev Biochem. 2014;83:5.1-5.30, which are incorporated herein by reference).

[0062] Examples of unnatural amino acids that may be used in the method of this embodiment include: unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thio acid, boric acid, boronic acid, phosphoric acid, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or any combination thereof; amino acids containing photoexcitable crosslinking agents; spin-labeled amino acids; fluorescent amino acids; amino acids containing novel functional groups; and others. Examples include amino acids that interact with molecules covalently or non-covalently; metal-linked amino acids; metal-containing amino acids; radioactive amino acids; photocage and / or photoisomerized amino acids; amino acid-containing biotin or biotin-analogs; glycosylated or carbohydrate-modified amino acids; amino acid-containing ketos; amino acids containing polyethylene glycol or polyethers; heavy atom-substituted amino acids; chemically cleavable or photodegradable amino acids; amino acids with elongated side chains; amino acids containing toxic groups; sugar-substituted amino acids, such as sugar-substituted serine; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothio acid-containing amino acids; α,α-disubstituted amino acids; β-amino acids; and cyclic amino acids other than proline.

[0063] Particularly preferred nnAAs for use in the present invention are those that can be incorporated during translation (in cells or cell-free systems) and provide a functional group not found in any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into polypeptides are known; see, for example, Young & Schultz (2010) J Biol Chem 285:11039-44, Maza et al. (2015) BioconjugateChem. 26:1884-9, and Zimmerman et al. (2014) BioconjugateChem. 25:351-61.

[0064] Specifically, nnAA residues optionally contain chemical groups suitable for "click" chemical reactions with the corresponding groups of separate antigen molecules or haptens. Chemical groups suitable for "click" chemistry include azide (N3) groups, alkyne (C≡C) groups, alkene (C=C) groups, and 1,2,4,5-tetrazine ( [ka] This includes, but is not limited to, the ) group.

[0065] The conjugate comprises a polypeptide and an antigen, the polypeptide being a carrier protein comprising at least one T cell-activating epitope and at least one nnAA, preferably at least two nnAAs, and the antigen being conjugated to at least one nnAA. In some embodiments, the at least one nnAA is a 2,3-disubstituted propanoic acid having an amino substituent at position 2 and an azide-containing substituent, a 1,2,4,5-tetradinyl-containing substituent, or an ethynyl-containing substituent at position 3.

[0066] In another related embodiment, the conjugate comprises a polypeptide and an antigen, the polypeptide being a carrier protein comprising at least one T cell activation epitope and at least one nnAA residue, the antigen conjugated to nnAA, and furthermore, the nnAA residue corresponds to an amino acid having the structure of formula XII. [ka] During the ceremony, Ar optionally comprises a 5-membered or 6-membered aromatic ring containing at least one heteroatom. W 5 C1~C 10 Selected from alkylene, -NH-, -O-, and -S-, Q1 is either zero or 1. W 6 It is selected from azide, 1,2,4,5-tetradinyl which is optionally substituted with a lower alkyl group, and ethynyl. As a result, the nnAA residue in the polypeptide has the structure of formula XIII, [ka] In the formula, R 3 R is an OH or an amino acid residue of the carrier protein. 4 This is an amino acid residue of H or a carrier protein.

[0067] In one embodiment, the disclosure provides a polypeptide comprising at least one nnAA that is replaced with a naturally occurring amino acid in the natural polypeptide according to SEQ ID NO: 1, wherein at least one nnAA is replaced with K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527 of SEQ ID NO: 1, and the nnAA comprises a linking moiety. In another embodiment, nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. In another embodiment, K265 of SEQ ID NO: 1 is replaced. In another embodiment, K386 of SEQ ID NO: 1 is replaced. In another embodiment, K265 and K386 of SEQ ID NO: 1 are replaced. In another embodiment, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs. In another embodiment, the nnAAs are selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof.

[0068] In another embodiment, the nnAA in the polypeptide is a 2,3-disubstituted propanoic acid having an amino substituent at position 2 and an azide-containing substituent, a 1,2,4,5-tetradinyl-containing substituent, or an ethynyl-containing substituent at position 3. In a preferred embodiment, the substituent at position 3 is an azide-containing substituent, and in a more preferred embodiment, the azide-containing substituent includes a terminal azide group bonded to the carbon atom at position 3 via a linking group. For example, the linking group may include an arylene moiety that is optionally substituted and optionally contains heteroatoms. For example, the linking group may include a 5-membered or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 nonhydrogen ring substituents.

[0069] In a more preferred embodiment, nnAA has the structure of formula XII, [ka] During the ceremony, Ar optionally comprises a 5-membered or 6-membered aromatic ring containing at least one heteroatom. W 5 C1~C 10 Selected from alkylene, -NH-, -O-, and -S-, Q1 is either zero or 1. W 6 The compounds are selected from azides, 1,2,4,5-tetradinyl which is optionally substituted with a lower alkyl group, and ethynyl.

[0070] In this case, the corresponding nnAA residue in the polypeptide has the structure of formula XIII, [ka] In the formula, R 3 R is an OH or an amino acid residue of the carrier protein. 4 It will be understood that this is an amino acid residue of H or a carrier protein.

[0071] In some embodiments, Ar does not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene group (i.e., Ar is -C6H4-). In other embodiments, Ar contains a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described below, and Ar may be, for example, pyridine or pyridazine. In particularly preferred embodiments, Q1 is 1 and W 5 It is a lower alkylene, W 6 It is Azid.

[0072] Azide-containing amino acids In some embodiments, the nnAA residue comprises azide-containing nnAA. In certain embodiments, the nnAA residue comprises azide-containing nnAA of formula I, [ka] During the ceremony, D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-, Ar is [ka] And, Each of W1, W2, and W3 is independently a single bond or lower alkylene. Each X1 is independently -NH-, -O-, or -S-. Each Y1 is independently a single bond, -NH- or -O-. Each Y2 is independently a single bond, -NH-, -O-, or N-linked or C-linked pyrrolidine ylene. One of Z1, Z2, and Z3 is -N-, and the other of Z1, Z2, and Z3 are independently -CH-.

[0073] In other embodiments, the nnAA residue comprises an azide-containing amino acid of formula II, [ka] During the ceremony, W4 is C1~C 10 It is alkylene.

[0074] In one embodiment, the nnAA residue comprises an azide-containing amino acid selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In a further embodiment, the nnAA residue comprises 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF). pAMF provides a very favorable reaction kinetics for conjugate formation (for example, it is much faster than using pAF when reacting with alkyne-containing carbohydrate antigens in the SPAAC method).

[0075] Preparation of azide-containing amino acids according to formulas I and II can be found, for example, in Stafford et al. US2014-0066598A1, particularly in paragraphs

[0331] -

[0333] , which are incorporated by reference. This process involves substitution of the hydroxyl group of the corresponding aryl amino acid derivative with the chloride using thionyl chloride, followed by nucleophilic substitution of the chloride with the azide. Aryl side chains containing suitable amino acids are also commercially available.

[0076] 1,2,4,5-Tetradinyl-containing amino acids In some embodiments, the non-natural amino acid residue comprises 1,2,4,5-tetrazine containing nnAA. In certain embodiments, the non-natural amino acid comprises 1,2,4,5-tetrazine containing nnAA of formula III, [ka] During the ceremony, Ar is [ka] And, V is a single bond, a lower alkylene, or -W1-W2-, One of W1 and W2 is absent or a lower alkylene, and the other is -NH-, -O-, or -S-. One of Z1, Z2, and Z3 is -CH- or -N-, the other of Z1, Z2, and Z3 are each independently -CH-, and X1 is independently -NH-, -O-, or -S-. R is a lower alkyl group. Arbitrarily, Ar [ka] And if V is -NH-, then one of Z1, Z2, and Z3 is a non-natural amino acid. [ka] Given that it is not -N-, the answer is -N-.

[0077] The preparation of 1,2,4,5-tetrazine-containing amino acids by formula III is found, for example, in Yang et al. US2016-0251336A1, particularly in paragraphs

[0341] to

[0377] , which is incorporated by reference. This process involves the Negishi coupling of an amino / carboxyl-protected derivative of (R)-2-amino-3-iodopropanoic acid with an aminopyridyl bromide to introduce Ar, followed by the reaction with a methylthio-1,2,4,5-tetrazine derivative to introduce the tetrazine moiety into the amino acid.

[0078] Alkyne-containing amino acids In some embodiments, the nnAA residue includes alkyne-containing nnAAs. In one embodiment, this is a propargyl group. Various propargyl-containing amino acids, including their synthesis, are found in Beatty et al. Angew. Chem. Int. Ed. 2006, 45, 7364-7; Beatty et al. J. Am. Chem. Soc. 2005(127):14150-1; Nguyen et al. J Am Chem Soc. 2009(131):8720-1. Such propargyl-containing amino acids are suitable for incorporation into proteins as nnAAs using cell-based systems. In some embodiments, the nnAA residue includes propargyl-containing nnAAs selected from the group consisting of homopropargylglycine, ethynylphenylalanine, and N6-[(2-propynyloxy)carbonyl]-L-lysine.

[0079] 5. Modified carrier protein In one embodiment, a polypeptide comprising at least one nnAA residue is a native carrier protein (e.g., eCRM), or a modified polypeptide comprising one or more T-cell activating epitopes of a native carrier protein. Suitable carrier proteins for such modification include, but are not limited to, Corynebacterium diphtheriae toxin, Tetanus tetanus tetanospasmin, Haemophilus influenzae protein D (PD, HiD), Serogroup B meningococcus (OMPC) outer membrane protein complexes, or proteins used in conjugate vaccines such as CRM197.

[0080] The amino acid and nucleic acid sequences of many native carrier proteins are publicly available. However, as described above, such unmodified (or native) carrier proteins have limitations, including non-discriminatory antigen conjugation to any surface-exposed amino acids. As a result, T cell activation epitopes are often the sites where antigen conjugation occurs. In preferred embodiments of this disclosure, the immunogenic polypeptide is a carrier protein modified by the inclusion of at least one nnAA residue for use as a conjugation site. As considered above, nnAAs can be substituted for native residues, or added to the polypeptide by being added before, after, or inserted into the polypeptide sequence. As described herein, the use of non-native amino acids allows for the selective placement of non-native amino acids for conjugation, and as a result, enhanced T cell activation epitopes of carrier proteins at antigen conjugation can be avoided.

[0081] Table 1 shows the amino acid and nucleic acid sequences (SEQ ID NOs: 1 and 2) of an exemplary native carrier protein: CRM197. Those skilled in the art will recognize the addition of an N-terminal methionine to the amino acid sequence of conventional CRM197 produced by fermentation of Corynebacterium diphtheriae, and the resulting addition of 1 to the conventional amino acid residue numbering. The methionine is present because the start codon in the cell-free protein synthesis method used to produce these carriers is included herein. In some embodiments, the enhanced carrier protein containing the nnAA residue has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the same native or non-toxic carrier protein used in the conjugate vaccine.

[0082] Carrier proteins with sequence identity to Sequence ID No. 1 (CRM197) include other mutant diphtheria toxin proteins, such as the non-toxic K51E / E148K double mutant, which are also used as carrier proteins in the zygote (Pecetta et al. 2016 Vaccine 34:1405-11). In all of these mutants of Sequence ID No. 1, the natural toxicity of wild-type diphtheria toxin is absent (via the G52E mutation in CRM197, or the K51E / E148K mutation in Pecetta et al.).

[0083] Table 1 also shows the amino acid sequence of protein D (SEQ ID NO: 8) derived from Haemophilus influenzae. Enhanced carrier proteins containing nnAA residues may have at least 80% sequence identity with SEQ ID NO: 8. At least one Lys residue in SEQ ID NO: 8 may be replaced by nnAA. Since there are 36 Lys residues in SEQ ID NO: 8, some may be replaced by nnAA and then used for conjugation.

[0084] When sequence identity for diphtheria or tetanus toxin is to be determined, it should be determined with respect to the treated heavy chain sequence, for example, amino acids 226-567 of P00588-1 or amino acids 458-1315 of P04958-1 (UniProt sequence).

[0085] In some embodiments, the enhanced carrier protein containing nnAA residues contains a sub-complete native sequence of the carrier protein, or instead contains at least one or more T-cell activating epitopes of Corynebacterium diphtheriae toxin, Tetanus tetanus tetanospasmin, Haemophilus influenzae protein D (PD, HiD), Serogroup B meningococcus (OMPC) outer membrane protein complex, CRM197, Pfs25, or another preferred native or non-toxic carrier protein molasses. In some embodiments, the toxicity of the enhanced carrier protein is limited to treatment with paraformaldehyde (or formaldehyde or glutaraldehyde), followed by treatment with a quenching coolant. In one embodiment, the enhanced carrier protein containing nnAA residues is a polypeptide containing multiple T-cell activating epitopes of native CRM197. [Table 1]

[0086] CRM197 containing 5a.nnAA As mentioned above, Table 1 shows the amino acid sequence of CRM197 (SEQ ID NO: 1). CRM197 ("Cross-reactive substance 197", CRM 197 (also known as ) is a non-toxic variant of diphtheria toxin used in many approved complex carbohydrate vaccines (see, e.g., Broker et al. (2011) Biologicals 39:195-204). Preferred carrier proteins for use herein contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1. For example, a carrier protein may contain the amino acid sequence of SEQ ID NO: 1, except for the presence of one or more nnAAs (which may be inserted into SEQ ID NO: 1 or substituted for one or more amino acid residues in SEQ ID NO: 1, e.g., substituted for Lys and / or Phe).

[0087] In some embodiments, at least one Lys and / or at least one Phe residue in SEQ ID NO: 1 is replaced by an nnAA residue. Preferably, more than one residue in SEQ ID NO: 1 having an nnAA is replaced, and ideally, only one species of residue in SEQ ID NO: 1 is replaced by an nnAA, for example, only a Lys residue is replaced. If more than one residue in SEQ ID NO: 1 is replaced with an nnAA, it is preferable that the same nnAA is used at each position, for example, in the pAMF at each substitution site.

[0088] Carrier proteins having 2 to 9 nnAA residues in SEQ ID NO: 1 are preferred, and ideally, 4 to 9, 4 to 8, or 4 to 6 nnAA residues, for example, 4, 5, or 6 nnAA residues. This allows for broader antigen adhesion to the antibody than using a single nnAA, thereby increasing the antigen:carrier ratio while avoiding excessive disruption of the native sequence and structure, which can lead to insolubility.

[0089] Studies of CRM197 identified T cell epitopes within residues P272-D291, V322-G384, and Q412-I458. Therefore, it is preferable to avoid introducing nnAA into these regions of SEQ ID NO: 1. These regions include F274, F356, F361, F369, K420, K441, K446, K448, and K457, and these are Phe and Lys residues that are undesirable due to nnAA substitution in CRM197. Preferred Lys residues for nnAA substitution in SEQ ID NO: 1 are K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527. Other Lys residues useful for substitution with nnAA are K11, K38, K83, K104, K105, K126, K158, K173, K222, K237, K243, K475, and K499. Preferred Phe residues for substitution with nnAA are F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532.

[0090] Structural studies of CRM197 reveal two common 3D regions: the first region extends from the N-terminus to Asn-374, and the second region extends from Ser-375 to the C-terminus. Ideally, the carrier used in this invention contains at least one nnAA in the first region and at least one nnAA in the second region, for example, at least two nnAAs in each region, or at least three nnAAs in each region. This allows for spatial separation of the ligated antigen when attached to the carrier. A carrier having three nnAAs in the first region and three nnAAs in the second region is useful.

[0091] The first region contains 27 Lys residues, and the second region contains 12 Lys residues. Therefore, one or more (e.g., three) Lys residues in the 374 amino acids at the N-terminus of SEQ ID NO: 1, and one or more (e.g., three) Lys residues in the 162 amino acids at the C-terminus, can be substituted by nnAA, for example, within pAMF.

[0092] A preferred embodiment of an nnAA-containing carrier based on CRM197 has the amino acid sequence of SEQ ID NO: 1, where one or more residues K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, and / or K527 are replaced by nnAA. One such sequence is SEQ ID NO: 9, where each X represents an nnAA (preferably the same nnAA such as pAMF). [ka]

[0093] This carrier protein was found to maintain good solubility and provide a good immunogenic response when conjugated to the pneumococcal capsular polysaccharide, while being expressed very well in a cell-free protein synthesis system.

[0094] The present invention also provides a composition comprising multiple different conjugates (e.g., different serotypes of Streptococcus pneumoniae), each conjugate comprising a carrier protein having the amino acid sequence of SEQ ID NO: 9 (ideally, each X residue is the same nnAA, preferably pAMF).

[0095] Sequence ID 1 has an N-terminal methionine (typically which would be formylated) that is not present in wild-type CRM197 but is included to initiate translation without requiring the entire natural leader sequence. In some embodiments, the carrier proteins used herein lack an N-terminal methionine, and may be, for example, the N-terminal methionine of Sequence ID 1 or Sequence ID 9 is absent. In some embodiments, the CRM197-based carrier proteins do not contain (and more preferably contain) a natural amino acid upstream of the N-terminus of Sequence ID 1 or downstream of the C-terminus of Sequence ID 1.

[0096] These nnAA-containing CRM197 carrier proteins are particularly useful for conjugating to pneumococcal capsular polysaccharides. These conjugates can be combined to form polyvalent compositions, as discussed elsewhere in this specification.

[0097] The present invention also provides a protein for preparing an immunogenic polysaccharide-protein conjugate, wherein the protein has an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, or at least 95%) with SEQ ID NO: 1, and contains at least one nnAA, and the protein has an N-terminal methionine. The present invention also provides an immunogenic polysaccharide-protein conjugate prepared by conjugating a polysaccharide to at least one nnAA in the protein.

[0098] The present invention also provides a protein for preparing an immunogenic polysaccharide-protein conjugate, wherein the protein comprises the amino acid sequence of SEQ ID NO: 1, except that at least one (e.g., 2 to 9) lysine residues are nnAAs. The nnAAs are ideally azide-containing nnAAs (preferably pAMF, etc.), 1,2,4,5-tetradinyl-containing nnAAs, or alkenyl-containing nnAAs. The present invention also provides a conjugate comprising a protein that conjugates to a polysaccharide antigen via at least one of its nnAAs.

[0099] The present invention also provides an immunogenic polysaccharide-protein conjugate in which the protein is CRM197 having an N-terminal methionine.

[0100] nnAA-containing CRM197 carriers typically exist in monomeric form when used to prepare conjugates, rather than being associated with other CRM197 subunits to form CRM197 polymers.

[0101] 6. Method for generating carrier proteins General methods for polypeptide generation The enhanced carrier protein is produced by any method described for polypeptide production. Preferred methods for polypeptide production include, but are not limited to, solid-phase chemical peptide synthesis, cell-based recombinant protein expression (in E. coli or in a native host), and cell-free protein expression, as well as any combination thereof (e.g., ligation of an expressed protein using a combination of synthetic and recombinant peptide components).

[0102] In one embodiment of a method for generating enhanced carrier proteins, the enhanced carrier protein having nnAA is generated by a method comprising "codon reassignment". In one variation of this embodiment, nnAA is used, which is a close structural analog of 20 standard amino acids (e.g., homoallylglycine, fluorinated leucine, azidohomoalanine). nnAA is loaded onto its corresponding tRNA using wild-type aminoacyl-tRNA synthetase, and nnAA completely replaces one of the 20 standard amino acids identified in the template DNA sequence. To prevent interference with native amino acids, this generally requires the use of bacterial expression strains that are nutrient-dependent for the native amino acids being replaced. This strategy is not residue-specific, as all AA residues of a particular type are replaced by nnAA.

[0103] In another embodiment of the method for generating enhanced carrier proteins, enhanced carrier proteins with nnAAs are generated by a strategy involving "nonsense repression." In this approach, non-natural amino acids are identified in the template DNA sequence by rare or "nonsense" codons that do not typically identify amino acids in nature. One variation of the nonsense repression approach, pioneered by Schultz (Noren et al. Science. 1989(244):182-188.) and Chamberlin (Bain et al. J Am Chem Soc. 1989(111):8013-8014.), involves the use of a rare stop codon TAG ("amber" codon, UAG in RNA coding) along with its tRNA and its corresponding aminoacyl-tRNA synthetase (aaRS) to incorporate nnAAs into the polypeptide in a site-specific manner.

[0104] In one embodiment, the “nonsense suppression” approach involves isolating a tRNA / aaRS pair, modifying the tRNA in the anticodon loop to recognize an orthogonal codon (e.g., amber codon TAG, opal codon TGA, or another codon or nucleotide sequence not commonly used to identify amino acids in translation), and modifying the aaRS to prefer nnAA over aminoacyl-tRNA native amino acids. In some variations of this embodiment, the tRNA / aminoacyl-tRNA synthetase pair is from the same microorganism as the translation mechanism used for polypeptide synthesis. In other embodiments, the tRNA / aminoacyl-tRNA synthetase pair is from a different species than the translation mechanism used for polypeptide synthesis. Methods for modifying the tRNA anticodon loop and aaRS active site are described as examples of engineered orthogonal tRNA / aaRS pairs.

[0105] In another embodiment of the “nonsense suppression” approach, the production of enhanced carrier proteins does not involve the use of engineered aminoacyl-tRNA synthetases. In this embodiment, only orthogonal tRNAs are isolated and modified with anticodons to recognize orthogonal codons (e.g., amber codon TAG, or another codon or nucleotide sequence not commonly used to identify amino acids in translation). The engineered orthogonal tRNAs are then acylated in vitro by a preferred chemical method (e.g., the method of Heckler et al. Biochemistry. 1984 Mar 27;23(7):1468-73, involving the use of T4 RNA ligase and mutant tRNAPhe) and captured in a cell-free protein synthesis extract. Because this embodiment uses chemically acylated tRNAs, it is only compatible with cell-free protein synthesis methods.

[0106] Cell-free protein synthesis A particularly useful technique for generating nnAA-containing carrier proteins is cell-free protein synthesis. Several cell-free protein expression techniques are known in the art and can incorporate various nnAAs in this way while avoiding the potential cytotoxic effects of nnAAs (see, for example, Table 1 in Quast et al. (2015) FEBS Letters 589:1703-12). In some embodiments, the enhanced carrier protein is produced by cell-free extract-based protein synthesis. In some embodiments, the cell-free extract includes rabbit reticulocytes, wheat germ, or E. coli extracts. In further embodiments, the cell-free extract is supplemented with amino acids, energy sources, energy regeneration systems, or cation cofactors, and any combination thereof. In some embodiments, the extract includes exogenously supplemented mutant tRNA or mutant aaRS (aminoacyl-tRNA synthetase), and any combination thereof. In some embodiments, the extract includes lysates derived from E. coli strains genetically encoding the mutant tRNA or mutant aaRS, and any combination thereof. In some embodiments, the E. coli strain used for the lysate is the RF-1 attenuated strain. Compatible cell-free protein synthesis systems are described for the insertion of formulas I, II, and III into recombinant polypeptides (e.g., US8715958B2, US2016 / 0257946A1, and US2016 / 0257945A1).

[0107] For example, US8715958B2 is a tRNA derived from Methanococcus yanashii (Wang et al. (2001) Science 292(5516):498-500). TyrWe have demonstrated a regenerating cell-free E. coli-based system that uses a tyrosine synthase pair to introduce the non-natural amino acid p-azido-L-phenylalanine (pAF) into recombinant chloramphenicol acetyltransferase (CAT), GM-CSF, and TetA. Using this system, the tRNA / synthase pair is either captured within the extract or transmuted into the bacteria used to produce the extract.

[0108] In another example, US2016 / 0257946A1 demonstrates a method for (a) adapting the above-mentioned methanococcus yanashii tyrosine synthase using mutagenicity to preferentially load p-azidomethyl-L-phenylalanine (pAMF) onto amber-recognizing tRNA, and (b) selectively incorporating pAMF into antibodies such as trastuzumab using a cell-free synthesis system containing a modified synthase / tRNA pair.

[0109] In a further example, US2016 / 0257945A1 demonstrates a method of adapting the above-mentioned methanococcus yanashii tyrosine synthase using mutagenicity to preferentially load (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridine-2-yl)propanoic acid (pyridyltetrazine amino acid derivative) onto amber-recognizing tRNA, and (b) a method of selectively incorporating (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridine-2-yl)propanoic acid into recombinant GFP using a cell-free synthesis system containing the modified synthase / tRNA pair.

[0110] In further embodiments, the disclosure provides a method for producing polypeptides in a cell-free extract containing two or more non-natural amino acids. In this embodiment, the polypeptides also have bioactivity comparable to that of natural proteins. In other embodiments, the polypeptides have improved or enhanced bioactivity comparable to that of natural proteins.

[0111] The specific activity of proteins in a composition is determined by optionally assessing the level of activity in a functional assay, quantifying the amount of protein present in a non-functional assay (e.g., immunostaining, ELISA, Coomassie, or silver-stained gel quantification), and determining the ratio of biologically active or non-aggregated proteins to total protein. Generally, a specific activity as defined in this way is at least about 5% of the specific activity of the native protein, typically at least about 10% of the specific activity of the native protein, and optionally about 20%, about 40%, or about 60% or more.

[0112] In some embodiments, methods for generating nnAA-containing polypeptides involve modifications to the concentration of nnAA-specific tRNA, nnAA-specific synthase, nnAA itself, or translation temperature, and any combination thereof. Such conditions optionally allow for a reduction in translation error, an improved rate of nnAA incorporation, improved activity of chaperones required for protein folding by nnAA incorporation, a decrease in the activity of cellular factors that interfere with nnAA incorporation, or any combination of the aforementioned mechanisms.

[0113] In some embodiments of the enhanced polypeptide production method, the nnAA-specific tRNA concentration is increased to a concentration greater than approximately 20 μM, resulting in an increase in the fraction of soluble or active polypeptides. In further modifications of this embodiment, the tRNA concentration is increased while the nnAA concentration is kept below approximately 2 mM and the nnAA synthase concentration is maintained below approximately 5 μM.

[0114] In some embodiments of the enhanced polypeptide production method, the culture temperature of the translation mixture is 20°C to 30°C, about 20°C, or below 20°C. In some modifications, these temperature modifications are independently combined with the nnAA-specific tRNA concentration, nnAA concentration, or nnAA synthase concentration as described in the preceding paragraphs.

[0115] 7. Sequence variants The improved carrier proteins of this disclosure include one or more nnAAs that are substituted at any position within the polypeptide, as long as the immunogenicity mechanism of one or more T cell epitopes of the polypeptide is preserved. When the improved carrier proteins are based on known carriers, it is usually preferable to substitute some or all of the nnAAs with existing naturally occurring amino acids in the known carriers to minimize the chance of adversely affecting the carrier properties. However, it is understood that nnAAs may be inserted internally as an addition to the start carrier sequence or inserted at the end. In some embodiments, at least one nnAA in the improved carrier protein (e.g., eCRM) is not present in one or more regions of the protein containing the T cell epitope. In another embodiment, the nnAA in the enhanced immunogenic polypeptide is not present in one or more regions of the protein containing the T cell epitope.

[0116] In some embodiments, the nnAA residue is substituted with one or more of 20 naturally encoded amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some other embodiments, the nnAA residue is substituted with one or more of a specific type of natural amino acid residue, such as aliphatic, aromatic, acidic, base, hydroxyl, sulfur-containing, or amide (containing an amide group). In some cases, only one specific amino acid (e.g., lysine) is substituted for nnAA in the polypeptide at one or more positions. In other cases, two or more different amino acids (e.g., lysine, phenylalanine, etc.) are substituted for nnAA in the polypeptide at two or more positions. Lysine and phenylalanine are preferred for substitution with nnAAs because (i) lysine is often used for binding to existing carrier proteins, so that nnAA-containing carriers can be maintained at the same attachment site, and (ii) many useful nnAAs are phenylalanine-based, so that carriers having nnAAs can have minimal structural modifications compared to the natural sequence. Polypeptides in which only a single amino acid is substituted with an nnAA, for example, only residues are substituted, are preferred.

[0117] In some embodiments, the nnAA residue is substituted with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 native amino acid residues of the carrier protein. SEQ ID NO: 1.

[0118] In a further embodiment, nnAA is substituted with one or more amino acid residues in the carrier protein. The specific amino acid residues selected to produce the single or multiple substitution nnAA mutants described herein are determined by selectively dividing the protein into subdomains and selecting single amino acids or sets of amino acid residues for substitution that do not sterically occlude each other (e.g., such that there are multi-angstrom distances between substitution sites). The division of CRM197 into two structural regions is considered below.

[0119] In some embodiments, nnAA is substituted with a charged amino acid residue. Thus, nnAA may be substituted with an aspartic acid, glutamic acid, lysine, arginine, or histidine amino acid residue. In some embodiments, nnAA is substituted with a negatively charged amino acid residue, such as an aspartic acid or glutamic acid residue. In some embodiments, nnAA is substituted with a positively charged amino acid residue, such as a lysine, arginine, or histidine residue.

[0120] In some embodiments, nnAA is substituted with one or more lysine residues in the immunogenic polypeptide. For example, the enhanced form of SEQ ID NO: 1 is generated by substituting nnAA with lysine in the following way: 1) one residue from the group consisting of K25, K34, K38, and K40; 2) one residue selected from the group consisting of K213 and K215; and 3) two to four residues selected from the group consisting of K228, K245, K265, K386, K523, and K527. In further embodiments, one or more of the specific types of native amino acid residues substituted are selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K265, K386, K523, and K527 of SEQ ID NO: 1, and any combination thereof. In other embodiments, the nnAA substitution in SEQ ID NO: 1 is selected from one or more of K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, and K527. In one embodiment, the nnAA substitution comprises six residues consisting of K25, K215, K228, K265, K386, and K523 in SEQ ID NO: 1. In some embodiments, the nnAA substitution in SEQ ID NO: 1 includes K265. In other embodiments, the nnAA substitution in SEQ ID NO: 1 includes K386. In yet another embodiment, the nnAA substitution in SEQ ID NO: 1 includes K265 and K386. In further embodiments, nnAA is substituted with phenylalanine. Preferred phenylalanine substitutions include F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. Due to their proximity, substitution is generally preferred not to occur with both F531 and F532.

[0121] The binding epitopes to diphtheria toxin for human CD4+ cells, recognized by the majority of subjects tested, encompass residues 271-290, 321-340, 331-350, 351-370, 411-430, or 431-450 (see Raju et al., Eur J Immunol. 1995 Dec;25(12):3207-14). Therefore, in some embodiments, one or more substituted nnAAs are not found within residues 271-290, 321-340, 331-350, 351-370, 411-430, and / or 431-450 of SEQ ID NO: 1. In one embodiment, one or more substituted nnAAs are not found within residues 331-350 of SEQ ID NO: 1. In another embodiment, one or more substituted nnAAs are not found within residues 321-340 of SEQ ID NO: 1. In another embodiment, one or more substituted nnAAs are not present within residues 431-450 of SEQ ID NO: 1.

[0122] The binding epitopes to human CD4+ cells recognized by all subjects tested include heavy chain residues H176-195, IDKISDVSTIVPYIGPALNI [SEQ ID NO: 3], and H491-510, NNFTVSFWLRVPKVSASHLE [SEQ ID NO: 4] (see Diethelm-Okita et al., J Infect Dis. 1997 Feb;175(2):382-91). Therefore, in some embodiments, one or more substituted nnAAs are not within residues 176-195 and / or 491-510 of the heavy chain peptide component of the tetanus toxin precursor protein. In another embodiment, one or more substituted nnAAs are not within residues 176-195 of the heavy chain peptide component of the tetanus toxin precursor protein. In yet another embodiment, one or more substituted nnAAs are not within residues 491-510 of the heavy chain peptide component of the tetanus toxin precursor protein.

[0123] The binding epitopes to the meningococcal outer membrane protein (OMP or PorA) for human CD4+ cells, recognized by the majority of subjects tested, are mostly located outside the variable region and are conserved among different meningococcal (and gonorrhea) strains, for example, encompassing immunodominant T cell epitopes corresponding to the conserved putative trans-membrane region of OMP (Wiertz et al. J Exp Med. 1992;176(1):79-88). Therefore, in some embodiments, one or more substituted nnAAs are not located within the conserved region of OMP.

[0124] The binding epitope to human CD4+ cells for the carrier protein BB, derived from the G protein of Streptococcus strain G148, recognized by the majority of subjects tested, includes amino acids 25-40 (VSDYYKNLINNAKTVE [SEQ ID NO: 5]), 63-78 (DGLSDFLKSQTPAEDT [SEQ ID NO: 6]), and 74-89 (AEDTVKSIELAEAKVL [SEQ ID NO: 7]) in the BB sequence (Goetsch et al., Clin Diagn Lab Immunol. 2003 Jan;10(1):125-32). Therefore, in some embodiments, one or more substituted nnAAs are not located within residues 25-40, 63-78, and / or 74-89 of the BB sequence.

[0125] In some embodiments, an immunogenic polypeptide comprising at least one non-natural amino acid residue further comprises at least one antigen. In some embodiments, an immunogenic polypeptide comprising at least one non-natural amino acid is an enhanced carrier protein and further comprises at least one antigen. In some embodiments, an immunogenic polypeptide comprising at least one non-natural amino acid is an enhanced carrier protein and further comprises at least one antigen.

[0126] 8. T cell epitopes The T cell epitopes of carrier proteins are determined, optionally, by any known method. To assist in the design of improved carrier proteins in this disclosure, T cell-binding epitopes in proteins are predicted using algorithms that consider various factors such as the protein's amphipathic profile, sequence motifs, quantification matrices (QMs), artificial neural networks (ANNs), support vector machines (SVMs), quantification structure-activity relationships (QSARs), and molecular docking simulations (see Desai et al. Methods Mol Biol. 2014;1184:333-64). For example, the T cell-binding epitope in diphtheria toxin / CRM has been predicted using the DeLisi & Berzofsky algorithm (see Bixler et al. WO89 / 06974 and PNAS 82:7848, 1985). The predicted T cell epitopes can be experimentally confirmed. For example, the T cell epitopes of the immunogenic polypeptide in question can be experimentally determined by synthesizing partially overlapping peptide fragments corresponding to the complete sequence of the immunogenic polypeptide (or predicted region) and performing proliferation assays of CD4+ cell lines (e.g., peripheral blood mononuclear cells (PBMCs)) in the presence of each fragment. This general approach has been used to map T cell epitopes in diphtheria toxin (Raju et al., Eur J Immunol. 1995 Dec;25(12):3207-14), tetanus toxin (Diethelm-Okita et al., J Infect Dis. 1997 Feb;175(2):382-91), meningococcal outer membrane protein (OMP) (J Exp Med. 1992 Jul 1;176(1):79-88), and BB, a carrier protein derived from the G protein of Streptococcus strain G148 (Goetsch et al., Clin Diagn Lab Immunol. 2003 Jan;10(1):125-32).To construct the presence of T cell epitopes that are not inactivated by the presence of one or more nnAAs, improved carrier proteins of this disclosure can be directly screened for CD4+ cell proliferation and / or cytokine response.

[0127] 9. Method for generating a juxtaposition body In one embodiment, the disclosure provides a method for synthesizing a polypeptide containing nnAA in a cell-free expression mixture maintained at a temperature of about 10°C to about 30°C. In another embodiment, the temperature is above about 20°C. In another embodiment, the temperature is below about 20°C. In another embodiment, the temperature is between about 14°C and about 18°C. In another embodiment, the polypeptide is encoded by a nucleic acid containing a repressor codon. In another embodiment, the cell-free expression mixture contains an orthogonal tRNA / aminoacyl-tRNA synthetase pair specific to nnAA. In another embodiment, the concentration of tRNA is at least 20 μM. In another embodiment, the concentration of nnAA is less than about 2 mM, and the concentration of aminoacyl-tRNA synthetase is less than about 5 μM. In another embodiment, the method includes conjugating the polypeptide to an active moiety. In another embodiment, the active moiety is selected from the group consisting of haptens, bacterial antigens, viral antigens, tumor-derived glycans, peptide toxins, macrolides, polyethers, and any combination thereof. In another embodiment, the polypeptide is selected from the group consisting of growth hormone, coagulation factor, plasma protein, interleukin, extracellular domain of T cell receptor, extracellular domain of growth factor, bacterial antigen, viral antigen, and any combination thereof. In another embodiment, the expression mixture comprises cell extracts of Escherichia coli, wheat germ, or rabbit reticulocytes. In another embodiment, the expression mixture comprises at least 30% cell extract. In another embodiment, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs.In another embodiment, nnAA is selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In another embodiment, the resulting polypeptide comprises both a soluble fraction and an insoluble fraction, with the ratio of the soluble fraction to the insoluble fraction being at least 40% (w / w). In another embodiment, the polypeptide produced comprises both a soluble fraction and an insoluble fraction, with a ratio of at least 60% (w / w) of soluble to insoluble fractions. In one embodiment, the polypeptide produced by cell-free expression comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs, with a ratio of at least 20% (w / w), at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w).

[0128] antigen The immunogenic antigens described herein are optionally further derivatized with chemical handles to enhance adhesion to carrier proteins. In one embodiment, the antigen is any purified natural, synthetic, or recombinantly produced polymer or fragment thereof. Examples include, but are not limited to, lipids, polysaccharides, nucleic acids, or polypeptides, and any combination thereof (e.g., glycoproteins, glycolipoproteins, glycolipids). For example, the glycolipid is optionally glycosylphosphatidylinositol. In another embodiment, the antigen is a T-independent or T-activated antigen (usually a weakly T-activated antigen) selected from the group consisting of bacterial polysaccharides, bacterial lipopolysaccharides, tumor-derived glycans, or haptens.

[0129] In some embodiments, polysaccharide-containing antigens include bacterial-derived polysaccharides such as capsular polysaccharides. Such capsular polysaccharides are high molecular weight polymers of Gram-positive or Gram-negative bacteria that perform a function of protecting the microorganism against immune responses, and thus represent attractive vaccine targets when the goal is the production of neutralizing antibodies. Such capsular polysaccharides are generally prepared from the whole cell lysate or culture supernatant of the corresponding bacteria via processes involving diafiltration, protein removal, ethanol precipitation, nucleic acid removal, and lyophilization. Examples include, but are not limited to, the Merieux protocol (Institut Merieux (1980) Brevet Belge 80:26320) and the Yavordios protocol (Yavordios et al. EP0071515A1 (1983)).

[0130] In some embodiments, the capsular polysaccharide of Streptococcus pneumoniae includes capsular polysaccharides derived from Streptococcus pneumoniae. Streptococcus pneumoniae is an encapsulated, Gram-positive bacterium that can cause pneumonia, bacterial infections, and meningitis. There are 90 distinct documented serotypes of Streptococcus pneumoniae (outlined, e.g., Kalin, M. Thorax 1998;53:159-162) that possess capsular polysaccharides with serotype-specific repeating unit structures. Therefore, in some cases the antigens are 1, 2, 3, 4, 5, 6A, 6B, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 1 These are capsular polysaccharides of Streptococcus pneumoniae selected from 9C, 20, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48 (Henrichsen J Clin Microbiol 1995;33:2759-2762). However, only a subset of these serotypes generally cause bacterial infections, including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. Serotypes 6C, 7C, 15A, 15C, 16F, 23A, 23B, 31, 34, 35B, 35F, 37, and 38 are also of clinical concern, as they also have serotypes 20A, 20B, and 24B. In another embodiment, the antigen is a capsular polysaccharide of Streptococcus pneumoniae selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.In another embodiment, the antigen is a capsular polysaccharide of Streptococcus pneumoniae selected from serotypes 6C, 7C, 15A, 15C, 16F, 23A, 23B, 31, 34, 35B, 35F, 37, and 38. Embodiments described herein may also additionally include one or more capsular polysaccharides of Streptococcus pneumoniae selected from serotypes 20A, 20B, and 24B.

[0131] As described above, the compositions of the present invention may comprise conjugates of capsular polysaccharides derived from at least 14, 15, 20, 21, 24, or 25 different serotypes of Streptococcus pneumoniae. If the composition comprises 14 or more serotypes, these preferably comprise 13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In addition to these 13 serotypes, the composition preferably comprises one or more of serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and / or 33F. Alternatively, in addition to the 13 serotypes mentioned above, the composition preferably includes one or more serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38. Useful combinations of 15 or more (e.g., 16 or more) serotypes include each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and may also include serotype 8. A useful combination of 20 or more serotypes (e.g., 21 or more) includes each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. A useful combination of 24 or more serotypes includes each of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0132] The structure of the repeating unit of the capsular polysaccharide of common pneumococcal serotypes is described by Jones et al. (Jones C et al. An Acad Bras Cienc. 2005 Jun;77(2):293-324). Type 1 [→3)-D-AAT-α-Galp-(1→4)-α-D-GalpA(2 / 3OAc)-(1→3)-α-D-GalpA-(1→] Type 2 [→4)-β-D-Glcp-(1→3)-[α-D-GlcpA-(1→6)-α-D-Glcp-(1→2)]-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)β-L-Rhap-(1→] Type 3 [→3)-β-D-GlcA-(1→4)-β-D-Glcp-(1→] Type 4 [→3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp2,3(S)Py-(1→] Type 5 [→4)-β-D-Glcp-(1→4)-[α-L-PnepNAc-(1→2)-β-D-GlcpA-(1→3)]-α-L-FucpNAc-(1→3)-β-D-Sugp-(1→] Type 6B [→2)-α-D-Galp-(1→3)-α-D-Glcp-(1→3)-α-L-Rhap-(1→4)-D-Rib-ol-(5→P→] Type 9N [→4)-α-D-GlcpA-(1→3)-α-D-Glcp-(1→3)-β-D-ManpNAc-(1→4)-β-D-Glcp-(1→4)-α-D-GlcpNAc-(1→] Type 9V [→4)-α-D-GlcpA(2 / 3OAc)-(1→3)-α-D-Galp-(1→3)-β-D-ManpNAc(4 / 6OAc)-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→] Type 12F [→4)-[α-D-Galp-(1→3)]α-L-FucpNAc-(1→3)-β-D-GlcNAc-(1→4)-[α-D-Glc-(1→2)-α-D-Glc-(1→3)]-β-D-ManNAcA-(→] 14-inch [→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→] 18C type [→4)-β-D-Glcp-(1→4)-[α-D-Glcp(6OAc)(1→2)][Gro-(1→P→3)]-β-D-Galp-(1→4)-α-D-Glcp-(1→3)-β-L-Rhap-(1→] 19F type [→4)-β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap-(1→P→] 23F type [→4)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→]

[0133] A more comprehensive discussion of polysaccharides can be found in Geno et al. (2015) Clin. Microbiol. Rev. 28:871-99, where Table 1 shows the structures for 97 known serotypes. This table also reveals the ratio of monosaccharide residues that are acetylated when acetylation is incomplete.

[0134] Capsular polysaccharides are optionally O-acetylated. In some embodiments, capsular polysaccharides derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F contain monosaccharides having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or about 100%. The degree of O-acetylation of the polysaccharide is optionally determined, for example, by proton NMR (see, e.g., Lemercinier & Jones (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247). In some embodiments, the presence of O-acetyl groups is determined by ion-HPLC analysis. Typically, the polysaccharide in the conjugate will retain the O-acetylation level found in the initial polysaccharide purified from bacteria.

[0135] In one embodiment, the capsular polysaccharides derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F have molecular weights ranging from 10 kDa to 4,000 kDa. In another such embodiment, the polysaccharides have molecular weights ranging from 50 kDa to 4,000 kDa. In yet another such embodiment, the polysaccharides have molecular weights ranging from 50 kDa to 1,400 kDa. In further such embodiments, the polysaccharides are 50kDa~3,500kDa, 50kDa~3,000kDa, 50kDa~2,500kDa, 50kDa~2,000kDa, 50kDa~1,750kDa, 50kDa~1,500kDa, 50kDa~1,250kDa, and 50kDa~1,000k Da, 50kDa~750kDa, 50kDa~500kDa, 100kDa~4,000kDa, 100kDa~3,500kDa, 100kDa~3 ,000kDa, 100kDa~2,500kDa, 100kDa~2,000kDa, 100kDa~2,000kDa, 100kDa~1,750kDa a, 100kDa~1,500kDa, 100kDa~1,250kDa, 100kDa~1,000kDa, 100kDa~750kDa, 100kDa a~500kDa, 200kDa~4,000kDa, 200kDa~3,500kDa, 200kDa~3,000kDa, 200kDa~2,500 The molecular weight is kDa, 200kDa to 2,000kDa, 200kDa to 2,000kDa, 200kDa to 1,750kDa, 200kDa to 1,500kDa, 200kDa to 1,250kDa, 200kDa to 1,000kDa, 200kDa to 750kDa, or 200kDa to 500kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0136] Capsular polysaccharides are selectively chemically modified from naturally occurring capsular polysaccharides. For example, polysaccharides are selectively de-O-acetylated (partially or completely), de-N-acetylated (partially or completely), or N-propionated (partially or completely). Deacetylation can occur selectively before, during, or after conjugation to a chemical handle or polypeptide, but typically occurs before conjugation.

[0137] Polysaccharides of Streptococcus pyogenes In some embodiments, the polysaccharide-containing antigen includes polysaccharides derived from Streptococcus pyogenes. Streptococcus pyogenes is a Gram-positive bacterium (Group A Streptococcus, also known as "GAS") that causes a variety of infections in humans, including pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, post-streptococcal glomerulonephritis, gangrenous fasciitis, myonecrosis, and lymphangitis. In embodiments, the polysaccharide is the capsular polysaccharide of Streptococcus pyogenes. The capsular polysaccharide of Streptococcus pyogenes is composed of hyaluronic acid, a high molecular weight polymer whose repeating units have the following structure: [→4)-β-D-GlcUAp-(143)-β-D-GlcpNAc-(→] This appears to be invariant among serotypes of Streptococcus pyogenes.

[0138] In one embodiment, the capsular polysaccharide derived from Streptococcus pyogenes has a molecular weight of 10 kDa to 4,000 kDa. In another such embodiment, the polysaccharide has a molecular weight of 50 kDa to 4,000 kDa. In yet another such embodiment, the polysaccharide has molecular weights of 50 kDa to 3,500 kDa, 50 kDa to 3,000 kDa, 50 kDa to 2,500 kDa, 50 kDa to 2,000 kDa, 50 kDa to 1,750 kDa, 50 kDa to 1,500 kDa, 50 kDa to 1,250 kDa, and 50 kDa to 1,000 kDa. Da, 50kDa~750kDa, 50kDa~500kDa, 100kDa~4,000kDa, 100kDa~3,500kDa, 100kDa~3 ,000kDa, 100kDa~2,500kDa, 100kDa~2,000kDa, 100kDa~2,000kDa, 100kDa~1,750kDa a, 100kDa~1,500kDa, 100kDa~1,250kDa, 100kDa~1,000kDa, 100kDa~750kDa, 100kDa a~500kDa, 200kDa~4,000kDa, 200kDa~3,500kDa, 200kDa~3,000kDa, 200kDa~2,500 The molecular weight is kDa, 200kDa to 2,000kDa, 200kDa to 2,000kDa, 200kDa to 1,750kDa, 200kDa to 1,500kDa, 200kDa to 1,250kDa, 200kDa to 1,000kDa, 200kDa to 750kDa, or 200kDa to 500kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0139] In another embodiment, the polysaccharide is a non-capsular polysaccharide derived from Streptococcus pyogenes. The non-capsular polysaccharide includes a cell wall polysaccharide of Group A Streptococcus, comprising a poly-L-rhamnopyranosyl unit backbone linked by alternating α-L-(1→3) and α-L-(1→2) linkages, with an N-acetyl-β-D-glucoseamine residue attached to the 3-position of the rhamnose backbone.

[0140] In one embodiment, the cell wall polysaccharides of Group A Streptococcus derived from Streptococcus pyogenes have a molecular weight of 10 kDa to 4,000 kDa. In another such embodiment, the polysaccharides have a molecular weight of 50 kDa to 4,000 kDa. In yet another such embodiment, the polysaccharides have molecular weights of 50 kDa to 3,500 kDa, 50 kDa to 3,000 kDa, 50 kDa to 2,500 kDa, 50 kDa to 2,000 kDa, 50 kDa to 1,750 kDa, 50 kDa to 1,500 kDa, 50 kDa to 1,250 kDa, and 50 kDa to 1,000 kDa. Da, 50kDa~750kDa, 50kDa~500kDa, 100kDa~4,000kDa, 100kDa~3,500kDa, 100kDa~3 ,000kDa, 100kDa~2,500kDa, 100kDa~2,000kDa, 100kDa~2,000kDa, 100kDa~1,750kDa a, 100kDa~1,500kDa, 100kDa~1,250kDa, 100kDa~1,000kDa, 100kDa~750kDa, 100kDa a~500kDa, 200kDa~4,000kDa, 200kDa~3,500kDa, 200kDa~3,000kDa, 200kDa~2,500 The molecular weight is kDa, 200kDa to 2,000kDa, 200kDa to 2,000kDa, 200kDa to 1,750kDa, 200kDa to 1,500kDa, 200kDa to 1,250kDa, 200kDa to 1,000kDa, 200kDa to 750kDa, or 200kDa to 500kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0141] Capsular Polysaccharides of Streptococcus agalactia In some embodiments, the polysaccharide-containing antigens include capsular polysaccharides derived from Streptococcus agalactia. Streptococcus agalactia (also known as Group B Streptococcus, or GBS) is a Gram-positive bacterium that is generally commensal to mammals, causing sepsis, pneumonia, and meningitis in immunologically vulnerable humans, as well as bovine mastitis in dairy cows. There are at least 10 serotypes of Streptococcus agalactia, each with distinct capsular polysaccharide repeating units (Ia, Ib, II-IX), but only a subset of serotypes generally cause disease. These include serotypes Ia, Ib, II, III, and V, and conjugates of capsular polysaccharides derived from these serotypes can be prepared. The structures of the capsular polysaccharide repeating units of common Streptococcus agalactia serotypes have been determined and are as follows: Type Ia [ka]

[0142] Type Ib [ka]

[0143] Type II [ka]

[0144] V type [ka]

[0145] In some embodiments, the polysaccharide-containing antigen includes a capsular polysaccharide derived from Haemophilus influenzae. Haemophilus influenzae is a Gram-negative, anaerobic pathogen that causes a wide range of localized and infiltrative infections, including pneumonia, bacterial infections, meningitis, epiglottitis, cellulitis, and infectious arthritis. There are at least six serotypes of Haemophilus influenzae with distinct capsular polysaccharide chemical structures (types a-f). However, only types a and b are considered "highly pathogenic" strains of Haemophilus influenzae, and it is believed that the majority of childhood infections are caused by type b (Jin et al. Infect.Immun.June 2007 vol.75 no.6 2650-2654), which is therefore the preferred type of Haemophilus influenzae polysaccharide for use in the present invention. The structure of the repeating unit of type b capsular polysaccharide has been determined and is as follows: [→3)-β-D-Ribf-(1→1)-D-Ribitol-(5→OPO3 - →]

[0146] Capsular Polysaccharides of Meningococcus In some embodiments, the polysaccharide-containing antigens include capsular polysaccharides derived from Meningococcus. Meningococcus is a Gram-negative bacterium that is the primary causative pathogen of meningitis and meningococcal septic infection. There are at least 13 serogroups of Meningococcus with distinct capsular polysaccharide chemical structures (serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z'(29E)). However, only six serogroups (A, B, C, W-135, X, Y) are thought to cause life-threatening diseases. The structures of the repeating units of capsular polysaccharides for the five main life-threatening serogroups targeted for zygote preparation have been determined and are as follows: Type A [→6)-α-D-ManpNAc(3 / 4OAc)-(1→OPO3→] C type [→9)-α-D-Neup5Ac(7 / 8OAc)-(2→] W-135 type [→6)-α-D-Galp-(1→4)-α-D-Neup5Ac(9OAc)-α-(2→] X type [→4)-α-D-GlcpNAc-(1→OPO3→] Y type [→6)-α-D-Glcp-(1→4)-α-D-Neup5Ac(9OAc)-α-(2→]

[0147] In a different embodiment of the Porphyromonas gingivalis capsular polysaccharide, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6). See Van Winkelhoff et al. (1993) Oral Microbiol. Immunol. 8:259-265 and Laine et al. (1996) J. Periodontal Res. 31:278-84.

[0148] In a specific embodiment of the capsular polysaccharide of Salmonella typhi, the antigen is the Vi polysaccharide. Vi is the capsular polysaccharide of Salmonella typhi (formerly classified as the species itself, but now also called the typhi serotype of the genus Salmonella). Vi can also be found in other serotypes of Salmonella (such as Salmonella serotype paratyphi C or serotype dublin) and in other bacteria, such as Citrobacter species (e.g., C. freundii and C. youngae). The Vi polysaccharide is a linear homopolymer of hexosaminuronic acid, which is α1,4-N-acetylgalactose-aminouronic acid acetylated 60-90% at the C-3 position. O-acetyl substitution of Vi is a factor in its ability to induce a protective immune response. The immunogenicity of Vi is closely related to the degree of O-acetylation. Partial de-o-acetylation may slightly increase immunogenicity, while complete de-o-acetylation eliminates the immunogenicity of Vi. The Vi polysaccharides used in this invention can be chemically modified from naturally occurring capsular polysaccharides. For example, Vi polysaccharides can be partially de-o-acetylated, de-N-acetylated (partially or completely), or N-propionated (partially or completely). Deacetylation may occur before, during, or after conjugation, but preferably before conjugation. The effects of deacetylation and other modifications can be evaluated by steady-state assays.

[0149] In embodiments of Staphylococcus aureus based on sugar types, the antigen is a polysaccharide derived from Staphylococcus aureus. The polysaccharide may be a Staphylococcus aureus exopolysaccharide, such as poly-N-acetylglucoseamine (PNAG), or a Staphylococcus aureus capsular polysaccharide, such as type 5, type 8, or type 336.

[0150] In a different embodiment of the Clostridium difficile surface polysaccharide, the antigen is a Clostridium difficile-derived surface glycan such as PS-I or PS-II.

[0151] In a different embodiment of the glucan, the antigen is a glucan containing β-1,3-links and / or β-1,6-links. These conjugated glucans may be useful in enhancing an antifungal immune response against, for example, Candida albicans. Glucans are glucose-containing polysaccharides found, among other things, in the cell walls of fungi. β-glucans contain one or more β-links between glucose subunits. The glucans used in the present invention contain β-links and may contain only β-links (i.e., no α-links). The glucan may contain one or more β-1,3-links and / or one or more β-1,6-links. It may also contain one or more β-1,2-links and / or β-1,4-links, but usually only its β-links will be β-1,3-links and / or β-1,6-links. The glucan may be branched or linear. The glucan may be a fungal glucan. "Fungal glucans" are generally obtained from fungi, but certain glucan structures are found in both fungi and nonfungi (e.g., bacteria, lower plants, or algae), and therefore nonfungal microorganisms can be used as alternative sources. Thus, glucans may be derived from the cell walls of Candida fungi such as Candida albicans, or from Coccidioides imitis, Trichophyton vercosum, Blastomyces dermatichidis, Cryptococcus neoformans, Histoplasma capsulatum, Saccharomyces cerevisiae, Paracoccidioides brasiliensis, or Pythium fungi. There are various sources of fungal β-glucans. For example, pure β-glucans are commercially available, and for instance, Pusturan (Calbiochem) is a β-1,6-glucan purified from Umbilicaria papullosa. β-glucans can be purified from fungal cell walls by various methods. In some embodiments, the glucan is a β-1,3 glucan with some β-1,6 branching, such as that found in laminarin. Laminarin is found in brown algae and seaweed. The β(1-3):β(1-6) ratio of laminarin varies between different sources, for example, it is as low as 3:2 in laminarin from Epinephelus fuscoguttatus, but as high as 7:1 in laminarin from Laminaria japonica.Therefore, the glucans used herein may have a β(1-3):β(1-6) ratio of 1.5:1 to 7.5:1, for example, about 2:1, 3:1, 4:1, 5:1, 6:1, or 7:1. In other embodiments, the glucans have β-1,3 linkages exclusively or primarily, as seen in curdlan. Thus, glucans can be made solely from β-1,3-linked glucose residues (e.g., linear β-D-glucopyranose having exclusively 1,3 linkages). However, optionally, glucans may contain monosaccharide residues other than β-1,3-linked glucose residues, for example, β-1,6-linked glucose residues. The ratio of β-1,3-linked glucose residues to these other residues should be at least 8:1 (for example, ≥9:1, ≥10:1, ≥11:1, ≥12:1, ≥13:1, ≥14:1, ≥15:1, ≥16:1, ≥17:1, ≥18:1, ≥19:1, ≥20:1, ≥25:1, ≥30:1, ≥35:1, ≥40:1, ≥45:1, ≥50:1, ≥75:1, ≥100:1, etc.).

[0152] Tumor-derived glycans: In some embodiments, antigens containing polysaccharides have developmentally inappropriate cell surface glycan properties in tumor cells. Among others, Danishevskygen (scheduled in Zhu et al. Expert Rev Vaccines. 2009(10):1399-1413) discovered that certain oligosaccharide motifs (time-specific embryonic antigens, SSEAs) are originally expressed on the cell surface during embryogenesis and are "reactivated" in adult tumors. Since these are short polysaccharides, they are accessed mainly through chemosynthesis (scheduled in Zhu above). Among these oligosaccharides, the most clearly associated with carcinogenesis (e.g., prostate cancer and breast cancer) are Globo-H, Le y These are STn, TF, and Tn.

[0153] In some embodiments, the hapten contains a non-polymeric synthetic moiety with a molecular weight of less than 1,000 Da. Uses of haptens in therapeutic protein conjugates include those mimicking abused drugs, such as nicotine or cocaine (see, e.g., Berkowitz & Spector. Science. 1972(178):1290-1292 for morphine, Kosten et al. Vaccine. 2002(20):1196-1204 for cocaine, and Hatsukami et al. Clin Pharmacol Ther. 2005(78):456-467). Alternatively, the conjugation of small molecules that are not sufficiently immunogenic to immunogenic polypeptides allows for the enhancement of drug-specific antibodies, thereby sequestering the abused drug from the central nervous system.

[0154] Methods for the derivatization and preparation of antigens and compositions resulting from this process. This specification describes antigens containing chemical handles that can react with corresponding groups introduced into non-natural amino acids of polypeptides as described earlier herein. In some embodiments, the chemical handles include groups suitable for a “click” chemical reaction with the corresponding group to the polypeptide. Chemical groups suitable for “click” chemistry include azide (-N3) groups, alkyne (C≡C) groups, phosphine (e.g., -P(Ph)2) groups, alkene (C=C) groups, and 1,2,4,5-tetrazine ( [ka] This includes, but is not limited to, the ) group.

[0155] A chemical handle is introduced through a general process comprising three steps: (a) activation of the antigen, (b) optionally, reaction of the antigen with a linker or nucleophilic group to introduce reactivity not normally present in the antigen, and (c) conjugation of the antigen to the chemical handle. In some embodiments, two or more of steps (a) to (c) occur simultaneously, for example, when the chemical handle is modified by the addition of a reactive moiety such as N-hydroxysuccinimide. In some embodiments, two or more of steps (a) to (c) are separate by optional purification of the antigen between steps. In some embodiments, step (a) additionally includes a step to remove protecting groups from the antigen so that specific functional groups (e.g., hydroxyl, amine, thiol) are accessible by activation.

[0156] Chemical handles are introduced at various locations on the antigen, optionally. In some embodiments, chemical handles are introduced at terminal locations (e.g., reducing and non-reducing ends of polysaccharides, N-terminus and C-terminus of polypeptides, or terminals of acyl chains of glycerides). In some embodiments, chemical handles are introduced at internal locations (e.g., internal amino acids, or internal hydroxyls, amines, or activated hydroxyls of polysaccharides) of polypeptides. In some embodiments, chemical handles are introduced at one or more terminal locations in addition to internal locations. The specific method of activation used for the antigen will affect the site activated for conjugation, and therefore the final site of the conjugated chemical handle on the antigen. It is preferable to introduce multiple chemical handles to the antigen so that multiple conjugations with the carrier can be achieved.

[0157] In a preferred embodiment, a method for conjugating a polypeptide to an antigen via a chemical handle is as follows: The antigen is activated to incorporate at least one first chemical handle internally, where the first chemical handle can conjugate to a second chemical handle of an nnAA in the polypeptide. The activated antigen is conjugated to a polypeptide containing at least one nnAA with a second handle under conditions that the first and second chemical handles react to form an antigen-polypeptide conjugate. The reaction thus enabled is a non-catalytic covalent bioconjugation reaction. The reactive site of the antigen acting as the “first chemical handle” is preferably an alkynyl group, which can be incorporated into a molecular environment that increases reactivity. For example, the alkynyl group can be incorporated into a ring, such as a cyclooctinyl ring, e.g., diaryl-strained cyclooctin. The preferred reactive site in the polypeptide provided by the nnAA residue, i.e., the “second chemical handle,” is an azide group. As is known in the art, the reaction in this case is a [3+2]loading cyclization, referred to in the art as a “strain-accelerated azide-alkyne loading cyclization” (SPAAC), which will be discussed in more detail below.

[0158] Antigen activation Antigens are optionally activated using any chemical method described for the generation of biozygotes. Such methods include, but are not limited to, periodate oxidation, demasking of specific aldehydes (e.g., the reducing end of polysaccharides), activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroboric acid (CDAP), or activation of the hydroxyl with 1,1'-carbonyldiimidazole (CDI), followed by nucleophilic addition. Further chemical strategies for monosaccharide derivatization are described in Hermanson (Hermanson, Greg. Bioconjugate Techniques (2008)). For activation, cyanidating agents (such as p-nitrophenyl cyanate, CDAP, or N-cyanotriethylammonium tetrafluoroboric acid), active esters, carbodiimides, hydrazides, norbornene, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc. may be used.

[0159] The present invention provides antigens activated by any of the chemicals discussed below (specifically, polysaccharide antigens such as pneumococcal capsular polysaccharide antigen as disclosed herein), for example, the products of reactions between an antigen and one or more DBCO groups and DIFO groups discussed below.

[0160] Activation of periodate In some embodiments, the antigen is activated by periodate oxidation. In some embodiments, periodate oxidation is used to introduce an aldehyde group into the antigen and is useful for 1) polysaccharides and 2) the addition of an aldehyde to the N-terminal residue of a polypeptide to produce an activated antigen. Periodates cleave carbon-carbon bonds that hold a primary or secondary hydroxyl or amine at either end, thus activating carbohydrate sugar residues with adjacent hydroxyls or amino acids (N-terminal threonine or serine residues) containing a 2-amino alcohol moiety. Because the aldehyde moiety has a long half-life, antigens activated in this manner are optionally purified and / or lyophilized by chromatography after activation.

[0161] For the periodate oxidation of antigens, (a) the antigen is dissolved in solution, (b) a periodate source is added to the antigen from a concentrated stock solution to form an oxidation mixture, (c) the reaction mixture is cultured, and (d) (optionally) excess periodate is removed.

[0162] Deionized water or a suitable buffer is optionally used for the oxidation reaction. In some embodiments, the solution in step (a) is deionized water. In some embodiments, the solution in step (a) contains an effective amount of a buffer having a pKa of approximately physiological pH. In some embodiments, the solution in step (a) contains an effective amount of a buffer having a pKa of approximately physiological pH, and the buffer does not contain amine groups. Examples of non-amine buffers include, but are not limited to, acetic acid, formic acid, and phosphoric acid.

[0163] The periodate source in step (b) is optionally selected from any periodate source having adequate stability in aqueous solution. Examples of periodate sources include, but are not limited to, sodium periodate, potassium periodate, tetrabutylammonium (meth)periodate, barium periodate, sodium hydrogen periodate, sodium (para)periodate, and tetraethylammonium (meth)periodate.

[0164] In some embodiments, the level of periodate addition and reaction conditions is adjusted to convert all available diols of the polysaccharide to aldehydes. For example, a significant excess of sodium periodate (an excess of >1000x with respect to the molar concentration of the polysaccharide, or sodium periodate in a 10 mM solution), combined with cultivation at room temperature, is preferable to the total conversion of diols rather than aldehydes.

[0165] In some embodiments, the level of periodate addition and reaction conditions is adjusted to introduce low amounts of oxidation / aldehyde formation into the polysaccharide chain. Less than stoichiometric amounts of sodium periodate (e.g., <1.0 equivalent) in the chemical reaction are preferred for low amounts of polysaccharide chain oxidation. For example, bacterial monosaccharides are activated by 0.001-0.7, 0.005-0.5, 0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0, or 0.4-0.9 molar equivalents of periodate (see WO2011 / 110531). In another embodiment, 0.4 molar equivalents of periodate are added to a pH 6.0 solution containing pneumococcal capsular polysaccharide and incubated at 25°C for 17 hours (see WO2011 / 110531).

[0166] In one embodiment, less than 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 30%, and 50% of bacterial monosaccharide vicinaldiols, for example, 5-10%, are oxidized during periodate activation (see WO2011 / 110531). Lower reaction temperatures are also preferable for lower polysaccharide chain oxidation. In some embodiments, low periodate concentrations (<0.1 equivalents) are combined with an overnight reaction at 4°C to minimize polysaccharide chain oxidation of certain capsular polysaccharides, such as those of Streptococcus pneumoniae 19F.

[0167] In some embodiments, the level of periodate addition and reaction conditions is adjusted to direct cleavage to selective sugar and polysaccharide chains. For example, 1 mM NaIO4 at 4°C has been used in the literature to selectively oxidize sialic acid residues at carbon 7, 8, or 9, while 10 mM NaIO4 at room temperature has been used to oxidize a wide variety of sugar residues, including sialic acid, galactose, and mannose residues.

[0168] For the oxidation of N-terminal serine or methionine residues in protein antigens, milder oxidation conditions (lower periodate concentrations and reaction times) are generally used to avoid oxidative damage to the inner side chains of the antigen. In embodiments, step (b) comprises adding sodium periodate to a final concentration of 2.5 mM, and step (c) comprises incubating the reaction mixture at 25°C for 3 minutes.

[0169] Excessive unreacted periodates can cause higher-than-desirable oxidation levels or damage to the immunogenic moiety in the antigen; therefore, excess periodates are optionally removed in step (d). For large antigens (>10 kDa), excess periodates are removed in some embodiments by size exclusion, dialysis, or diafiltration against water or buffer solution using a medium with a suitable molecular weight exclusion limit. For small antigens (short-chain peptides or oligosaccharides), size-based purification is undesirable, and the removal of periodates in step (d) involves adding a quenching coolant. Excess periodates are optionally quenched by the addition of glycerol (10% (v / v)), molar excess sodium sulfite, or molar excess N-acetylmethionine.

[0170] In some embodiments, polysaccharide or protein antigens are deprotected to increase the proximity of hydroxyl or amine groups to periodate activation. In one embodiment, O-acetyl or N-acetyl groups of polysaccharides are removed to increase the reactivity of adjacent hydroxyls to periodate. For polysaccharide antigens, de-O-acetylation or de-N-acetylation is achieved optionally by culturing in a weakly acidic (e.g., low concentration HCl) or alkaline (e.g., sodium bicarbonate) solution, followed by optional heating and adjustment back to physiological pH. In some embodiments, high molecular weight polysaccharides are partially hydrolyzed ("sizing") using weakly acidic treatment (<0.1 M HCl or <0.2 M AcOH), followed by heating and neutralization. In some embodiments, a weak acid treatment (e.g., <0.1 M HCl or <0.2 M AcOH), followed by heating (45-95°C) and neutralization (to pH 5.5-6.0) is used to simultaneously partially hydrolyze ("size") high molecular weight polysaccharides and deprotect them. In some embodiments, serotypes 3, 4, 18C, and 11A are treated by such acid / heat / neutralization processes to deprotect the polysaccharides, size the polysaccharides, or both. In one embodiment, serotype 3 polysaccharides of Streptococcus pneumoniae are treated with 0.18 M acetic acid followed by heating at 85°C for 1 hour. In one embodiment, serotype 4 polysaccharides of Streptococcus pneumoniae are treated with 0.01 M HCl followed by heating at 45°C for 1 hour. In one embodiment, 18C polysaccharides of Streptococcus pneumoniae are treated with 0.18 M acetic acid followed by heating at 95°C for 40 minutes. In one embodiment, Streptococcus pneumoniae serotype 11A polysaccharide is treated with 0.18 M acetic acid, followed by heating at 80°C for 1 hour.

[0171] In another embodiment, the N-formyl group of the purified protein is removed and the amine group is deformylated by treatment with formyl-L-methionyl peptide amidohydrolase in deionized water or physiological pH buffer. In yet another embodiment, the N-formyl group of the purified protein is removed by treatment of the freeze-dried protein with anhydrous hydrazine vapor at -5°C (Miyataki et al. Eur.J.Biochem.212,785-789(1993)).

[0172] CDAP activation In some embodiments, the antigen is activated by forming a transient adduct with cyano-4-dimethylaminopyridinium tetrafluoroboric acid (CDAP) (see, e.g., WO2011 / 110531 and US2012 / 0321658). In some embodiments, the hydroxyl group of a protein or polysaccharide antigen is activated by reaction with CDAP to form a transient cyanato(-OCN) adduct, which is then reacted with a suitable nucleophile of a chemical handle or linker to form a carbamidate linkage. In this embodiment, the CC linkage of the antigen is not cleaved (as opposed to activation with periodate). In some embodiments, certain capsular polysaccharides are preferentially activated using CDAP. In certain embodiments, the capsular polysaccharides of Streptococcus pneumoniae serotypes 3, 7F, or 10A are activated using CDAP.

[0173] Regarding the activation of the antigen by CDAP, (a) the antigen is dissolved in a suitable solvent, (b) CDAP is added to the antigen from the stock solution, and (c) a buffer is added.

[0174] CDAP activation is optionally carried out in any suitable solvent. In some embodiments, the solvent (a) includes distilled water. In further embodiments, the solvent (a) additionally includes an organic solvent such as DMSO or acetonitrile. In certain embodiments, the capsular polysaccharide of Streptococcus pneumoniae serotype 3, 7F, or 10A is activated in water.

[0175] In some embodiments, hyperstoichiometric (with respect to polysaccharides) amounts of CDAP are used for activation. In some embodiments, about 0.1 to about 3 equivalents of CDAP are used for the activation of polysaccharides. In some embodiments, about 0.2 to 0.8 equivalents of CDAP are used for the activation of polysaccharides. In one embodiment, the capsular polysaccharide of Streptococcus pneumoniae serotype 3 is activated using 2.0 equivalents of CDAP. In one embodiment, the capsular polysaccharide of Streptococcus pneumoniae serotype 10A is activated using 0.8 equivalents of CDAP.

[0176] In some embodiments, the efficiency of CDAP activation is dramatically increased by the addition of the buffer in (c) (Lees et al. Vaccine. 1996(14):190-198). In some embodiments, the buffer in (c) is triethanolamine (TEA). In some embodiments, about 1 to about 4 equivalents of TEA (with respect to polysaccharides) are used as the buffer. In one embodiment, about 1 to about 4 equivalents of TEA are used as a buffer for the CDAP activation reaction with polysaccharides of Streptococcus pneumoniae serotype 7F. In some embodiments, 2.5 equivalents of TEA are used as the buffer. In one embodiment, 2.5 equivalents of TEA are used as a buffer for the CDAP activation reaction with polysaccharides of Streptococcus pneumoniae serotype 7F. In some embodiments, the buffer is sodium borate, sodium carbonate, or sodium hydroxide, or any combination thereof. In some embodiments, the buffer has a pKa of about 8.0 to about 11.0, or the buffer is used to adjust the pH of the reaction solution to about 8.0 to about 11.0. In some embodiments, the buffer has a pKa of about 9.0 to about 9.5, or the buffer is used to adjust the pH of the reaction solution to about 9.0 to about 9.5. In one embodiment, the adjustment of sodium hydroxide to pH 9.5 is used for the CDAP activation reaction with polysaccharides of Streptococcus pneumoniae serotype 3. In one embodiment, the adjustment of sodium hydroxide to pH 9.5 is used for the CDAP activation reaction with polysaccharides of Streptococcus pneumoniae serotype 10A.

[0177] Activation of carbonyldiimidazole (CDI) / carbonylditriazole (CDT) In some embodiments, the antigen is activated by carbonyl diimidazole (CDI) or carbonyl ditriazole (CDT). CDI and CDT, such as CDAP, can activate the hydroxyl group of the antigen to form a transient reactive moiety, in which case it is an unstable carbamate (CDI). [ka] and about CDT [ka] ) is then optionally reacted with an amine or thiol of a chemical handle or linker to form a carbamate or carbonothioate linkage. Activation should be carried out in a dry organic solvent. In some embodiments, CDI / CDT activation is carried out in dimethyl sulfoxide (DMSO). In some embodiments, CDI / CDT activation is carried out by adding a molar excess of CDI / CDT with respect to the antigen. In other embodiments, CDI / CDT activation is carried out by adding a molar amount of CDI / CDT that is approximately equal to the molar amount of the antigen.

[0178] No chemical activation In some embodiments, a given polysaccharide is conjugated to a chemical handle or carrier protein using an endogenous amine or other nucleophilic moiety (e.g., a primary amine) that is either naturally occurring or a result of a deprotection process (e.g., as considered above). Such a nucleophilic moiety can conveniently react with a variety of common electrophilic junctioning reagents (e.g., N-hydroxysuccinimide (NHS) or sulfo-NHS esters), such as succinic acid derivatives. In such embodiments, it may be beneficial to treat the antigen-contaminated portion, such as the C-polysaccharide of Streptococcus pneumoniae, with a periodate protocol, as in (i). In this embodiment, the periodate treatment is followed by treatment with a vast excess of sodium borohydride to quench any chemically introduced aldehyde groups. In one embodiment, the polysaccharides of Streptococcus pneumoniae serotype 1 are treated with approximately 0.05 to 0.25 equivalents of sodium periodate for approximately 12 to 14 hours at room temperature, followed by treatment with approximately 5 to 15 equivalents of sodium borohydride. In another embodiment, the polysaccharides of Streptococcus pneumoniae serotype 1 are treated with 0.15 equivalents of sodium periodate for 18 hours at room temperature, followed by treatment with 10 equivalents of sodium borohydride.

[0179] Bonding to chemical handles In some embodiments, the antigen is conjugated to a chemical handle using any chemical method compatible with the activation methods described above ("Activation of Antigen"). Such methods include, but are not limited to, Schiff base formation with synthetic antigen aldehydes followed by reductive amination, hydrazone formation, oxime formation, direct nucleophilic addition, and Schiff base formation with natural antigen aldehydes followed by reductive amination. In some embodiments, the absolute polysaccharide concentration in the conjugation reaction with the chemical handle is important to minimize polysaccharide aggregation or cross-reactivity. In some embodiments, the absolute polysaccharide / antigen concentration in the conjugation reaction with DBCO (dibenzocyclo-octin) or DBCO derivatives is important for the activation of the polysaccharide by periodate or CDAP. In some embodiments, the polysaccharide concentration in the DBCO / DBCO-derivative conjugation reaction is less than 2, less than 5, less than 7, less than 10, less than 15, less than 17.5, or less than 20 μmol / mL. In some embodiments, the polysaccharide concentration in the DBCO / DBCO-derivative conjugation reaction is approximately 1.5 to approximately 17.5 μmol / mL.

[0180] Reaction with periodate-activated antigen In some embodiments, the chemical handle is conjugated to a polypeptide or polysaccharide antigen that has been activated by periodate as described above ("activated antigen"). In these embodiments, the chemical handle, which contains a functional group that forms a stable or metastable adduct with an aldehyde, is conjugated to the periodate-activated antigen and subsequently conjugated to an optional reduction to convert the metastable adduct to a stable adduct (see, e.g., WO2014 / 111344; Wu et al. Vaccine 31(2013):5623-2626; Hermanson, GT, Bioconjugate Techniques, Second Edition, 2008). In some variations of these embodiments, the chemical handle is added in a large molar excess with respect to the aldehyde group of the activated antigen, so that all of the aldehyde is consumed in the chemical handle / antigen conjugation reaction. In other variations of these embodiments, the chemical handle is added in a lower molar ratio with respect to the aldehyde group of the activating antigen, and the excess unreacted aldehyde of the activating antigen is consumed by further reaction with an excess of an inexpensive aldehyde-reactive nucleophile (e.g., ethanolamine) or by treatment with a reducing agent strong enough to reduce the aldehyde to a hydroxyl group (e.g., NaBH4).

[0181] In one embodiment, the chemical handle is conjugated to the antigen by Schiff base formation with a synthetic antigen aldehyde, followed by reductive amination. This embodiment results in a secondary amine conjugation between the chemical handle and the antigen: a final product having a direct NC bond between the amine of the chemical handle and the carbon atom of the antigen. In this embodiment, the chemical handle contains an amine. In this embodiment, the conjugation method comprises combining a periodate-activated antigen and an amine-containing handle in DI water or buffer containing DMSO, culturing to form a Schiff base, reducing the Schiff base to a secondary amine using sodium cyanoborohydroxide (NaBH3CN), and optionally quenching the unreacted aldehyde with NaBH4. In some embodiments of this method, the chemical handle and antigen are combined in a 1:1 or near 1:1 stoichiometric ratio. In some embodiments of this method, the chemical handle and antigen are combined with a molar excess of the chemical handle. In some embodiments of this method, the chemical handle and antigen are combined with a molar excess of the antigen. In some embodiments, sodium cyanoborohydride is substituted with another reducing agent, such as sodium triacetoxyborohydride, by similar selection for reducing C=N bonds.

[0182] In one embodiment, the chemical handle is conjugated to the antigen via hydrazone formation. In this embodiment, the chemical handle contains a hydrazide (-C(=O)-NH-NH2) group. This embodiment yields a final product having a hydrazone (-C(=O)-NH-N=C-) or N'-alkylhydrazide (-C(=O)-NH-NH-C-) linkage between the chemical handle and the antigen carbon. In this embodiment, the conjugation method includes combining a molar excess hydrazide-containing chemical handle with the antigen in a solution at pH 6.0-8.5 and culturing to form a hydrazone (-C(=O)-NH-N=C-). In some further embodiments of this method, sodium cyanoborohydroxide or sodium triacetoxyborohydride is included in the reaction mixture to reduce the N=C bond, thereby generating an N'-alkylhydrazide (-C(=O)-NH-NH-C-).

[0183] In one embodiment, the chemical handle is conjugated to the antigen by oxime formation. In this embodiment, the chemical handle contains an aminooxy (-O-NH2) group. This embodiment yields a final product having an oxime (-ON=C-) linkage between the chemical handle and the antigen carbon. In this embodiment, the conjugation method comprises combining a molar excess aminooxy-containing chemical handle with the antigen in a solution at pH 6.0 to 8.5 to form an oxime linkage (-ON=C-). In some further embodiments of this method, sodium cyanoborohydride or sodium triacetoxyborohydride is included in the reaction mixture to reduce the N=C bond and improve stability, thereby generating an N'-alkylhydroxylamine linkage (-ONC-).

[0184] Reaction with CDAP-activating antigen In some embodiments, the chemical handle is conjugated to a polypeptide or polysaccharide antigen that has been activated by CDAP as described above ("CDAP activated"). In these embodiments, the transient cyanate (-OCN) group generated via CDAP activation is further reacted with the amine-containing chemical handle to form a carbamidate linkage (-NH-C(=NH)-O-) between the chemical handle and the antigen carbon.

[0185] Regarding the CDAP conjugation of a chemical handle, the hydroxyl group of the antigen is activated as described above ("CDAP activation"), and a chemical handle containing an amine is additionally added to the activation mixture. Since the cyanato group is unstable, the chemical handle is generally added immediately after (within a few minutes) the activation of the antigen. In some embodiments, the antigen is added 2.5 minutes after CDAP is introduced. In some embodiments, a large molar excess of the amine-containing chemical handle is added with respect to the activated hydroxyl group of the antigen. In other embodiments, the chemical handle is added at a concentration closer to a 1:1 molar ratio with respect to the activated hydroxyl group of the antigen, and the excess unreacted cyanato groups are reduced by the addition of an excess of an inexpensive amine (e.g., ethanolamine or hexanediamine).

[0186] Reaction with CDI / CDT-activated antigen In some embodiments, the chemical handle is conjugated to a polypeptide or polysaccharide antigen that has been activated by CDI / CDT as described above ("carbonyldiimidazole (CDI) / carbonylditriazole (CDT) activation"). In these embodiments, the unstable carbamate generated by the CDI / CDT activation of the antigen hydroxyl group (for CDI

Chemical formula

Chemical formula

[0187] Reaction with inactivated antigen In some embodiments, the chemical handle is conjugated to an endogenous amine or other nucleophilic moiety (e.g., a primary amine) derived from a polypeptide or polysaccharide antigen as described above, either naturally occurring or as a result of a deprotection process. In one embodiment, the electrophile group of the chemical handle (e.g., NHS or sulfo-NHS ester) reacts with the primary amine group of the antigen to form an amide linkage (-C(=O)-NH-) between the chemical handle and the antigen amine. In another embodiment, the carboxylic acid group of the chemical handle reacts with the primary amine group in the presence of a standard peptide coupling reagent and conditions to form an amide between the chemical handle and the antigen amine.

[0188] Alkyne-containing handle In some embodiments, the chemical handle includes a moiety that enables a "click" chemical reaction with the corresponding group of the nnAA residue of the polypeptide. One such moiety is an alkyne group that can react with an nnAA group containing an azide group. In the simplest embodiment, this is a propargyl group, and so the alkyne group of the antigen has the structure of formula IV, [ka] During the ceremony, L 22 C1~C 10 It is alkyl, U1 is at least one part of the antigen.

[0189] In other embodiments, the alkyne group of the antigen comprises the structure of formula IVa, [ka] During the ceremony, L 22 is -(CH2CH2O) 1-10 -and, U1 is at least one part of the antigen.

[0190] In some embodiments, the alkyne group further includes additional features that promote or facilitate the reaction with the alkyne's azide group. One example of such a feature is an eight-membered ring structure (e.g., cyclo-octin), so the alkyne group of the antigen further includes a DIFO group or a DBCO group. In some embodiments, the alkyne group of the antigen includes the structure of formula V, formula VI, or VIa. [ka] During the ceremony, L1 independently binds to -NH-, -O-, -S-, and -NH(L 12 )-,-O(L 12 )-, or -S(L 12 )- and, L2 is independent, bonded, -C(=O)-, -S(=O)2-, -C(=O)L 12 -, -S(=O)2L 12 And, L 12 Independently, L 22 or L 22 It is NH-, L 22 Independently, C 1-10 Alkyl or -(CH2CH2O) 1-10 -and, U1 is independently at least one part of the antigen.

[0191] In some embodiments, the structures of formulas V and VIa are conveniently formed from an antigen comprising a nucleophilic group (e.g., a primary amine) and the NHS or sulfo-NHS ester of the corresponding DIFO or DBCO carboxylic acid of structures V and VIa. In some embodiments, the structure of formula VI is conveniently formed from an activating antigen and a DBCO derivative such as DBCO-NH2 or DBCO-PEGn-NH2. In some embodiments, DBCO-PEGn-NH2 is DBCO-PEG4-NH2. [ka]

[0192] In "PEGn", the value of "n" represents, for example, the number of repeating units of oxyethylene in the structure shown above, or in formula VII, formula VIIb, formula XI, or in part "A", or in the poly(alkyloxy) of L 22 and represents the number of repeating units of oxyethylene in the poly(alkyloxy) of L. The value of n is within the range of 1 to 20, for example, within 2 to 18, 3 to 16, or 4 to 14. Thus, n can be any one of, for example, 4, 5, 11, 12, or 13.

[0193] In some embodiments of formula IV, V, or VI, the moiety U1 is at least one polyol of a polysaccharide. In some embodiments, the moiety U1 is at least one polyol of a lipopolysaccharide. In some embodiments, the moiety U1 is at least one amino acid of an antigen polypeptide.

[0194] In a further embodiment, the antigen containing an alkyne comprises the structure of formula VII or VIIa,

Chemical formula

[0195] When a group (e.g., X, Y, or U1) is described as being a polyol, this can refer to a chemical attachment to a polyol within a polysaccharide (e.g., to a monosaccharide within a polysaccharide where the monosaccharide is a polyol). The attachment itself can be an attachment to any suitable functional group (e.g., to an aldehyde resulting from the oxidation of a vicinal diol).

[0196] In a further embodiment, the antigen containing an alkyne comprises the structure of formula VIIb or VIIc, <(

Chemical formula

[0197] In some embodiments, the antigen containing alkynes is (AX) z -Contains polysaccharides by Y, in the formula, A is [ka] And, X is independently at least one polyol, Y is independently at least one polyol of a polysaccharide, n is at least 1, z is greater than 1.

[0198] In some embodiments, the antigen comprises a polysaccharide further comprising DBCO groups, comprising a polysaccharide containing at least 1.5%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (w / w) of covalently attached DBCO. In some embodiments, the antigen contains more than about 1.5% (w / w) of DBCO. In some embodiments, the antigen contains more than 3% (w / w) of DBCO. In some embodiments, the antigen contains at most 20%, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3.5%, at most 3.0%, at most 2.5%, at most 2.0%, or at most about 1.7% (w / w) of covalently attached DBCO. In some embodiments, the antigen contains less than 20% (w / w) of covalently attached DBCO. In other embodiments, the antigen contains less than 10% (w / w) of covalently attached DBCO. In some embodiments, the antigen contains covalently attached DBCO in amounts of approximately 1.5–20%, 3–20%, 3–18%, 3–16%, 3–14%, 3–12%, 3–10%, 3–8%, 3–6%, or 3–4%, or 1.5–9% (w / w).

[0199] In some embodiments, the antigen comprises a polysaccharide further comprising DBCO groups, wherein each 100 repeating units of the polysaccharide contains at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% DBCO molecules. In some embodiments, the antigen contains more than 3% DBCO molecules per 100 repeating units of the polysaccharide. In some embodiments, the antigen contains at most 20%, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, or at most 3.5% covalently attached DBCO molecules per 100 polysaccharide repeating units. In some embodiments, the antigen contains less than 20% covalently attached DBCO per polysaccharide repeating unit. In other embodiments, the antigen contains less than 10% covalently attached DBCO molecules per 100 polysaccharide repeating units. In some embodiments, the antigen contains covalently attached DBCO molecules at a rate of approximately 3%–20%, 3%–18%, 3%–16%, 3%–14%, 3%–12%, 3%–10%, 3%–8%, 3%–6%, or 3%–4% per 100 repeating polysaccharide units.

[0200] In embodiments, the polysaccharide-containing antigen is optionally an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5 to 15 repeating units) and are typically obtained synthetically or by hydrolysis of higher molecular weight polysaccharides.

[0201] In one embodiment, the antigen containing the polysaccharide has a molecular weight of approximately 10 kDa to approximately 10,000 kDa. In another such embodiment, the polysaccharide has a molecular weight of 50 kDa to 10,000 kDa. In further such embodiments, the polysaccharides are 50kDa~10,000kDa, 50kDa~9,500kDa, 50kDa~9,000kDa, 50kDa~8,500kDa, 50kDa~8,000kDa, 50kDa~7,500kDa, 50kDa~7,000kDa, 50kDa~6,500kDa, 50kDa~6,000kDa, 50kDa~5,500kDa, 50kDa~5,000kDa, 50kDa~4,500kDa, 50kDa~4,000kDa, 50kDa~3,500 kDa, 50kDa~3,000kDa, 50kDa~2,500kDa, 50kDa~2,000kDa, 50kDa~1,750kDa, 50kDa~1,500kDa, 50kDa~1,250kDa, 50kDa~1,000kDa, 50kDa ~750kDa, 50kDa~500kDa, 100kDa~10,000kDa, 100kDa~9,500kDa, 100kDa~9,000kDa, 100kDa~8,500kDa, 100kDa~8,000kDa, 100kDa~7,500k Da, 100kDa~7,000kDa, 100kDa~6,500kDa, 100kDa~6,000kDa, 100kDa~5,500kDa, 100kDa~5,000kDa, 100kDa~4,500kDa, 100kDa~4,000kDa , 100kDa~3,500kDa, 100kDa~3,000kDa, 100kDa~2,500kDa, 100kDa~2,000kDa, 100kDa~2,000kDa, 100kDa~1,750kDa, 100kDa~1,500kDa, 1 00kDa~1,250kDa, 100kDa~1,000kDa, 100kDa~750kDa, 100kDa~500kDa, 200kDa~10,000kDa, 200kDa~9,500kDa, 200kDa~9,000kDa, 200kDa ~8,500kDa, 200kDa~8,000kDa, 200kDa~7,500kDa, 200kDa~7,000kDa, 200kDa~6,500kDa, 200kDa~6,000kDa, 200kDa~5,500kDa, 200kDa~5,It has a molecular weight of 000kDa, 200kDa~4,500kDa, 200kDa~4,000kDa, 200kDa~3,500kDa, 200kDa~3,000kDa, 200kDa~2,500kDa, 200kDa~2,000kDa, 200kDa~2,000kDa, 200kDa~1,750kDa, 200kDa~1,500kDa, 200kDa~1,250kDa, 200kDa~1,000kDa, 200kDa~750kDa, or 200kDa~500kDa. Any integer within any of the above ranges is considered an embodiment of this disclosure.

[0202] In the embodiment, the antigen containing polysaccharides has a molecular weight of approximately 50 kDa to approximately 1,400 kDa. In the embodiment, the antigen containing polysaccharides has a molecular weight of approximately 500 kDa to approximately 3,000 kDa.

[0203] Azide-containing handle In some embodiments, the chemical handle includes a moiety that enables a "click" chemical reaction with the corresponding group of the nnAA residue of the polypeptide. One such moiety is an azide group that can react with the nnAA residue containing an alkyne group or phosphine of the polypeptide. In some embodiments, the azide group of the antigen includes the structure of formula VIII, [ka] L 22 is a bond, alkyl, or poly(alkyloxy), U1 is independently at least one part of the antigen.

[0204] Alken-containing handle In some embodiments, the chemical handle includes a moiety that enables a "click" chemical reaction with the corresponding group of the nnAA residue of the polypeptide. One such moiety is an alkene group that can react with the nnAA residue containing a 1,2,4,5-tetrazine group. In the simplest embodiment, this is a vinyl group. In one such embodiment, the alkene group of the antigen has the structure of formula IX, [ka] During the ceremony, U1 is independently at least one part of the antigen.

[0205] In other embodiments, the alkene group of the antigen comprises the structure of formula IXa, [ka] During the ceremony, L 22 C 1-10 Alkyl or -(CH2CH2O) 1-10 -and, U1 is independently at least one part of the antigen.

[0206] In one embodiment, the present disclosure provides a method for producing a complex carbohydrate, comprising: (a) providing a nucleic acid encoding a carrier protein and comprising a repression codon; (b) preparing a reaction mixture by combining the nucleic acid with a cell-free bacterial extract comprising 4-azidomethylphenylalanine (pAMF), a tRNA complementary to the repression codon, and aminoacyl-tRNA synthetase; (c) culturing the reaction mixture from (b) under conditions sufficient to selectively incorporate the pAMF into a site corresponding to the repression codon in the carrier protein; and (d) conjugating the pAMF to a polysaccharide by [2+3] cyclization. In another embodiment, the [2+3] cyclization comprises a reaction between an azide and an alkyne group. In another embodiment, step (c) comprises culturing the reaction mixture at a temperature below 20 degrees Celsius. In another embodiment, the method further comprises purifying the carrier protein immediately after (c). In another embodiment, the repressor codon is selectively substituted with codons 25, 34, 38, 40, 213, 215, 228, 245, 265, 386, 523, or 527 of SEQ ID NO: 2. In another embodiment, the reaction mixture of (b) further comprises biocomponents necessary for protein synthesis. In another embodiment, the tRNA of (b) can be charged with pAMF. In another embodiment, the aminoacyl-tRNA synthetase of (b) preferentially aminoacylates the tRNA with pAMF compared to 20 native amino acids. In another embodiment, the alkyne group comprises a DBCO moiety conjugated to a polysaccharide. In another embodiment, the polysaccharide is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, or any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).In another embodiment, the disclosure provides a complex carbohydrate prepared by a process comprising steps (a) to (d). In another embodiment, pAMF is conjugated to a polysaccharide to generate a conjugate of formula X, Xa, XI, or XIa. In one embodiment, the disclosure provides a vaccine comprising a complex carbohydrate prepared by steps (a) to (d).

[0207] polypeptide antigen zygote This specification describes polypeptide-antigen conjugates that can be formed between an immunogenic polypeptide as described above and an antigen as described above. In some embodiments, the polypeptide-antigen conjugate comprises an enhanced carrier protein and an antigen, where the antigen is linked to nnAAs in the enhanced carrier protein. In one embodiment, the antigen is not linked to any native amino acids of the immunogenic polypeptide. In another embodiment, the antigen is not linked to any lysine in the immunogenic polypeptide. For example, the antigen is not linked to the lysine of SEQ ID NO: 1. In yet another embodiment, the antigen is linked to only one or more nnAAs of the immunogenic polypeptide. The one or more nnAAs are optionally located at the N-terminus, C-terminus, or anywhere between the N-terminus and C-terminus of the immunogenic polypeptide. In some cases, the antigen is linked to only one or more pAMFs in the immunogenic polypeptide. For example, the antigen is linked to only one or more pAMFs of SEQ ID NO: 1.

[0208] In another embodiment, at least one antigen is linked to an amino acid located outside the T cell epitope of the immunogenic polypeptide. In yet another embodiment, the antigen is not linked to an amino acid located inside the T cell epitope of the immunogenic polypeptide.

[0209] The amino acids selected for conjugation within the immunogenic polypeptide optionally include one or more surface-accessible residues based on the polypeptide's crystalline structure (or other 3D structure such as NMR structure). Additionally or alternatively, to evaluate the efficacy of polypeptide-specific sites for conjugation, a comprehensive exchange of native amino acids with nnAAs is performed in the immunogenic polypeptide, followed by conjugation.

[0210] In one embodiment, the antigen is indirectly conjugated to the enhanced carrier protein (for example, by first combining the enhanced carrier protein or antigen with a reactive linker, and then combining the enhanced carrier protein-linker or antigen-linker adduct with the antigen or enhanced carrier protein, respectively). In another embodiment, the antigen is directly conjugated to the enhanced carrier protein (for example, by combining two components, the enhanced carrier protein and the antigen, together in a single reaction). If the conjugate contains a linker, any suitable group can be used. For example, the conjugate may contain a linker selected from adipic acid, adipic acid dihydrazide (ADH), β-propionamide, nitrophenyl-ethylamine, haloacyl halides, glycoside linkers, 6-aminocaproic acid, N-succinimidyl-3-(2-pyridyldithio)-propionic acid (SPDP), C4-C12 moieties, etc. For example, linkers derived from the DBCO and DIFO groups discussed above, including residues of the diarylcyclooctin moiety such as diarylcyclooctene, may also be used. The linker will generally be attached to the antigen for conjugation rather than to a carrier.

[0211] Because antigen-polypeptide conjugates can form from large cross-linking complexes, it may not be possible to directly measure or determine the precise location and other physical characteristics of some or all conjugates by available analytical methods. However, it is understood that such locations or physical characteristics can be reliably inferred from the synthetic scheme that is consistent with its expectations, its expected product, and the design of the analytical results.

[0212] Antigen-polypeptide conjugation reaction In some embodiments, the antigen is conjugated to a carrier protein enhanced using any chemical method suitable for conjugating non-natural amino acids and chemical handles as described herein. Such methods include, but are not limited to, copper(I)-catalyzed alkyne-azide cyclization (CuAAC), strain-enhanced azide-alkyne cyclization (SPAAC), and tetrazine-alkene ligation. All of these reactions can be carried out in aqueous solution as "click" reactions. Staudinger ligation between phosphine and azide can also be used.

[0213] CuAAC: In some embodiments, the antigen is conjugated to a carrier protein enhanced by copper(I)-catalyzed alkyne-azide cyclization (CuAAC). In one variation of this embodiment, the enhanced carrier protein contains propargyl-containing nnAA and the antigen contains an azide group. In another variation of this embodiment, the enhanced carrier protein contains azide-containing nnAA and the antigen contains a propargyl group. Preferred conditions for CuAAC conjugation of biomolecules are found, for example, in Presolski et al. Curr Protoc Chem Biol. 2011;3(4):153-162, all of which involve the addition of Cu2+. In some embodiments, the reaction is accelerated by the addition of a Cu-coordinating ligand such as THPA. In some embodiments, the reaction is accelerated by the addition of a reducing agent to maintain the oxidized state of Cu2+. Preferred reducing agents include sodium ascorbate, DTT, or TCEP.

[0214] SPAAC: In some embodiments, the antigen is conjugated to a carrier protein enhanced by strain-enhanced azide-alkyne cyclization (SPAAC). In one variation of these embodiments, the enhanced carrier protein contains azide-containing nnAA and the antigen contains a cyclooctin group. In another variation of these embodiments, the enhanced carrier protein contains cyclooctin-containing nnAA and the antigen contains an azide group. Since SPAAC does not require additional catalysts or cofactors, this reaction can be carried out in distilled water, 0.9% saline, PBS, or physiological buffer. In one embodiment, the enhanced carrier protein and antigen are combined in a mass ratio of 1.20:1 (w / w).

[0215] In some embodiments, the antigen is linked to an azide-containing nnAA in a carrier protein enhanced via the structure of formula X or Xa, [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the enhanced carrier protein. R2 is independently at least one amino acid of the OH or enhanced carrier protein. D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-, Ar is [ka] And, Each of W1, W2, and W3 is independently a single bond or lower alkylene. Each X1 is independently -NH-, -O-, or -S-. Each Y1 is independently a single bond, -NH- or -O-. Each Y2 is independently a single bond, -NH-, -O-, or N-linked or C-linked pyrrolidine ylene. One of Z1, Z2, and Z3 is -N-, and the others of Z1, Z2, and Z3 are independently -CH-. L22 is independently bonded, alkyl, or poly(alkyloxy). X is at least one polyol of a polysaccharide.

[0216] In some embodiments, the antigen is linked to an azide-containing nnAA in a carrier protein enhanced via a structure of formula XI or XIa, [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the enhanced carrier protein. R2 is independently at least one amino acid of the OH or enhanced carrier protein. W is either C or N, y is at least 1, n is at least 1, X is independently at least one polyol of the capsular polysaccharide.

[0217] The value of "n" is considered above in relation to "PEGn". The value of "y" is in the range of 1 to 10 according to formula XII, and is preferably a lower alkylene, such as C1 to C4 alkylene.

[0218] Tetrazine-alkyne ligation In some embodiments, the antigen is conjugated to a carrier protein enhanced by tetrazine-alkyne ligation. In one variation of these embodiments, the enhanced carrier protein contains 1,2,4,5-tetrazine-containing nnAA, and the antigen contains an alkene group. Similar to the SPAAC reaction, the tetrazine-alkyne ligation proceeds without the addition of cofactors, and this and the reaction can be carried out in distilled water, 0.9% saline, PBS, or physiological buffer.

[0219] Characterization of the joint Following the conjugation reaction, the carrier protein conjugate, enhanced with the target antigen, is optionally purified by methods including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, and size exclusion), molecular size exclusion (dialysis, diafiltration, tangential flow filtration, deep filtration), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or SDS-PAGE (see, e.g., Protein Purification, JC Janson & Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain a substantially pure conjugate.

[0220] The target conjugation proteins are optionally quantified by methods including, but not limited to, microfluidic electrophoresis, gel electrophoresis, Western blotting, immunoassays (e.g., ELISA), and other assays to evaluate the activity of the conjugation proteins.

[0221] Exemplary physical parameters One important parameter for antigen-enhanced carrier-protein conjugates is the molecular weight of the conjugate. Because conjugates contain a variable number of antigen molecules conjugated to each protein molecule in an optional manner, as well as variable higher-order crosslinks (e.g., protein-antigen-protein linkages), the output molecular weight of the conjugate is not necessarily predictable from the input molecular weights of the enhanced carrier protein and antigen. A vast amount of literature (e.g., Howard et al. Immunology. 1971(21):535-545 and Kabat & Bezer. Arch Biochem Biophys. 1958(78)306-18) suggests that antigen particle size has a significant effect on immunogenicity. Wessels et al. (1998) Infect Immun 66:2186-92 reported that conjugate size and crosslinking can affect the immunogenicity and protective efficacy of type III conjugates of GBS.

[0222] In general terms, a conjugate can be formed by linking a carrier protein to an antigen having one or more handles per antigen. A crosslinked conjugate can be formed with protein-antigen-protein linkages, having multiple handles per antigen. This type of conjugate lattice, having a single handle per antigen (e.g., a terminal group in a polysaccharide), is not formed because a single antigen cannot bind to multiple carrier protein molecules. Crosslinked conjugates are preferred herein (particularly for Streptococcus pneumoniae) where a higher molecular weight is desired and therefore antigens with multiple handles are preferred.

[0223] In some embodiments, the antigen-enhanced carrier protein conjugates have molecular weights of approximately 750 kDa, 1,000 kDa, 1,500 kDa, 2,000 kDa, 2,500 kDa, 3,000 kDa, 3,500 kDa, 4,000 kDa, 4,500 kDa, 5,000 kDa, 5,500 kDa, 6,000 kDa, 6,500 kDa, 7,000 kDa, 7,500 kDa, or 8,000 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 750 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 800 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 850 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 900 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 950 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of approximately 1,000 kDa to approximately 2,800 kDa.

[0224] Another important parameter for the conjugate vaccines of this disclosure is the ratio of antigen (e.g., polysaccharide) to immunogenic polypeptide carrier (e.g., carrier protein of this disclosure). Using the polysaccharide-carrier protein conjugate as an example of a general principle, the polysaccharide-to-protein (PS:PC) ratio of the purified conjugate is generally expressed in terms of weight-to-weight (w / w) ratio. Such a ratio is conventionally expressed as including any purified free polysaccharides together with the individual complex carbohydrates. A higher PS:PC ratio of the polysaccharide-carrier protein conjugate allows for the delivery of more polysaccharide antigens together with a lower amount of enhanced carrier protein. For pneumococcal conjugate vaccines, this ratio is typically in the range of 0.3 to 3.0, but this can vary depending on the serotype and the nature of the conjugate chemistry (Appendix 2: Recommendations for the production and control of pneumococcal conjugate vaccines; WHO Technical Report Series, No. 927, 2005). The ratio of the commercially available vaccine Prevnar-13 is 0.9 (see Prevnar 13 Package Insert, M / 2016 Revision, pg.24; www.fda.gov / downloads / biologicsbloodvaccines / vaccines / approvedproducts / ucm201669.pdf), suggesting a preferred range of 1.0 to 3.0. When formulating vaccines with more than 13 serotypes, it may be preferable to achieve a ratio of 1.5 to 3.0, and a ratio of approximately 1.5 to approximately 2.0 may be particularly preferable. This may be the average ratio for all conjugates in the composition, which can be achieved by ensuring that all individual conjugates have this ratio, or by ensuring that any conjugates outside this range on one side are balanced by conjugates outside this range on the opposite side.

[0225] In another embodiment, the ratio (in units of weight) of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate is 0.5 to 4.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0). In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate is 0.7 to 2.8. In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate is 1.0 to 2.8. In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate is at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, or at least 1.5. In another embodiment, the ratio of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate is greater than 0.9 (w / w). In another embodiment, the ratio of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate is about 0.9 to about 3.0 (w / w). Mixing individual mixtures with such PS:PC ratios may result in combinations having the desired overall PS:PC ratio.

[0226] Presence of Contaminants (Free Polysaccharides, C-Polysaccharides) An important parameter for polysaccharide-enhanced carrier protein conjugates is the level of free polysaccharides present in the conjugate composition, even though they are not covalently bound to the enhanced carrier protein. For example, in certain cases, free polysaccharides are non-covalently associated with the polysaccharide-enhanced carrier protein conjugate (i.e., non-covalently bound and adsorbed to it, or incorporated within or with it). In some embodiments, the polysaccharide-enhanced carrier protein conjugates described herein contain free polysaccharides at about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or less than 10% of the total amount of polysaccharides. In another embodiment, the polysaccharide-enhanced carrier protein described herein contains free polysaccharides at about 10% of the total amount of polysaccharides. In yet another embodiment, the polysaccharide-enhanced carrier protein described herein contains free polysaccharides at about 25% of the total amount of polysaccharides. In another embodiment, the polysaccharide-enhanced carrier protein described herein contains less than 30% free polysaccharides relative to the total amount of polysaccharides. In another embodiment, the polysaccharide-enhanced carrier protein described herein contains less than 15% free polysaccharides relative to the total amount of polysaccharides. Free polysaccharides are optionally measured by any preferred method, including the method of Lei et al. (Dev Biol (Basel). 2000:103:259-64), which uses an HCl / deoxycholic acid-based precipitation method to distinguish the pool of polysaccharides. In a preferred composition, the amount of unconjugated bacterial polysaccharides is less than 5% by weight of the total amount of bacterial polysaccharides in the composition. In a composition containing multiple pneumococcal conjugates, the amount of unconjugated bacterial polysaccharides for each serotype is preferably less than 5% by weight of the total amount of bacterial polysaccharides for that serotype in the composition.

[0227] A key parameter for pneumococcal capsular polysaccharide-enhanced carrier protein conjugates is the level of C-polysaccharide contaminants present in the conjugate preparation. C-polysaccharides are immunologically unproductive but highly immunogenic cell wall components of Streptococcus pneumoniae, "moving along" in many pneumococcal capsular polysaccharide preparation methods. Since the immune response to C-polysaccharides generally does not produce neutralizing antibodies, C-polysaccharide contamination can interfere with the proper evaluation of the efficacy of antigen-enhanced carrier protein conjugates when administered to animals.

[0228] The level of C-polysaccharides is optionally indicated by total acid hydrolysis of the polysaccharide conjugate preparation, chromatography of the hydrolysate, and detection of the electrical conductivity of choline. Alternatively, non-hydrolyzed polysaccharides are analyzed by NMR for choline. NMR techniques use the ratio of the choline signal to the rhamnose-methyl signal (for rhamnose-containing capsular polysaccharides; different signals for other capsular polysaccharides) to calculate the C-polysaccharide content. Chromatographic methods use the polysaccharide content determined by an electrical conductivity assay to calculate the C-polysaccharide content, or the ratio of the choline signal to one of the component peaks of the capsular polysaccharide. In either method, using the theoretical repeating structure of C-polysaccharides [Hermans, et al., Recl. Trav. Chim. Pays-Bas, 107, 600 (1988)] based on a known concentration of choline, once the choline concentration is known and the concentration of C-polysaccharides in the polysaccharide preparation is known, it becomes possible to directly calculate the level of choline present in the polysaccharide preparation.

[0229] The polysaccharide concentration in polysaccharide-enhanced carrier protein conjugate samples is optionally measured by various techniques. For example, the total polysaccharide concentration is optionally determined by total polysaccharide hydrolysis and subtotal measurement of specific monosaccharide concentrations. The degree of C-polysaccharide contamination (w / w) is determined by comparing the C-polysaccharide concentration with the total polysaccharide concentration. A C-polysaccharide level of less than 3% (w / w) of total polysaccharides is considered acceptable. In some embodiments, the C-polysaccharide level is less than 1%.

[0230] In one embodiment, the disclosure provides a conjugate comprising a carrier protein and an antigen, wherein the antigen is linked to nnAA in the carrier protein. In another embodiment, the carrier protein holds diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (PD), serogroup B meningococcal (OMPC) outer membrane protein complex, or a T cell-binding epitope of CRM197. In another embodiment, nnAA is 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, 2-amino-5-azidopentaic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof. In another embodiment, nnAA is not a T-cell activation epitope of the carrier protein. In another embodiment, nnAA is substituted for one or more lysine residues in the carrier protein. In another embodiment, the apparent molecular weight of the conjugate is about 900 kDa to about 5 MDa. In another embodiment, one or more lysine residues to be substituted are selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K265, K386, K523, and K527 of SEQ ID NO: 1, and any combination thereof. In another embodiment, nnAA is not a T cell activation epitope of the carrier protein. In another embodiment, the antigen is ligated to the carrier protein by formula XI or XIa, [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the carrier protein. R2 is independently an OH group, or at least one amino acid of the carrier protein. W is either C or N, y is at least 1, n is at least 1, X is independently at least one polyol of the capsular polysaccharide.

[0231] In another embodiment, the antigen is a polysaccharide. In another embodiment, the polysaccharide is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).

[0232] In one embodiment, the disclosure provides a method for identifying the optimal placement of an antigen on a carrier protein to improve the host immune response, comprising: i) introducing an nnAA substitution into a carrier protein; ii) conjugating a polysaccharide to the nnAA to form a complex carbohydrate; and iii) measuring the apparent molecular weight of the complex carbohydrate. In another embodiment, the nnAA substitution is not present in the T cell activation epitope of the carrier protein. In another embodiment, the carrier protein holds the T cell binding epitope of diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (PD), serogroup B meningococcus (OMPC) outer membrane protein complex, or CRM197. In another embodiment, the antigen is a polysaccharide. In another embodiment, at least one polysaccharide is conjugated to the carrier protein according to formula XI or XIa. [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the carrier protein. R2 is independently an OH group, or at least one amino acid of the carrier protein. X is independently at least one polyol of the capsular polysaccharide.

[0233] In another embodiment, the at least one non-natural amino acid to be substituted is a pAMF. In another embodiment, the present disclosure provides a carrier protein having an optimal arrangement of the antigen identified by the processes of i) to iii). In another embodiment, the substitution is introduced at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine times. In another embodiment, the polysaccharide is a bacterial capsular polysaccharide. In another embodiment, the bacterium is Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the bacterium is Streptococcus pneumoniae. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).

[0234] Modified polypeptides and polysaccharides In one embodiment, the present disclosure relates to a modified polypeptide comprising at least one compound comprising formula XI or XIa, or a salt thereof, [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the carrier protein. R2 is independently an OH group, or at least one amino acid of the carrier protein. X independently provides a modified polypeptide which is at least one polyol of a polysaccharide.

[0235] In another embodiment, the carrier protein holds diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), or the T cell binding epitope of CRM197. In another embodiment, the polysaccharide is the capsular polysaccharide of the bacterial species. In another embodiment, the bacterial species is Streptococcus pneumoniae. In yet another embodiment, the bacterial species is Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, R1 and R2 are not amino acids that occur in the T cell epitopes of the carrier protein. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).

[0236] In one embodiment, the present disclosure relates to a modified polysaccharide comprising at least one compound comprising formula VII or VIIa, or a salt thereof, [ka] During the ceremony, X is independently at least one polyol of the capsule polysaccharide, The present invention provides a modified polysaccharide in which n is at least 1.

[0237] In another embodiment, the modified polysaccharide of formula VII is further conjugated to a carrier protein containing at least one nnAA. In another embodiment, the modified polysaccharide is conjugated by [2+3]-loading cyclization. In another embodiment, the polysaccharide is derived from a bacterial species. In another embodiment, the bacterial species is Streptococcus pneumoniae. In another embodiment, the bacterial species is Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the polysaccharide is a bacterial capsular polysaccharide. In another embodiment, the molar ratio of DBCO to the repeating units of the capsular polysaccharide is greater than 1. In another embodiment, the capsular polysaccharide is a Streptococcus pneumoniae serotype comprising 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6).

[0238] In one embodiment, this disclosure is (AX) z - Provides modified polysaccharides with Y, During the ceremony, A is [ka] And, X is independently at least one polyol, Y is independently at least one polyol of a polysaccharide, n is at least 1, z is greater than 1.

[0239] In another embodiment, the polysaccharide is derived from a bacterial species. In another embodiment, the bacterial species is Streptococcus pneumoniae. In another embodiment, the bacterial species is Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the polysaccharide is a bacterial capsular polysaccharide. In another embodiment, the capsular polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6). In another embodiment, the polysaccharide is further conjugated to a carrier protein. In another embodiment, the polysaccharide is conjugated to the carrier protein via a linkage of formula II. In another embodiment, the carrier protein holds the T cell binding epitope of CRM197. In another embodiment, the polysaccharide is conjugated by [2+3]-loading cyclization. In another embodiment, the carrier protein contains one or more non-natural amino acids. In another embodiment, the carrier protein holds diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), or the T cell binding epitope of CRM197. In another embodiment, the carrier protein is further conjugated to the antigen. In another embodiment, the antigen is conjugated via a carrier protein, ligated by formula II. In yet another embodiment, the ratio (w / w) of polysaccharide to carrier protein (PS:PC) is approximately 1.5 to approximately 4.

[0240] Polypeptide antigen conjugate composition This disclosure describes an immunogenic composition comprising at least one enhanced carrier protein-antigen conjugate together with at least one excipient, wherein the antigen is conjugated to a polypeptide via an nnAA residue in the enhanced carrier protein. In one embodiment, this disclosure provides a vaccine composition comprising the complex carbohydrate described herein. In some embodiments, a conjugate vaccine composition comprising at least one enhanced carrier protein-antigen conjugate as described herein induces a reduction in carrier suppression in the subject compared with a conjugate vaccine composition comprising a native carrier protein. In some embodiments, a conjugate vaccine composition comprising at least one enhanced carrier protein-antigen conjugate as described herein improves the overall immune response and / or increases the T cell-dependent response in the subject compared with a conjugate vaccine composition comprising a native carrier protein.

[0241] In some embodiments, the immunogenic composition comprises a single carrier-protein-antigen conjugate (e.g., a single serotype of Streptococcus pneumoniae). In some embodiments, the immunogenic composition comprises multiple carrier-protein-antigen conjugates (e.g., multiple serotypes of Streptococcus pneumoniae). In further embodiments, the multiple carrier-protein-antigen conjugates optionally comprise (a) multiple antigens conjugated to a common enhanced carrier protein, or (b) multiple antigens conjugated to different enhanced carrier proteins. In further embodiments, the multiple enhanced carrier-protein-antigen conjugates comprise antigens derived from different serotypes of the same microorganism (e.g., Streptococcus pneumoniae). When the composition comprises multiple different antigens (e.g., capsular polysaccharides derived from multiple serotypes of Streptococcus pneumoniae, or capsular polysaccharides derived from multiple serogroups of Neisseria meningitidis), it is preferable that the same type of carrier protein is used for each antigen, for example, each antigen is individually conjugated to the same nnAA-containing CRM197 variant, and then the individual antigen-protein conjugates are combined to give a multi-antigen composition.

[0242] In some embodiments, the overall (weight) ratio of all serotype polysaccharides to carrier proteins (PS:PC) in the polyvalent serotype polysaccharide conjugate composition is within a specific range. In another embodiment, the ratio (in units of weight) of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate (or in the overall polyvalent composition) is 0.5 to 4.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0). In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate (or in the overall polyvalent composition) is 0.7 to 2.8. In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate (or in the overall polyvalent composition) is 1.0 to 2.8. In another embodiment, the (w / w) PS:PC ratio in the carrier protein conjugate (or in the overall polyvalent composition) is at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, or at least 1.5 (w / w). In another embodiment, the ratio of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate (or in the overall polyvalent composition) is greater than 0.9 (w / w). In another embodiment, the ratio of polysaccharides to enhanced carrier protein in the polysaccharide-enhanced carrier protein conjugate (or in the overall polyvalent composition) is about 0.9 to about 3.0 (w / w). A preferred composition includes protein-monosaccharide conjugates of capsular polysaccharides derived from multiple serotypes of Streptococcus pneumoniae, having an overall excess of polysaccharides relative to the protein, for example, a protein:polysaccharide ratio of 1:1.1 to 1:2 (w / w), for example, 1:1.5 to 1:1.9.

[0243] In some embodiments, the overall molecular weight range of all serotype polysaccharide-carrier protein conjugates in a polyvalent serotype polysaccharide-carrier protein conjugate composition is within a specific range. In some embodiments, the antigen-enhanced carrier protein conjugates have molecular weights of approximately 750 kDa, approximately 1,000 kDa, approximately 1,500 kDa, approximately 2,000 kDa, approximately 2,500 kDa, approximately 3,000 kDa, approximately 3,500 kDa, approximately 4,000 kDa, approximately 4,500 kDa, approximately 5,000 kDa, approximately 5,500 kDa, approximately 6,000 kDa, approximately 6,500 kDa, approximately 7,000 kDa, approximately 7,500 kDa, or approximately 8,000 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 750 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 800 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 850 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 900 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of about 950 kDa to about 2,800 kDa. In some embodiments, the antigen-enhanced carrier protein conjugate has a molecular weight of approximately 1,000 kDa to approximately 2,800 kDa.

[0244] In a further embodiment, the immunogenic composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 distinct enhanced carrier protein-antigen conjugates.

[0245] In any composition comprising multiple conjugates (e.g., conjugates for each of several pneumococcal serotypes), in some examples, it may be preferable that the carrier proteins in each conjugate be identical. In alternative embodiments of such compositions having multiple conjugates, it may be preferable to use more than one carrier. While each antigen (e.g., a capsular polysaccharide derived from a different pneumococcal serotype) can be conjugated to a different carrier, typically there will be only 2 to 4 (e.g., 2 or 3) different carriers represented in the individual conjugates in such a composition. In the exemplary method, but not in an exemplary method, in a composition of 24 different conjugates, each conjugate comprising a capsular polysaccharide derived from a different pneumococcal serotype, some but not all of the 24 conjugates comprise a first carrier protein (e.g., based on CRM197), and the remaining 24 conjugates comprise a second protein carrier (e.g., based on HiD). Therefore, again by the illustrative method, and not by the limiting method, 12, 13, 15, or 20 of the 24 conjugates may contain the first carrier protein, and the remaining 12, 11, 9, or 4 conjugates may contain the second carrier protein, respectively.

[0246] In some embodiments, at least one excipient contains an ingredient suitable for parenteral administration.

[0247] In further embodiments, at least one excipient optionally comprises a buffer or pH adjuster. In specific embodiments, the buffer or pH adjuster is selected from the group consisting of sodium borate, sodium phosphate, sodium citrate, ammonium sulfate, or succinate esters, and any combination thereof. Other examples of suitable buffers include acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate / glucose, sodium bicarbonate, and ammonium chloride. Histidine buffers are also useful in immunogenic compositions.

[0248] In further embodiments, at least one excipient optionally includes an isotonic agent to bring the osmolality of the composition to an acceptable range. In certain embodiments, the isotonic agent is selected from the group consisting of sodium chloride, glucose, and glycerin, and any combination thereof. Other examples of buffers suitable for parenteral administration include salts having sodium, potassium, or ammonium cations and chlorides, and anions of citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfuric acid, thiosulfate, or bisulfite, with preferred salts including potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0249] In further embodiments, at least one excipient optionally comprises a surfactant. In certain embodiments, the surfactant is polyoxyethylene sorbitan monolaurate (Polysorbate 20 or "Tween 20"), polyoxyethylene sorbitan monooleate (Polysorbate 80 or "Tween 80"), Brij 35, Triton X-10, Pluronic F127, or sodium dodecyl sulfate (SDS). In some embodiments, the surfactant is present at a concentration of 0.0003% to 0.3% (w / w).

[0250] In some embodiments, at least one excipient optionally includes an adjuvant, which is an agent that increases the stimulation of the immune system by enhancing antigen presentation (depot formulation, delivery system) and / or by providing a co-stimulatory signal (immunostimulant). In some variations of this embodiment, the adjuvant is aluminum salt-based. In certain embodiments, the adjuvant is aluminum potassium phosphate, aluminum hydroxyphosphate, aluminum hydroxide, or aluminum phosphate, and any combination thereof. In other variations, the adjuvant is an oil-in-water emulsion. In certain embodiments, the adjuvant is AS03, MF59, or AF03, and any combination thereof. In yet another variation, the adjuvant is a TLR4 agonist. In certain embodiments, the adjuvant is RC529. Preferred adjuvants for use in the present invention are aluminum salts such as aluminum phosphate adjuvants (e.g., aluminum hydroxyphosphate adjuvants). When the composition contains an aluminum salt adjuvant, the Al in the composition 3+ The concentration of is preferably ≤1.25 mg per dose, for example ≤1.25 mg per 0.5 ml, and ideally ≤0.85 mg per dose. The conjugate in the composition can be adsorbed onto the aluminum salt adjuvant. For the mixed composition, the conjugate can be individually adsorbed onto the aluminum salt and then mixed, or added to the aluminum salt to achieve sequential adsorption, thereby forming a mixed conjugate composition.

[0251] Preferred compositions include (i) one or more conjugates as defined herein, for example, capsular polysaccharides derived from multiple serotypes of Streptococcus pneumoniae conjugated to an nnAA-containing carrier protein, and (ii) an aluminum phosphate adjuvant.

[0252] In one embodiment, the disclosure provides a method for increasing the polysaccharide-to-protein carrier ratio (w / w) (PS:PC) of an immunogenic composition, comprising (a) introducing one or more nnAA substitutions into a carrier protein, and (b) conjugating the polysaccharide to the carrier protein via one or more non-natural amino acid substitutions. In another embodiment, the one or more substitutions comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine substitutions. In another embodiment, the nnAA substitution is not present in the T cell activation epitope of the carrier protein. In another embodiment, nnAA is pAMF. In another embodiment, the carrier protein holds the T cell binding epitope of diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus protein D (PD), serogroup B meningococcus (OMPC) outer membrane protein complex, or CRM197. In another embodiment, the non-natural amino acid substitution occurs at a lysine residue. In another embodiment, the lysine residue is selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K265, K386, K523, and K527 of SEQ ID NO: 1, and any combination thereof. In another embodiment, the polysaccharide is conjugated to the carrier protein via a linkage of formula XI or XIa, [ka] During the ceremony, R1 is independently H, formyl, or at least one amino acid of the carrier protein. R2 is independently an OH group, or at least one amino acid of the carrier protein. X is independently at least one polyol of the capsular polysaccharide.

[0253] In another embodiment, the PS:PC ratio is approximately 1.5 to approximately 4. In another embodiment, the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F, and any combination thereof. In another embodiment, the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., K1, K2, K3, K4, K5, and / or K6). In another embodiment, the polysaccharide is a capsular polysaccharide of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, or Streptococcus agalactia. In another embodiment, the present disclosure provides a glycoprotein prepared by a process comprising steps (a) to (b).

[0254] 10. Increased immune response This specification provides a method for inducing an immunoprotective antibody response to an antigen in a subject by administering a conjugate or composition as described herein. The conjugate or composition will typically be combined with an excipient suitable for parenteral administration.

[0255] The invention also provides conjugates and compositions for use in inducing an immunoprotective antibody response against an antigen. Furthermore, it provides the use of conjugates and compositions for the manufacture of agents for inducing an immunoprotective antibody response against an antigen.

[0256] The immunoprotective antibody response means that, using conjugates and compositions, it is possible to provide, for example, active immunization for the prevention of infiltrative diseases caused by Streptococcus pneumoniae, for the prevention of otitis media caused by Streptococcus pneumoniae, for the prevention of pneumonia caused by Streptococcus pneumoniae, and for active immunization of subjects at risk of exposure to meningococcus to prevent infiltrative diseases.

[0257] The present invention is illustrated in the following examples. Materials, methods, and examples are illustrative and not intended to limit. Numerous variations, modifications, and substitutions will arise for those skilled in the art without departing from the present invention. The examples are performed using the art that is well known to those skilled in the art and is in common use, unless otherwise described in detail. [Examples]

[0258] Example 1: Synthesis of one-part eCRM portions: K11TAG, K25TAG, K34TAG, K38TAG, K40TAG, K52TAG, K60TAG, K77TAG, K83TAG, K91TAG, K96TAG, and K103TAG. eCRM was expressed in cell-free protein synthesis (CFPS) extracts provided by Sutro Biopharma, Inc. (South San Francisco, California, USA). The characterization and preparation of such extracts are described in other publications, in which case the extracts were prepared as described in Zawada et al., 2011, Biotechnol. Bioeng., 108(7), 1570-1578, with the following modifications from US2016 / 0257946. (1) Cell-free extracts were prepared from an OmpT-sensitive RF-1 attenuated strain engineered to overexpress E. coli DsbC; (2) Cell-free extracts were prepared from a similar RF-1 attenuated strain engineered to produce orthogonal CUA-coding tRNA for insertion of a non-native amino acid at an amber stop codon; (3) Cell-free extracts from (1) and (2) were mixed (in a ratio of 85:15) and treated with 50 μM iodoacetamide for 30 minutes at room temperature (20°C); and (4) the mixed extracts were added to a premixture containing all other components of a cell-free protein synthesis system except for the DNA encoding eCRM. The final concentrations in the cell-free protein synthesis reaction were 30% (per volume) of cell extract, 2 mM para-methylazide-L-phenylalanine (pAMF) (Shirley, Massachusetts RSP Amino Acids), 5 μM pAMF-specific tRNA synthetase ("RS"), 2 mM GSSG (oxidized glutathione), 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (excluding 0.5 mM for tyrosine and phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, and 2.5 μM eCRM plasmide encoding the nnAA variant. The cell-free synthesis reaction was initiated by the addition of plasmide DNA encoding eCRM.

[0259] The reaction mixture was cultured in a 48-well flower dish (m2p-labs#MTP-48-B) with a stirrer at 650 rpm for 14 hours. After the culture period, the reaction mixture was kept at 4°C until processed for purification or analysis. Following the cell-free protein synthesis reaction, the mixture containing pAMF-eCRM was transferred to a 96-well dish (DyNa Block®, 2 mL; Labnet, Edison, NJ) and centrifuged at 5000 × g for 15 minutes at 40°C.

[0260] First, optimization experiments were performed to evaluate the optimal temperature and additives for CFPS generation of eCRM. CFPS reactions were carried out at 30, 25, and 20 degrees Celsius, each containing additional CUA-encoding tRNA supplements (0, 1, 2, 4, 8, 12% v / v) and nnAA / synthase mixtures (50, 100, 150, 200 μg / ml) at each of the three temperatures. Samples of the CFPS mixture were collected before and after centrifugation and analyzed by SDS-PAGE electrophoresis. Clump quantification was performed by concentration measurement to evaluate the amount of total protein (pre-centrifugation sample) and soluble protein (post-centrifugation sample) generated under each condition.

[0261] Figure 1 shows the yields of nnAA-eCRM produced under each condition, as evaluated by quantified concentration measurements. The CFPS reaction at 30°C produced a relatively small fraction of soluble protein (maximum of approximately 0.33 total across all conditions), but the yield of soluble protein was enhanced at 25°C (>approximately 0.40 soluble / total across all conditions) and further enhanced at 20°C (>approximately 0.60 soluble / total across all conditions). Under both of the "lower" temperature conditions, the yield of soluble protein was further enhanced by increasing the tRNA concentration (1–12x indicates an increase in yield), while increasing the nnAA / synthase concentration had no adverse effect on the soluble yield.

[0262] Based on the experiment in Figure 1, temperatures below 20 degrees Celsius and tRNA concentrations of at least 20 μM were selected for the synthesis of K11TAG, K25TAG, K34TAG, K38TAG, K40TAG, K52TAG, K60TAG, K77TAG, K83TAG, K91TAG, K96TAG, and K103TAG variants.

[0263] The CFPS reaction was performed as described above. For convenience of purification in these preliminary experiments, histidine labeling (GSGHHHHHH, SEQ ID NO: 10) was fused to the C-terminus of the carrier protein sequence via an expression vector, and purification of the eCRM mutant from the supernatant after centrifugation was performed using IMAC Phytips (San Jose, California, USA, Phynexus) containing 40 μL of resin. The resin bed was pre-equilibriumized in IMAC equilibrium buffer (1 × PBS and 10 mM imidazole), and the clarified supernatant was pipetted 10 times through an equilibriumized IMAC Phytips at a flow rate of 4.2 μL / min. The bound protein was washed with IMAC equilibrium buffer and then eluted with 125 μL of IMAC elution buffer (1 × PBS and 0.5 M imidazole). Histidine labeling is not essential and is omitted for large-scale purification.

[0264] The integration and reactivity of nnAA were evaluated by SDS-PAGE and fluorescence analysis after reaction with DBCO-fluorescein (Figure 2). 2–12 μM eCRMs were cultured in 50 μM DBCO-fluorescein for 16 hours, subjected to unreduced SDS-PAGE, and visualized using Coomassie Blue (visible light) and Sypro-ruby filter sets (fluorescence, fluorescein). Figure 2 shows corresponding Coomassie (left) and fluorescence (right) gel images illustrating the ability of pAMF to integrate into eCRM and react with DBCO. Amber substitutions of K25, K34, K38, and K40 showed high expression and binding efficiency, while others did not.

[0265] Example 2: Design of multiple nnAA eCRMs Several nnAA eCRM mutants were selected as described in the detailed description above. The mutants were synthesized via CFPS and tested according to Example 1. [Table 2]

[0266] Further variations were prepared, including those with different numbers of Lys→pAMF substitutions. Generally, a higher number of substitutions resulted in carriers leading to higher MW zygotes (e.g., for serotype 14, increasing from 998 kDa with 2 substitutions to 1238 kDa with 3 substitutions, to 1789 kDa with 4 substitutions, and to 2547 kDa with 5 substitutions), but the carriers were found to have lower solubility. Carriers with 6 pAMF residues generally provided both good solubility (>>50 mg / mL) and immunogenicity. The high solubility was surprising because the exchange of charged Lys residues in the native sequence with hydrophobic pAMF residues increased the hydrophobicity of CRM197, a protein whose hydrophobicity has already been reported to affect its solubility (Orr et al. 1999 Infect Immun 67:4290-4). Therefore, these results indicate that it is possible to maintain the same attachment site used in known CRM197 conjugates (i.e., Lys residues) without producing insolubility when the charged residue is lost.

[0267] Studies of CRM197 identified T cell epitopes within residues P272-D291, V322-G384, and Q412-I458 of SEQ ID NO: 1. These epitope regions contain lysine residues K420, K441, K446, K448, and K457, and therefore substitutions of these lysine residues may disrupt the T cell epitopes supporting the activity of CRM197. Preferred lysine residues for substitution with nnAA in SEQ ID NO: 1 are therefore K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, and K527, as shown in Table 2 above.

[0268] It is desirable that the conjugated polysaccharides do not become excessively strongly localized to a single region on the CRM197 surface. Therefore, within a cluster of residues, it is preferable to (i) isolate only one of K25, K34, K38, and K40, and (ii) isolate either K213 or K215. Furthermore, beyond its primary structure, studies of the 3D structure of CRM197 have identified two common regions (a first pathway to Asn-374 and a second pathway from Ser-375), and therefore it is also preferable to isolate residues in both of these regions, for example, three from each region for six substitutions. This general induction allows for spatial separation of the polysaccharides when attached to the CRM197 support.

[0269] One sequence particularly useful for producing pneumococcal conjugates is mutant 12 in Table 2, in which K34, K213, K245, K265, K386, and K527 are replaced by nnAA. This protein has the amino acid sequence of Sequence ID No. 9, where each X is pAMF (preferably nnAA). This protein is used to prepare the conjugates described below.

[0270] Lysine residues are useful because they are amino acids used in known CRM197 conjugates; therefore, nnAAs at these positions allow conjugation to occur at the same sites already known to be compatible with CRM197. However, as mentioned above, the loss of charged lysine can result in structural changes, increased hydrophobicity, and lower solubility. Modification of phenylalanine residues to phenylalanine-based nnAAs (such as para-azido-Phe, para-azido-methyl-Phe, para-fluoro-Phe, para-acetyl-Phe, or para-benzoyl-Phe) would reduce the risk of these changes. Therefore, residues F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 are also selected for substitution, either alone or in combination. Substitution of up to five Phe residues with pAMF was tested and provided soluble conjugates, but with a lower tendency toward MW conjugates than achieved by the same number of multiple Lys substitutions.

[0271] Rather than substituting amino acids within CRM197, it is also possible to insert nnAAs within the CRM197 sequence. For example, the TAG codon encoding pAMF can be directly inserted downstream of lysine residues K34, K213, K245, K265, K386, and K527, either individually (to create six point insertions) or in combination (to insert two, three, four, five, or six nnAAs). Carriers having these inserted nnAAs are useful for producing conjugates and polyvalent compositions as described above.

[0272] Example 3: Identification of T cell epitopes in Pfs25 The T-cell activation epitope of the malaria orkinate-specific surface protein Pfs25 is experimentally determined according to the method described, for example, Diethelm-Okita et al., J Infect Dis. 1997 Feb;175(2):382-91. Briefly, a 20-amino acid peptide fragment and a 5-amino acid duplication are synthesis corresponding to the fully expressed sequence of Pfs25. CD8+ degraded and CD4+-rich human peripheral blood lymphocytes (PBLs) are obtained from multiple subjects. PBLs are triple-plated and cultured with individual synthetic peptides spanning the Pfs25 sequence, which serves as an experimental stimulus. The proliferation of PBLs in response to each peptide fragment is used to convert the culture into [ 3 This is measured by pulsating with [H]-thymidine, and this is compared across cultures originating from different individuals. Pfs25 fragments that stimulate proliferation are identified as containing T cell epitopes. Fragments that stimulate PBL proliferation from multiple or all subjects are classified as containing generic or immunodominant T cell epitopes in Pfs25.

[0273] Example 4: General protocol for the activation of polysaccharides with sodium metaperiodate Serotype polysaccharides (approximately 30 μmol) were dissolved in an aqueous solution (10 mM HCl). The solution was then heated at 45°C for 30 minutes, then cooled, and NaOH solution was added to adjust the pH to 6.70. The reaction mixture was dialyzed against HPLC-grade water using an AMICON ultracentrifuge (30 kDa MWCO). The supernatant was transferred to a 50 mL Falcon tube, and acetate buffer (pH 5.35) was added to 25 mM, followed by the addition of 0.5 equivalents of NaIO4. The mixture was stirred at 25°C for 17 hours, after which the oxidized sample was optionally treated with an excess of sodium borohydride (10 equivalents) and purified against several changes in HPLC-grade water using an AMICON ultracentrifuge (30 kDa MWCO) to obtain the oxidized polysaccharide solution.

[0274] Example 5: General procedure for derivatization of periodate-oxidized polysaccharides using DBCO Oxidized polysaccharides (approximately 30 μmol) were combined with DBCO-PEG4-NH2 (approximately 30 μmol) or DBCO-NH2 (approximately 30 μmol) in 72 mM sodium phosphate at pH 6.79 containing 16% DMSO at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which sodium borohydroxide solution (16 mg / ml solution in water, 59.54 μmol, 20 equivalents) was added, and the mixture was stirred at 25°C for two nights. The reaction mixture was then washed three times with ethyl acetic acid and transferred to an AMICON ultracentrifuge (30 kDa MWCO). Dialysis was then performed using six exchanges of 20% ethanol in water, followed by three exchanges with water, to obtain a polysaccharide-DBCO derivative solution. The polysaccharide-DBCO derivative was then prepared with 10:1 (w / w) sucrose and freeze-dried to obtain a white powder for use in the subsequent conjugation reaction.

[0275] Example 6: General protocol for polysaccharide activation using CDAP Capsular polysaccharide (30 mg) (PS3) was dissolved in an aqueous solution (13.5 mL of H2O containing 1.5 mL of 2 M acetic acid). The mixture was heated at 85°C for 1 hour, cooled to ambient temperature, and then excess magnesium chloride was added from a 1 M solution. The resulting polysaccharide was purified by dialysis using an Amicon centrifuge 30 kDa MWCO with six water changes.

[0276] Next, the prepared polysaccharides were dissolved in water at pH 7.0, and the cyanide reagent CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroboric acid in acetonitrile) was added. The solution was then adjusted to pH 9.5, or trimethylamine (2.5 equivalents) was added. Next, DBCO-PEG4-NH2 or DBCO-NH2 was added to the solution. The solution was adjusted to 5% DMSO and stirred overnight at 25°C. The solution was washed 3 times with 20 mL of ethyl acetic acid and purified using an Amicon 30 kDa MWCO dialysis unit with 7 exchanges with 3% DMSO, 20% ethanol, and 0.9% sodium chloride, and 3 exchanges with water. Next, the polysaccharide-DBCO derivatives were prepared with sucrose in a 10:1 (w / w) ratio and lyophilized.

[0277] Example 7: General procedure for conjugating polysaccharide-DBCO with eCRM Lyophilized polysaccharide-DBCO samples, prepared (by the procedure of Example 4 or 5) with 10:1 w / w sucrose, were dissolved in 0.9% NaCl and mixed with eCRM in solution to obtain a 1:1 (w / w) PS:eCRM input mass ratio. The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 18 hours. The click reaction was quenched by the addition of an excess of sodium azide solution. The joined PS-eCRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO) and then dialyzed against five exchanges of 0.9% sodium chloride solution over 24 hours. The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a PS-eCRM conjugate solution.

[0278] Example 8: Preparation of pneumococcal PS serotype 1 conjugates in eCRM from Table 2 1. Oxidation Type 1 PS purity: 80% (Uron) Molecular weight: 625 g / mol -1 (per repeating unit) Reaction procedure Natural polysaccharides (19.7 mg, adjusted to 80%, 15.8 mg, 25.2 μmol) were dissolved in 9.85 mL of aqueous solution (7.0 mL of water and 2.85 mL of acetate buffer, 200 mM, pH 5.5). To this solution, 300 μL of sodium periodate solution (104 μg, 3.78 μmol, 0.15 equivalents) was added. The mixture was stirred at 25°C for 18 hours, after which a large molar excess of sodium borohydride (10 molar equivalents) was added. Oxidized PS was purified using Amicon centrifugation with 30 kDa MWCO dialysis using at least six water exchanges to obtain purified PS-1 solution. [Table 3]

[0279] 2. DBCO derivatization Reaction procedure Dissolve PS1-OX (15.8 mg, 25.2 μmol) in phosphate buffer (3.6 mL, 50 mM, pH 7.0), and add DBCO-PEG4-NHS ester (1.0 equivalent, 649.1 g / mol in DMSO) to this solution. -1 (0.35 mL) was added. The reaction mixture was stirred in a thermostat bath at 37°C for 2 days, and then extracted with ethyl acetic acid (3 × 20 mL). The DBCO derivative was purified by centrifugal dialysis unit (Amicon 30 kDa MWCO) using 6 exchanges with 20% ethanol in water, followed by 3 exchanges with water (12 mL each), to obtain the 1-DBCO derivative type. Sucrose solution (96 mg in 1 mL of water) was added to this solution (2.20 mL, 9.59 mg). The combined solution was divided into three equal parts, and each was freeze-dried to obtain three samples of white powder. Each sample contained 3.18 mg of 1 DBCO and 32 mg of sucrose for use in the subsequent conjugation reaction. [Table 4]

[0280] 3. Conjugation of PS 1-DBCO derivative with eCRM Lyophilized powder of PS 1-DBCO3.18 mg (containing 32 mg of sucrose) DBCO(%): 1.67% CRM concentration: 6.5 mg / mL solution PS:CRM (input ratio): 1:1 Reaction procedure 1-DBCO was dissolved in azide-functionalized eCRM solution (0.51 mL) to obtain a PS1:CRM input mass ratio of 1:1 (w / w). To reduce gel formation, 1.0 mg mL was added. -1Further dilution with a 0.9% sodium chloride solution (filtered to 0.22 μm) was required. The solution was mixed very gently by hand, and then gently mixed in a circular stirrer for 18 hours at room temperature (20°C). The click reaction was quenched by the addition of sodium azide solution (10 mg / mL, 50 μL). The CRM conjugate was transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with a 0.9% sodium chloride solution for 24 hours (three changes, 800 ml each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain the 1-CRM conjugate solution. [Table 5] *CJD = Dialyzed Joint

[0281] Example 9: Preparation of pneumococcal PS serotype 2 conjugates in eCRM from Table 2 1. Oxidation Type 2 PS purity: 80% Molecular weight: 960.84 g / mol -1 Reaction procedure Natural polysaccharides (25.5 mg, 26.5 μmol) were dissolved in 12.75 mL of aqueous solution (9.24 mL of water and 3.51 mL of acetate buffer, 200 mM, pH 5.5). 216 μL of sodium periodate solution (5.26 mg / ml, 0.20 equivalents) was added to this solution. The mixture was stirred at 25°C for 18 hours while monitoring by UV absorption at 222 nm relative to NaIO4. Oxidized PS was purified using Amicon centrifugation with 100 kDa MWCO dialysis with at least six water exchanges to obtain purified PS-2 solution. [Table 6]

[0282] 2. DBCO derivatization Reaction procedure PS2-OX (18.1 mg, 18.8 μmol) in 2.14 mL of water was diluted with phosphate buffer (1.95 mL, 200 mM, pH 6.0), and DBCO-PEG4-NH2 (9.85 mg, 1 equivalent, 0.197 mL in DMSO) was added. After 25 minutes, NaCNBH3 (2.36 mg, 59 μL from a solution in 2 equivalents of H2O) was added. The reaction mixture was stirred in a thermostat bath at 25°C for 2 days, followed by the addition of phosphate buffer (0.5 mL, 200 mM, pH 6). NaBH4 (60 μL, 10 mg / mL aqueous solution, 1 equivalent) was added. After stirring for 30 minutes, the mixture was extracted with ethyl acetic acid (4 × 5 mL). Residual ethyl acetic acid was removed by bubbling with nitrogen gas, and the mixture was transferred to a 100 kDa MWCO Amicon centrifuge filter. The DBCO derivative was purified by centrifugal dialysis using one exchange of water, followed by six exchanges with 20% ethanol in water, followed by three exchanges with water (12 mL each), to obtain the 2-DBCO derivative form. To this solution (2.14 mL, 14.3 mg), a sucrose solution (100 mg in 1 mL of water) was added. The combined solution was divided into three approximately equal parts, each of which was freeze-dried to obtain three samples of white powder (4.96 mg, 4.96 mg, and 4.4 mg). [Table 7]

[0283] 3. Conjugation of PS 2-DBCO derivative with eCRM Lyophilized powder containing PS 2-DBCO: 4.4 mg (including 32 mg of sucrose) %DBCO:4.03% CRM concentration: 3.18 mg / mL solution PS:CRM (input ratio): 1.5:1 Reaction procedure PS2-DBCO was dissolved in 0.9% NaCl (3.01 mL) and DMSO (0.44 mL) was added. Next, azide-functionalized eCRM solution (0.95 mL) was added to obtain a PS2:CRM input mass ratio of 1.5:1 (w / w). The solution was mixed very gently by hand and then gently mixed with a cyclic stirrer at room temperature (20°C) for 18 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 100 μL). The CRM conjugate was transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 24 hours (5 changes, 1000 ml each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 2-CRM conjugate solution. [Table 8]

[0284] Example 10: Preparation of Streptococcus pneumoniae PS serotype 3 conjugates in eCRM from Table 2 1. Hydrolysis Type 3 PS purity: 86% (Antron) Molecular weight: 360.3 g / mol -1 Reaction procedure Natural polysaccharide 3 (30.0 mg) was dissolved in 15.0 mL of aqueous solution (13.5 mL of water and 1.5 mL of acetic acid, 2 M). The mixture was heated at 85°C for 1 hour, then cooled to ambient temperature, and magnesium chloride solution (1.5 mL, 1 M) was added. The hydrolyzed PS was purified using Amicon centrifuge 30 kDa MWCO dialysis with at least 6 exchanges with water to obtain a purified PS-3 solution, which was then lyophilized in two equal portions. [Table 9]

[0285] 2. DBCO derivatization Reaction procedure Hydrolyzed PS3 (12.75 mg, 35.4 μmol) was dissolved in water (6.4 mL) and the pH was adjusted to 7.0 with sodium hydroxide solution (0.2 M, 100 μL). Next, the cyanide reagent CDAP was added dropwise (0.426 M, 0.2 equivalents, 16.7 μL in acetonitrile). After 90 seconds, the solution was rapidly adjusted to 9.5 pH with sodium hydroxide solution (0.2 M, 300 μL). DBCO-PEG4-NH2 (0.032 M, 0.1 equivalents, 523 gmol in DMSO) -1 0.110 mL of DMSO was immediately added dropwise. Additional DMSO was added to obtain 5% (v / v) DMSO (0.320 mL). The reaction mixture was stirred overnight in a thermostat bath at 25°C and then filtered through a 0.22 μm PES syringe filter. The filtrate was extracted with ethyl acetic acid (3 × 20 mL). The DBCO derivative was purified by centrifugal dialysis using an Amicon 30 kDa MWCO unit with a total of seven exchanges with 3% DMSO, 20% ethanol in water, and 0.9% sodium chloride, followed by three exchanges with water (12 mL each), to obtain the 3-DBCO derivative. The aqueous solution was then filtered through a 0.45 μm PVDF syringe filter. To this solution (3.84 mL, 8.52 mg), 10 times the mass of excess sucrose (85 mg in 0.85 mL of water) was added. The combined solution was broken down into three freeze-dried portions to obtain three samples of white powder. Two of the samples contained 5.0 mg of 3-DBCO and 50 mg of sucrose for use in the next conjugation reaction, with a total of 8.5 mg in the remaining sample for all three samples. [Table 10]

[0286] 3. Conjugation of PS3-DBCO derivative with eCRM PS 3-DBCO: 5.0 mg (containing 50 mg of sucrose) lyophilized powder DBCO percentage (%): 5.0% CRM concentration: 4.0 mg / mL solution PS:CRM (input ratio): 1:1 Reaction procedure 3-DBCO was dissolved in 0.9% sodium chloride solution (6.39 mL, filtered to 0.22 μm), phosphate buffer (pH 7.0, 0.5 M, 0.333 mL), and DMSO (0.833 mL). Azide-functionalized eCRM solution (0.770 mL) was added dropwise to obtain a 1:1 (w / w) PS3:CRM mass ratio. The solution was mixed very gently by hand and then gently mixed with a cyclic stirrer at room temperature (20°C) for 18 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 50 μL). The CRM conjugate was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 48 hours (four changes, 1 L each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 3-CRM conjugate solution. [Table 11] *CJF = Dialyzed and filtered joint

[0287] Example 11: Preparation of pneumococcal PS serotype 3 conjugates in eCRM from Table 2 1. Oxidation Type 3 PS purity: 86% (Antron) Molecular weight: 360.3 g / mol -1 Reaction procedure Natural polysaccharide 3 (14.4 mg, adjusted to 86%, 12.4 mg, 34.4 μmol) was dissolved in 7.2 mL of aqueous solution (5.9 mL of water and 1.3 mL of acetate buffer, 200 mM, pH 5.5). To this solution, 300 μL of sodium periodate solution (1.10 mg, 5.16 μmol, 0.15 equivalents) was added. The mixture was stirred at 25°C for 18 hours. Oxidized PS was purified using Amicon centrifugation 30 kDa MWCO dialysis with at least 6 exchanges with water to obtain purified PS3-OX solution. [Table 12]

[0288] 2. DBCO derivatization Reaction procedure Dissolve PS3-OX (9.05 mg, 25.1 μmol) in phosphate buffer (2.11 mL, 50 mM, pH 6.7), and add DBCO-PEG4-NH2 (1.0 equivalent, 523 gmol in DMSO) to this solution. -1 0.40 mL of the solution was added. The reaction mixture was stirred at 25°C for 25 minutes, then a solution of sodium cyanohydroxide (2 equivalents, 44.5 mg / mL, 35 μL) was added and stirred for 2 days. At this point, the reaction mixture was extracted with ethyl acetic acid (3 × 20 mL). The DBCO derivative was purified using a centrifugal dialysis unit (Amicon 30 kDa MWCO) with six exchanges with 20% ethanol in water, followed by three exchanges with water (12 mL each), to obtain the 3-DBCO derivative. To this solution (3.20 mL, 8.60 mg), 10 times the mass of excess sucrose (86 mg in 0.86 mL of water) was added. The combined solution was divided into four parts, and each was freeze-dried to obtain three samples of white powder. The three samples, for a total of four, contained 2.0 mg of 3-DBCO and 20 mg of sucrose for use in the next conjugate reaction, with 2.6 mg of the remaining samples in each. [Table 13]

[0289] 3. Conjugation of PS3-DBCO derivative with eCRM Lyophilized powder of PS 3-DBCO: 2.0 mg (containing 20 mg of sucrose) DBCO percentage (%): 2.3% CRM concentration: 4.0 mg / mL solution PS:CRM (input ratio): 1:1 Reaction procedure 3-DBCO was dissolved in 0.400 mL of 0.9% sodium chloride solution (filtered to 0.22 μm) and DMSO (0.100 mL). 0.330 mL of azide-functionalized eCRM solution was added dropwise to obtain a 1:1 (w / w) PS3:CRM mass ratio. The solutions were mixed very gently by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 48 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 50 μL). The CRM conjugates were transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 48 hours (four changes, 1 L each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 3-CRM conjugate solution. [Table 14]

[0290] Example 12: Preparation of pneumococcal PS serotype 4 conjugates in eCRM from Table 2 1. Oxidation Type 4 PS purity: 80% (Antron) Molecular weight: 825.78 Reaction procedure Type 4 PS (27.5 mg, 33.30 μmol) powder was dissolved in 13.75 mL of aqueous solution (12.38 mL of water and 1.37 mL of 0.1 M HCl). The solution was then heated at 45°C for 30 minutes, then cooled, and the pH was adjusted to 6.70 by adding 1.37 mL of 0.1 M NaOH solution. The reaction mixture was dialyzed using an AMICON ultracentrifuge (30 kDa MWCO 6-12 mL) by three exchanges (12 mL each) with HPLC-grade water. The supernatant was transferred to a 50 mL Falcon tube containing 9.84 mL of water. To this solution, 3.43 mL of 200 mM acetate buffer (pH 5.35) and 632 μL of NaIO4 solution (3.56 mg, 16.65 μmol, 0.5 equivalents) were added. The mixture was stirred at 25°C for 17 hours, and then the oxidized sample was purified using HPLC-grade water in an AMICON ultracentrifuge (6-12 mL of 30 kDa MWCO) with 6 exchanges (12 mL total) to obtain oxidized PS-4 solution. [Table 15]

[0291] 2. DBCO derivatization Reaction procedure To an oxidized (assuming a 10% oxidation level) type 4 PS solution (24.58 mg, 29.77 μmol, 2.4 mL of water), buffer solution (1.8 mL of 200 mM phosphate buffer, pH=6.79), DMSO (0.6 mL), and DBCO-PEG-4-NH2 solution (15 mg, 28.65 μmol, 9.6 equivalents in 200 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 224 μL of sodium cyanoborohydroxide solution (5.0 mg, 59.54 μmol, 20 equivalents in 300 μL of water) was added, and the mixture was stirred at 25°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 225 μL of sodium borohydride solution (0.01 mg / μL, 10 equivalents) was added to water. After stirring for 30 minutes, the reaction mixture was extracted with ethyl acetic acid (3 × 20 mL of ethyl acetic acid), then transferred to an AMICON ultracentrifuge (6-12 mL of 30 kDa MWCO), and dialyzed using six exchanges with 20% ethanol in water, followed by three exchanges with water (12 mL each), to obtain a 4 DBCO derivative type solution. Sucrose solution (153 mg in 1 mL of water) was added to this solution (4.75 mL, 15.25 mg). The combined solution was divided into three parts, each of which was freeze-dried to obtain three white powder samples. Two samples contained 5.35 mg of 4DBCO and 54 mg of sucrose, while one sample contained 4.55 mg of 4DBCO and 45 mg of sucrose for use in the subsequent conjugation reaction. [Table 16]

[0292] 3. Conjugation of PS 4-DBCO derivative with eCRM PS 4-DBCO: 5.35 mg (containing 54 mg of sucrose) - White powder DBCO percentage (%): 3.52% CRM concentration: 4 mg / mL solution PS:CRM (input ratio): 1.20:1 Reaction procedure A type 4 DBCO sample (5.35 mg of white powder containing 54 mg of sucrose) was dissolved in 0.67 mL of 0.9% NaCl solution, and then 0.74 mL of azide-functionalized eCRM solution was added. After 10 minutes, another portion of azide-functionalized eCRM (0.37 mL) was added to obtain a PS4:CRM mass ratio of 1.20:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 2 days. The joined PS-CRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO), and then dialyzed with 0.9% sodium chloride solution for 24 hours (5 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a type 4 PS-CRM conjugate solution. [Table 17]

[0293] Example 13: Preparation of Streptococcus pneumoniae PS serotype 5 conjugates in eCRM from Table 2 1. Oxidation Type 5 PS purity: 89% (uronic acid) Molecular weight: 919.32 Reaction procedure Type 5 PS (22.8 mg, 24.36 μmol) powder was dissolved in 8.26 mL of water, 3.14 mL of 200 mM acetate buffer (pH 5.26), and 163 μL of NaIO4 solution (1.3 mg, 6.1 μmol, 0.25 equivalents). The mixture was stirred at 25°C for 18 hours, and then the oxidized sample was purified using an AMICON ultracentrifuge (100 kDa MWCO 6-12 mL) with HPLC-grade water in six exchanges (12 mL) to obtain oxidized PS-5 solution. [Table 18]

[0294] 2. DBCO derivatization Reaction procedure To an oxidized (assuming a 10% oxidation level) type 5 PS solution (6.25 mg, 6.68 μmol, 0.992 mL of water), buffer solution (0.063 mL of 200 mM phosphate buffer, pH=6.74), DMSO (25 μL), and DBCO-PEG-4-NH2 solution (3.5 mg, 6.68 μmol, 10 equivalents in 100 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 37°C for 30 minutes, after which 84 μL of sodium cyanoborohydroxide solution (0.84 mg, 13.36 μmol, 20 equivalents in 84 μL of water) was added, and the mixture was stirred at 37°C for 24 hours. The reaction mixture was extracted with ethyl acetic acid (6 × 10 mL). The extract was transferred to an AMICON ultracentrifuge filter (6-12 mL of 30 kDa MWCO), and then dialyzed using eight exchanges (12 mL each) with 20% ethanol in water, followed by three exchanges (12 mL each) with water to obtain the 5 DBCO derivative. To this solution (5.35 mL, 6.0 mg), sucrose solution (60 mg in 0.6 mL of water) was added. The combined solution was divided into two equal parts, each of which was freeze-dried to obtain two samples of white powder. Each sample contained 3.0 mg of 5 DBCO and 30 mg of sucrose for use in the subsequent conjugation reaction. [Table 19]

[0295] 3. Conjugation of PS 5-DBCO derivative with eCRM PS 5-DBCO: 3.0 mg (containing 30 mg of sucrose) - White powder DBCO percentage (%): 5.17% CRM concentration: Solution with a concentration of 3.25 mg / mL PS:CRM (input ratio): 1:1 Reaction procedure A 5-DBCO derivative (3.0 mg of white powder containing 30 mg of sucrose) was dissolved in 0.9% sodium chloride solution (4.48 mL) and DMSO (0.6 mL). Azide-functionalized eCRM solution (0.92 mL) was added to obtain a 1:1 (w / w) PS5:CRM mass ratio. The reaction mixture was gently mixed and then gently stirred with a cyclic stirrer at room temperature (20°C) for 5 hours. Sodium azide solution (20 μL, 10 mg / mL in water) was added. After 30 minutes, the combined PS-CRM mixture was transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 24 hours (5 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 5PS-CRM conjugate solution. [Table 20]

[0296] Example 14: Preparation of pneumococcal PS serotype 6A conjugates in eCRM from Table 2 1. Oxidation Molecular weight of type 6A PS: 706 NaIO4 solution in water (10 mg / mL)

[0297] Reaction procedure PS-6A (15 mg, 21.2 μmol) powder was dissolved in 7.5 mL of aqueous solution (10 mM sodium acetate solution, pH 4.5). To this solution, 36.3 μL of NaIO4 solution (0.363 mg, 1.69 μmol, 0.08 equivalents) was added. The mixture was stirred at 4°C for 18 hours, and then the oxidized sample was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235057, 20K MWCO). Next, the sample was dialyzed with 50 ml of PB buffer at pH 6.8 for 24 hours (four changes, 600 ml each) to obtain an oxidized PS-6A solution. After dialysis, DMSO was added, and PS-6A was prepared in 10% DMSO with 50 ml of PB buffer at pH 6.8. [Table 21]

[0298] 2. DBCO derivatization Final PS concentration: 3.37 mg / ml Final buffer concentration: 10% DMSO in 50 mM PB (pH 6.8) Reaction procedure To a solution of oxidized type 6A PS (13.5 mg, 19.1 μmol, 4 mL in 10% DMSO, 50 mm PB, pH 6.8), a solution of DBCO-PEG4-NH2 (10.01 mg, 19.1 μmol, 10 equivalents in 100.1 μL of DMSO) was added at 25°C. Next, the reaction mixture was stirred at 25°C for 60 minutes, after which sodium cyanoborohydroxide solution (1.2 mg, 19.1 μmol, 10 equivalents in 120 μL of water) was added, and the mixture was stirred at 25°C for 24 hours. Next, the reaction mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235057, 20K MWCO), and then dialyzed using four exchanges with 20% ethanol in 50 mM PB buffer, followed by three exchanges with 50 mM PB buffer to obtain the 6A DBCO derivative. [Table 22]

[0299] 3. Conjugation of PS 6A-DBCO derivative with eCRM PS 6A-DBCO: 7.1 mg (containing 71 mg of sucrose) white powder DBCO: 9% CRM concentration: Solution with a concentration of 2.617 mg / mL PS:CRM (input ratio): 2:1 Final PS concentration: 5.2 mg / ml Reaction procedure A 1.4 mL azide-functionalized eCRM solution was mixed with a 6A DBCO derivative (7.1 mg of white powder containing 71 mg of sucrose) to obtain a PS 6A:CRM mass ratio of 2:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed with a circular stirrer at room temperature (23°C) for 17 hours. Next, the mixture was placed in an incubator (37°C) for 3 hours. After the reaction, the mixture was diluted 2-fold with a 0.9% sodium chloride solution and reduced with sodium borohydride (1.9 mg, 50.2 μmoles, 50 equivalents in 191 μL of water) for 3 hours. The combined PS-CRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235072, 300K MWCO) and then dialyzed with pH 7 PBS for 24 hours (3 changes, 1000 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 6A PS-CRM conjugate solution. [Table 23]

[0300] Example 15: Preparation of pneumococcal PS serotype 6B conjugates in eCRM from Table 2 1. Oxidation 6B type PS purity: 80% (Antron) Molecular weight: 706.18 NaIO4 solution in water (5.45 mg / mL) Reaction procedure PS-6B powder (27.28 mg, adjusted to 80%, 21.82 mg, 30.9 μmol) was dissolved in 14 mL of aqueous solution (9.5 mL of water and 4.5 mL of 0.2 M acetate buffer, pH=5.5). To this solution, 145 μL of NaIO4 solution (0.79 mg, 3.71 μmol, 0.12 equivalents) was added. The mixture was stirred at 25°C for 18 hours, and then the oxidized sample was purified using an AMICON ultracentrifuge (30 kDa MWCO 6-12 mL) with HPLC-grade water in 6 exchanges (12 mL) to obtain oxidized PS-6B solution. [Table 24]

[0301] 2. DBCO derivatization Final PS concentration: 3.5 mg / ml Final buffer concentration: 53 μM (pH 6.0) Reaction procedure To an oxidized (assuming a 10% oxidation level) 6B type PS solution (18.4 mg, 27.6 μmol, 3.35 mL of water), buffer solution (1.4 mL of 200 mM phosphate buffer, pH=6.01), DMSO (700 μL), and DBCO-PEG-4-NH2 solution (14.43 mg, 27.6 μmol, 10 equivalents in 295 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 75 μL of sodium cyanoborohydroxide solution (9.39 mg, 55.6 μmol, 20 equivalents in 200 μL of water) was added, and the mixture was stirred at 25°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 104 μL of sodium borohydride solution (0.01 mg / μL, 10 equivalents) was added to water. After stirring for 30 minutes, the reaction mixture was extracted with ethyl acetate (3 × 20 mL of ethyl acetate), then transferred to an AMICON ultracentrifuge (6-12 mL of 30 kDa MWCO), and dialyzed using six exchanges with 20% ethanol in water, followed by three exchanges with water (12 mL each) to obtain a solution of the 6B DBCO derivative type. To this solution (2.96 mL, 20.1 mg), sucrose solution (200 mg in 1 mL of water) was added. The combined solution was divided into three equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 6.7 mg of 6B DBCO and 67 mg of sucrose for use in the subsequent conjugation reaction. [Table 25]

[0302] 3. Conjugation of PS 6B-DBCO derivative with eCRM PS 6B-DBCO: 6.7 mg (containing 67 mg of sucrose) white powder DBCO percentage (%): 3.2% CRM concentration: Solution with a concentration of 2.617 mg / mL PS:CRM (input ratio): 2:1 Final PS concentration: 5.23 mg / ml Reaction procedure A 1.28 mL azide-functionalized eCRM solution was added to a 6B-DBCO derivative (6.70 mg of white powder containing 67 mg of sucrose) to obtain a PS6B:CRM mass ratio of 2:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 17 hours. Next, the mixture was placed in an oven (37°C) for 2 hours. The conjugated PS-CRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235071, 100K MWCO) and then dialyzed with a 0.9% sodium chloride solution for 24 hours (3 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 6B PS-CRM conjugate solution. [Table 26]

[0303] Example 16: Preparation of pneumococcal PS serotype 7F conjugates into eCRM from Table 2 1. CDAP activation and DBCO crosslinking Purifying PS 7F: Assuming nd% (Anthrone) -100%

[0304] Molecular weight: 1227 g / mol -1 (Repeating unit)

[0305] Reaction procedure PS7F (6.2 mg, 5.1 μmol) was dissolved in water (3.1 mL), and CDAP (2.0 equivalents, 100 mg / mL in acetonitrile, 24 μL) was added. The reaction mixture was stirred at room temperature for 30 seconds. At this point, triethylamine (TEA, 2.5 equivalents, 0.2 M, 63 μL) was added, and the reaction mixture was stirred for 120 seconds. DBCO-PEG4-NH2 (1.0 equivalent, 28.7 μmol / mL in DMSO, 180 μL) was added together with borate buffer (0.1 M, pH 8.5, 1.0 mL), and the mixture was stirred overnight at room temperature. DBCO-derivativeized PS7F was purified by ethanol precipitation and centrifugal dialysis (Amicon 100 kDa MWCO) using three water exchanges. After analysis by UV absorption spectroscopy, anthron assay, and SEC, this solution (3.61 mL, 3.05 mg) was diluted with sucrose solution (10 times the mass content, 100 mg / mL) and lyophilized to a white powder. [Table 27]

[0306] 2. Conjugation of PS 7F-DBCO derivative with eCRM PS 7F-DBCO: 2.62 mg (containing 26.2 mg of sucrose) lyophilized powder

[0307] DBCO percentage (%): 8.1%

[0308] CRM: 5.0 mg / mL in PBS buffer

[0309] PS:CRM (input mass ratio): 1.73:1

[0310] Reaction procedure Lyophilized 7F-DBCO was dissolved in saline solution (0.9% (w / v), 0.938 mL), phosphate buffer (0.5 M, pH 7.0, 58 μL), and DMSO (144 μL). To this, eCRM solution (0.300 mL) was added to obtain a PS7F:CRM mass ratio of 1.73:1.00 (w / w). The solution was very gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 17 hours. The CRM conjugates were transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 24 hours (three changes, 800 ml each). The dialyzed solution was sterile filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 7F-CRM conjugate solution. [Table 28]

[0311] Example 17: Preparation of Streptococcus pneumoniae PS serotype 8 conjugates in eCRM from Table 2 1. Oxidation 8-type PS purity 84% Molecular weight: 684.54 g / mol -1 Reaction procedure Natural polysaccharides (42 mg, 61.3 μmol) were dissolved in 21 mL of aqueous solution (14.7 mL of water and 6.3 mL of acetate buffer, 200 mM, pH 5.5). Sodium periodate solution (calculated for 2.63 mg, 0.20 equivalents) was added to this solution. The mixture was stirred at 25°C for 18 hours while monitoring by UV absorption at 222 nm relative to NaIO4. Oxidized PS was purified using Amicon centrifugation with 30 kDa MWCO dialysis using at least 6 exchanges with water to obtain a purified PS-8 solution. [Table 29]

[0312] 2. DBCO derivatization Reaction procedure PS8-OX (33.8 mg, 49.4 μmol) in 3.14 mL of water was diluted with phosphate buffer (789 μL, 0.5 M, pH 6.0), 1 mL of H2O, and DMSO (313 μL), to which DBCO-PEG4-NH2 (25 mg, 1 equivalent, 250 μL in DMSO) was added. After 10 minutes, NaCNBH3 (6.2 mg, 2 equivalents (by adding 132 μL from 9.43 mg in 200 μL of H2O)) was added. The reaction mixture was stirred in a thermostat bath at 25°C for 2 days, followed by the addition of phosphate buffer (0.5 mL, 200 mM, pH=6). NaBH4 (1 equivalent) was then added. After stirring for 30 minutes, the mixture was extracted with ethyl acetic acid (3 × 5 mL). Residual ethylacetic acid was removed by bubbling with nitrogen gas, and the mixture was transferred to a 100 kDa MWCO Amicon centrifuge filter. The DBCO derivative was purified by centrifugal dialysis using six exchanges with 20% EtOH and three exchanges with water (12 mL each) to obtain the 8-DBCO derivative. Sucrose solution was added to this solution (5.63 mL, 25 mg), and it was freeze-dried. [Table 30]

[0313] 3. Conjugation of PS 8-DBCO derivative with eCRM PS 8-DBCO: 3.77 mg (containing 38 mg of sucrose) lyophilized powder DBCO percentage (%): 3.57% CRM concentration: Solution with a concentration of 5.966 mg / mL PS:CRM (input ratio): 1.5:1 Reaction procedure PS8-DBCO was dissolved in 0.9% NaCl (2.28 mL), phosphate buffer (0.126 mL, 0.5 M, pH 7.0), and DMSO (0.314 mL). Next, azide-functionalized eCRM solution (0.42 mL) was added to obtain a PS8:CRM mass ratio of 1.5:1 (w / w). The solution was mixed very gently by hand, then gently mixed with a circular stirrer at room temperature (20°C) for 1 hour, and then left in an oven overnight at 37°C. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 100 μL). The CRM conjugate was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 48 hours (8 changes, 1000 ml each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain an 8-CRM conjugate solution. [Table 31]

[0314] Example 18: Conjugation of Streptococcus pneumoniae PS serotype 9N conjugates to eCRM from Table 2 1. Oxidation 9N type PS purity: 75% Molecular weight: 928.29 g / mol -1 Reaction procedure Natural polysaccharides (19.0 mg, 20.4 μmol) were dissolved in 9.49 mL of aqueous solution (7.12 mL of water and 2.37 mL of acetate buffer, 200 mM, pH 5.5). Sodium periodate solution (1.31 mg, 0.30 equivalents, 56 μL from 23.65 mg in 1.0 mL of aqueous solution) was added to this solution. The mixture was stirred at 25°C for 18 hours while monitoring by UV absorption at 222 nm relative to NaIO4. Oxidized PS was purified using Amicon centrifugation with 30 kDa MWCO dialysis using four water exchanges to obtain purified PS-9 solution. [Table 32]

[0315] 2. DBCO derivatization Reaction procedure PS9N-OX (12.6 mg, 13.6 μmol) in 1.643 mL of water was diluted with phosphate buffer (0.945 mL, 200 mM, pH 6.0, containing 94.5 mg of sucrose) and DMSO (0.33 mL), and DBCO-PEG4-NH2 (7.2 mg, 1 equivalent, 0.142 mL in DMSO) was added. After 10 minutes, NaCNBH3 (1.71 mg, 2 equivalents (by adding 47 μL from 7.36 mg in 200 μL of H2O)) was added. The reaction mixture was stirred in a thermostat bath at 25°C for 2 days, and then phosphate buffer (0.4 mL, 200 mM, pH=6) was added. NaBH4 (0.51 mg, 1 equivalent) was added. After stirring for 30 minutes, the mixture was extracted with ethyl acetic acid (5 × 5 mL). Residual ethylacetic acid was removed by bubbling with nitrogen gas, and the mixture was transferred to a 30 kDa MWCO Amicon centrifuge filter. The DBCO derivative was purified by centrifugation analysis using three exchanges with water (12 mL each), followed by six exchanges with 20% aqueous ethanol (12 mL each), and finally three exchanges with water (12 mL each) to obtain the 9N-DBCO derivative. Sucrose solution was added to this solution (2.388 mL, 9.05 mg), and it was freeze-dried. [Table 33]

[0316] 3. Bonding of PS 9N-DBCO derivative with eCRM Lyophilized powder containing PS 9N-DBCO: 4.5 mg (including 45 mg of sucrose) DBCO percentage (%): 4.9% CRM concentration: 3.0 mg / mL solution PS:CRM (input ratio): 1.5:1 Reaction procedure PS9N-DBCO was dissolved in 0.9% NaCl (1.30 mL) with pH=7 phosphate buffer (96 μL of 0.5 M), and DMSO (0.24 mL) was added. Next, azide-functionalized eCRM solution (0.60 mL) was added to obtain a PS:CRM mass ratio of 1.5:1 (w / w). The solution was mixed very gently by hand, and then gently mixed with a cyclic stirrer at room temperature (20°C) for 18 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 100 μL). The CRM conjugates were transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO), and then dialyzed for 24 hours (7 changes, 1000 ml each) with 0.9% sodium chloride solution containing 3 mL of pH=7 buffer added to the tubes. The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 9N-CRM conjugate solution. [Table 34]

[0317] Example 19: Preparation of pneumococcal PS serotype 9V conjugates in eCRM from Table 2 1. Oxidation 9V type PS purity: 85% (Antron) Molecular weight: 704 kDa (repeating unit = 971.8 g / mol) Reaction procedure 9V type PS (35.90 mg, 37.80 μmol) powder was dissolved using a stirring bar in 17.95 mL of aqueous solution (12.565 mL of water and 5.385 mL of 0.2 M acetate buffer, pH 5.5) in a 50 mL polystyrene sample tube. Once the PS was solubilized, 852 μL of NaIO4 solution (2.83 mg, 13.23 μmol, 0.35 molar equivalent) was added. The reaction tube was wrapped in foil and placed in a water bath at 24°C. The mixture was stirred at 24°C. After 18 hours, the reaction mixture was dialyzed using three AMICON® Ultra-15 centrifugal filter devices (30 kDa MWCO, 15 mL) with four exchanges (15 mL each) with HPLC-grade water to obtain oxidized PS-9V solution. [Table 35]

[0318] 2. DBCO derivatization Reaction procedure To a solution of oxidized 9V type PS (21.64 mg, 22.78 μmol, 4.27 mL), buffer solution (0.541 mL of 0.5 M phosphate buffer, pH 6.0), DMSO (66 μL), and DBCO-PEG4-NH2 solution (11.9 mg, 22.78 μmol, 1 molar equivalent in 475 μL of DMSO) were added. The reaction mixture was stirred at 25°C for 30 minutes, after which 140 μL of sodium borohydride solution (2.86 mg, 45.56 μmol, 2 molar equivalents in 140 μL of water) was added. The reaction mixture was wrapped in aluminum foil and stirred continuously in a water bath set at 25°C for 2 days. On the second day, the reaction was interrupted by adding 163 μL of sodium borohydride solution (1.72 mg, 45.56 μmol, 2 molar equivalents in 163 μL of water). After stirring for 30 minutes (until observable bubbling ceased), the reaction mixture was extracted with ethyl acetic acid (2 × 10 mL), followed by dichloromethane (2 × 10 mL). The extract was bubbling with N2 for 20 minutes to remove residual dichloromethane, and then transferred to a 2 AMICON® Ultra-15 centrifugal filter device (50 kDa MWCO, 15 mL). Dialysis was performed by three exchanges with 3% DMSO solution (15 mL each), three exchanges with 20% ethanol solution (15 mL each), and two exchanges with HPLC-grade water (15 mL each) to obtain the 9V DBCO derivative. This solution (4.40 mL, 12.144 mg) was added to sucrose solution (121.44 mg, 1.214 mL water). The combined solution was divided into three fractions (2 × 5 mg and 1 × 2.14 mg), and each was freeze-dried to obtain a fine white powder. All fractions were stored at 4°C until required for the bonding reaction. [Table 36]

[0319] 3. Junction of PS 9V-DBCO derivative with eCRM PS 9V-DBCO: 5 mg (containing 50 mg of sucrose) white powder DBCO percentage (%): 3.0% CRM concentration: Solution with a concentration of 6.009 mg / mL PS:CRM (input ratio): 1.5:1 Reaction procedure A 9V DBCO derivative (5.0 mg of white powder containing 50 mg of sucrose) was dissolved in 0.881 mL of 0.9% sodium chloride solution, 0.067 mL of pH 7 phosphate buffer (0.5 M), and DMSO (0.167 mL). A azide-functionalized eCRM solution (0.555 mL of solution) was added to obtain a PS9V:CRM mass ratio of 1.5:1 (w / w). The reaction mixture was gently mixed in a cyclic stirrer at room temperature (20°C) for 18 hours, followed by a further 2 hours at 37°C. After the total reaction time, a certain volume of sodium azide was added to the combined mixture (0.33 mg, 5.15 μmol). Next, the reaction mixture was diluted with 0.9% sodium chloride solution (2.83 mL) and transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO). The sample was dialyzed in 0.9% sodium chloride solution for 48 hours (8 exchanges, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 9V PS-CRM conjugate solution. [Table 37]

[0320] Example 20: Preparation of pneumococcal PS serotype 9V conjugates in eCRM from Table 2 1. Oxidation 9V type PS purity: 81% (Antron) Molecular weight: 949.83 NaIO4 solution in water (5.41 mg / mL) Reaction procedure PS-9V powder (21.15 mg, adjusted to 81%, 17.13 mg, 18.04 μmol) was dissolved in 10.57 mL of aqueous solution (7.4 mL of water and 3.17 mL of 0.2 M acetate buffer, pH=5.5). To this solution, 214 μL of NaIO4 solution (1.16 mg, 5.41 μmol, 0.3 equivalents) was added. The mixture was stirred at 25°C for 20 hours. The oxidized sample was purified through an AMICON ultracentrifuge filter (6-12 mL of 30 kDa MWCO) using six changes (12 mL) of HPLC-grade water to obtain an oxidized PS-9V solution. [Table 38]

[0321] 2. DBCO derivatization Reaction procedure To a solution of oxidized (assuming a 10% oxidation level) 9V type PS (15.36 mg, 16.17 μmol, 2.20 mL of water), buffer solution (1.4 mL of 200 mM phosphate buffer, pH=6.01), DMSO (500 μL), and DBCO-PEG-4-NH2 (8.46 mg, 16.17 μmol, 10 equivalents in 131 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 41 μL of sodium cyanoborohydroxide solution (15.5 mg, 32.34 μmol, 20 equivalents in 200 μL of water) was added, and the mixture was stirred at 25°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 62 μL of sodium borohydride solution (0.01 mg / μL, 10 equivalents) was added to water. After stirring for 30 minutes, the reaction mixture was extracted with ethyl acetic acid (3 × 20 mL). The extract was transferred to an AMICON ultracentrifuge filter (6-12 mL of 30 kDa MWCO) and dialyzed using six exchanges with 20% ethanol in water (12 mL each), followed by three exchanges with water (12 mL each) to obtain the 9V DBCO derivative. To this solution (4.0 mL, 10.08 mg), sucrose solution (100 mg in 1 mL of water) was added. The combined solution was divided into two equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 5.04 mg of 9V DBCO and 50 mg of sucrose for use in the subsequent conjugation reaction. [Table 39]

[0322] 3. Junction of PS 9V-DBCO derivative with eCRM PS 9V-DBCO: 5.04 mg (containing 50 mg of sucrose) white powder DBCO percentage (%): 3.42% CRM concentration: Solution with a concentration of 3.923 mg / mL PS:CRM (input ratio): 1.11:1 Reaction procedure An azide-functionalized eCRM solution (CRM in 0.1156 mL of solution) was added to a 9V DBCO derivative (5.04 mg of white powder containing 50 mg of sucrose) to obtain a PS9V:CRM mass ratio of 1.11:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 18 hours. The conjugated PS-CRM mixture was transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO) and then dialyzed with a 0.9% sodium chloride solution for 24 hours (5 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 9V PS-CRM conjugate solution. [Table 40]

[0323] Example 21: Preparation of pneumococcal PS serotype 10A conjugates into eCRM from Table 2 1. CDAP activation and DBCO crosslinking PS 10A purity: 77% (Anthron) Molecular weight: 1227 g mole-1 (repeating unit) Reaction procedure PS10A (18.7 mg, 15.2 μmol) was dissolved in water (7.9 mL), and CDAP (0.8 equivalents, 100 mg / mL in acetonitrile, 30 μL) was added. The reaction mixture was stirred at room temperature for 30 seconds. At this point, sodium hydroxide solution (0.2 M, 200 μL) was added to achieve a pH of 9.5, and the reaction mixture was stirred for 150 seconds. Next, DMSO (1.2 mL), followed by DBCO-PEG4-NH2 (0.5 equivalents, 32.0 μmol / mL in DMSO, 238 μL) was added, and the mixture was stirred overnight at room temperature. DBCO-derivativeized PS10A was purified by solvent extraction and centrifugal dialysis (Amicon 30 kDa MWCO) using three exchanges of 3% (v / v) DMSO, two exchanges of 0.9% (v / v) saline, and three exchanges of water. After analysis by UV absorption spectroscopy, anthron assay, and SEC, this solution (3.18 mL, 13.5 mg) was diluted with sucrose solution (10 times the mass content, 100 mg / mL) and lyophilized to a white powder. [Table 41]

[0324] 2. Conjugation of PS10A-DBCO derivative with eCRM PS 10A-DBCO: 5.00 mg (containing 50.0 mg of sucrose) lyophilized powder DBCO percentage (%): 8.8% CRM: 5.0 mg / mL in PBS buffer PS:CRM (input mass ratio): 1.75:1 Reaction procedure Lyophilized 10A-DBCO was dissolved in saline solution (0.9% (w / v), 3.759 ml), phosphate buffer (0.5 M, pH 7.0, 200 μL), and DMSO (500 μL). To this, eCRM solution (0.541 mL) was added to obtain a PS10A:CRM mass ratio of 1.75:1.00 (w / w). The solution was very gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 17 hours. The CRM conjugates were transferred to two pre-washed dialysis tubes (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 24 hours (three changes, 800 ml each). The dialyzed solution was sterile filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a 10A-CRM conjugate solution. [Table 42]

[0325] Example 22: Preparation of eCRM pneumococcal PS serotype 11A conjugates from Table 2 1. Hydrolysis Purity of 11A type PS: 69% (Antron) Molecular weight: 908.7 g / mol -1 Reaction procedure Natural polysaccharide 11A (35.0 mg) was dissolved in 17.5 mL of aqueous solution (15.75 mL of water and 1.75 mL of acetic acid, 2 M). The mixture was heated at 80°C for 1 hour, then cooled to ambient temperature, and sodium hydroxide solution was added to pH 5.5 (3.2 mL, 1 M). The hydrolyzed PS was purified using Amicon centrifuge 30 kDa MWCO dialysis with at least 6 exchanges with water to obtain a purified PS-3 solution, which was then freeze-dried in individual portions. [Table 43]

[0326] 2. Oxidation Reaction procedure To a hydrolyzed polysaccharide solution (5.027 mL, 28.75 mg, 31.6 μmol), water (5.75 mL) and acetate buffer (0.2 M, pH 5.5, 3.6 mL) were added. To this solution, 135 μL of sodium periodate solution (1.35 mg, 6.32 μmol, 0.20 equivalents) was added dropwise. The mixture was stirred at 25°C for 8 hours. Oxidized PS was purified using Amicon centrifugation 100 kDa MWCO dialysis with at least 6 exchanges with water to obtain a purified PS-11A-OX solution. [Table 44]

[0327] 3. DBCO derivatization Reaction procedure Add PS11A-OX (22.0 mg, 24.2 μmol, 2.235 mL) to phosphate buffer (1.37 mL, 200 mM, pH 6.0), and then add DBCO-PEG4-NH2 (1.0 equivalent, 523 gmol in DMSO). -1 100 mg / mL, 127 μL) and an additional volume of DMSO (560 μL) were added. The reaction mixture was stirred at 25°C for 25 minutes, then sodium cyanohydroxide solution (2 equivalents, 44.5 mg / mL, 68 μL) was added and stirred for 2 days. The reaction mixture was extracted with ethyl acetic acid (3 × 20 mL) and filtered through a 0.45 μm syringe filter. The DBCO derivative was purified by centrifugal dialysis using an Amicon 100 kDa MWCO unit with 7 exchanges with 20% ethanol in water, followed by 3 exchanges with water (12 mL each), to obtain the 11A-DBCO derivative type. Sucrose solution (150 mg in 1.5 mL of water) was added to this solution (2.535 mL, 15.00 mg). The combined solution was divided into three equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 5.00 mg of 11A-DBCO and 50 mg of sucrose for use in the subsequent conjugation reaction. [Table 45]

[0328] 4. Conjugation of PS 11A-DBCO derivative with eCRM PS 11A-DBCO: 5.0 mg (containing 50 mg of sucrose) lyophilized powder DBCO percentage (%): 5.77% CRM concentration: 5.42 mg / mL solution PS:CRM (input ratio): 1.5:1 Reaction procedure 11A-DBCO was dissolved in 0.9% sodium chloride solution (7.656 mL, filtered to 0.22 μm), phosphate buffer (pH 7.0, 0.5 M, 0.385 mL), and DMSO (0.962 mL). Azide-functionalized eCRM solution (5.42 mg / mL, 0.617 mL) was added dropwise to obtain a PS11A:CRM mass ratio of 1.5:1 (w / w). The solution was mixed very gently by hand, and then gently mixed in a cyclic stirrer at room temperature (20°C) for 17 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 50 μL). The CRM conjugate was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, 300K MWCO), and then dialyzed with a 0.9% sodium chloride solution for 48 hours (four changes, 1L each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain the 11A-CRM conjugate solution. [Table 46]

[0329] Example 23: Preparation of pneumococcal PS serotype 12F conjugates into eCRM from Table 2 1. Oxidation 12F type PS purity: 82% (Antron) Molecular weight: 1094 g mole-1 Reaction procedure 12F type PS (21.8 mg, 20 μmol) powder was dissolved using a stirring bar in 10.9 mL of aqueous solution (8.175 mL of water and 2.725 mL of 0.2 M acetate buffer, pH 5.5) in a 50 mL polystyrene sample tube. Once the PS was solubilized, 160 μL of NaIO4 solution (0.64 mg, 3 μmol, 0.15 molar equivalent) was added. The reaction tube was wrapped in foil and placed in a 25°C water bath. The mixture was stirred at 25°C. After 18 hours, the reaction mixture was dialyzed using two AMICON® Ultra-15 centrifugal filter devices (30 kDa MWCO, 15 mL) with six exchanges (15 mL each) with HPLC-grade water to obtain oxidized PS-12F solution. [Table 47]

[0330] 2. DBCO derivatization Reaction procedure Add PS12F-OX (13.1 mg, 12 μmol, 2.68 mL) to phosphate buffer (1.00 mL, 200 mM, pH 6.0), and then add DBCO-PEG4-NH2 (1.0 equivalent, 523 gmol in DMSO). -1 33 mg / mL, 199 μL) and an additional volume of DMSO (500 μL) were added. The reaction mixture was stirred at 25°C for 25 minutes, then sodium cyanohydroxide solution (2 equivalents, 52.5 mg / mL, 29 μL) was added and stirred for 2 days. The reaction mixture was extracted with ethyl acetic acid (3 × 20 mL) and bubbling without solvent. The DBCO derivative was purified by two centrifugal dialysis units (Amicon 30 kDa MWCO) using six exchanges with 20% ethanol in water, followed by three exchanges with water (12 mL each time) to obtain the 12F-DBCO derivative. To this solution (2.2 mL, 10.45 mg), sucrose solution (104.5 mg in 1.05 mL of water) was added. The combined solution was divided into two equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 5.0 mg of 12F-DBCO and 50 mg of sucrose for use in the conjugation reaction. [Table 48]

[0331] 3. Conjugation of PS 12F-DBCO derivative with eCRM PS 12F-DBCO: 5.0 mg (containing 50 mg of sucrose) lyophilized powder DBCO percentage (%): 2.0% CRM concentration: 5.29 mg / mL solution PS:CRM (input ratio): 1.5:1 Reaction procedure 12F-DBCO was dissolved in 0.9% sodium chloride solution (6.542 mL, filtered to 0.22 μm), phosphate buffer (pH 7.0, 0.5 M, 0.334 mL), and DMSO (0.834 mL). Azide-functionalized eCRM solution (5.29 mg / mL, 0.630 mL) was added dropwise to obtain a PS12F:CRM mixing ratio of 1.5:1 (w / w). The solutions were mixed very gently by hand and then gently stirred in a circular stirrer at room temperature (20°C) for 17 hours. The click reaction was quenched by adding sodium azide solution (10 mg / mL, 50 μL). The CRM conjugates were transferred to pre-washed dialysis tubing (SpectrumLab Float-A-Lyzer G2, 300K MWCO) and then dialyzed with 0.9% sodium chloride solution for 48 hours (four changes, 1L each). The dialyzed solution was filtered through a Millex-GP syringe filter (0.22 μm, 33 mm polyethersulfone) to obtain a sterile 12F-CRM conjugate solution. [Table 49]

[0332] Example 24: Preparation of pneumococcal PS serotype 14 conjugates in eCRM from Table 2 1. Oxidation Type 14 PS purity: 91% (Antron) Molecular weight: 689.25 NaIO4 solution in water (7.8 mg / mL) Reaction procedure PS-14 powder (28.3 mg, adjusted to 80%, 25.75 mg, 37.36 μmol) was dissolved in 14 mL of aqueous solution (10 mL of water and 4 mL of 0.2 M acetate buffer, pH=5.5). To this solution, 110 μL of NaIO4 solution (0.86 mg, 4.05 μmol, 0.13 equivalents) was added. The mixture was stirred at 25°C for 3 hours, and then the oxidized sample was purified using an AMICON ultracentrifuge (30 kDa MWCO 6-12 mL) with HPLC-grade water in 6 exchanges (12 mL) to obtain oxidized PS-14 solution. [Table 50]

[0333] 2. DBCO derivatization Reaction procedure To a solution of oxidized (assuming a 10% oxidation level) type 14 PS (20.5 mg, 29.74 μmol, 2.92 mL of water), buffer solution (1.3 mL of 200 mM phosphate buffer, pH=6.8), DMSO (550 μL), and DBCO-PEG-4-NH2 solution (11.68 mg, 22.3 μmol, 0.75 equivalents in 150 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 70 μL of sodium cyanoborohydroxide solution (6.39 mg, 59.48 μmol, 20 equivalents in 120 μL of water) was added, and the mixture was stirred at 25°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 100 μL of sodium borohydride solution (1.13 mg, 10 equivalents) was added to the water. After stirring for 30 minutes, the reaction mixture was extracted with ethyl acetate (3 × 20 mL of ethyl acetate), then transferred to an AMICON ultracentrifuge (6-12 mL of 30 kDa MWCO), and dialyzed using seven exchanges with 20% ethanol in water (12 mL each), followed by three exchanges with water (12 mL each) to obtain the 14DBCO derivative. To this solution (3.78 mL, 17.7 mg), sucrose solution (177 mg in 1.17 mL of water) was added. The combined solution was divided into three equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 5.9 mg of 14DBCO and 59 mg of sucrose for use in the subsequent conjugation reaction. [Table 51]

[0334] 3. Conjugation of PS 14-DBCO derivative with eCRM PS 6B-DBCO: 5.9 mg (containing 59 mg of sucrose) white powder DBCO percentage (%): 3.5% CRM concentration: 5.06 mg / mL solution PS:CRM (input ratio): 1.5:1 Reaction procedure A 0.779 mL azide-functionalized eCRM solution was added to a 14DBCO derivative (5.9 mg of white powder containing 59 mg of sucrose) to obtain a PS14:CRM mass ratio of 1.5:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 18 hours. The conjugated PS-CRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235071, 100K MWCO) and then dialyzed with a 0.9% sodium chloride solution for 48 hours (8 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 14 PS-CRM conjugate solution. [Table 52]

[0335] Example 25: Preparation of pneumococcal PS serotype 14 conjugates in eCRM from Table 2 1. Oxidation Type 14 PS purity: 91% (Antron) Molecular weight: 689.25 NaIO4 solution in water (10.19 mg / mL) Reaction procedure PS-14 powder (23.5 mg, adjusted to 80%, 21.38 mg, 31.02 μmol) was dissolved in 11.75 mL of aqueous solution (8.2 mL of water and 3.55 mL of 0.2 M acetate buffer, pH=5.5). To this solution, 97 μL of NaIO4 solution (0.95 mg, 4.03 μmol, 0.13 equivalents) was added. The mixture was stirred at 25°C for 18 hours, and then the oxidized sample was purified using an AMICON ultracentrifuge (30 kDa MWCO 6-12 mL) with HPLC-grade water in six exchanges (12 mL) to obtain oxidized PS-14 solution. [Table 53]

[0336] 2. DBCO derivatization Reaction procedure To a solution of oxidized (assuming a 10% oxidation level) type 14 PS (14.3 mg, 20.75 μmol, 3.46 mL of water), buffer solution (1.3 mL of 200 mM phosphate buffer, pH=6.8), DMSO (637 μL), and DBCO-PEG-4-NH2 solution (10.86 mg, 20.75 μmol, 10 equivalents in 263 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 51 μL of sodium cyanoborohydroxide solution (10.2 mg, 41.50 μmol, 20 equivalents in 200 μL of water) was added, and the mixture was stirred at 25°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 78 μL of sodium borohydride solution (0.01 mg / μL, 10 equivalents) was added to water. After stirring for 30 minutes, the reaction mixture was extracted with ethyl acetate (3 × 20 mL of ethyl acetate), then transferred to an AMICON ultracentrifuge (6-12 mL of 30 kDa MWCO), and dialyzed using six exchanges with 20% ethanol in water (12 mL each), followed by three exchanges with water (12 mL each) to obtain the 14DBCO derivative. Sucrose solution (12 mg in 1 mL of water) was added to this solution (4.12 mL, 12.24 mg). The combined solution was divided into three equal parts, each of which was freeze-dried to obtain three samples of white powder. Each sample contained 6.12 mg of 14DBCO and 6 mg of sucrose for use in the subsequent conjugation reaction. [Table 54]

[0337] 3. Conjugation of PS 14-DBCO derivative with eCRM PS 6B-DBCO: 6.12 mg (containing 62 mg of sucrose) white powder DBCO percentage (%): 4.42% CRM concentration: Solution with a concentration of 2.617 mg / mL PS:CRM (input ratio): 1.8:1 Reaction procedure 1.3 mL of azide-functionalized eCRM solution was mixed with 14DBCO derivative (6.12 mg of white powder containing 62 mg of sucrose) to obtain a PS14:CRM mass ratio of 1.8:1 (w / w). The reaction mixture was gently mixed by hand and then gently mixed in a circular stirrer at room temperature (20°C) for 17 hours. The conjugated PS-CRM mixture was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235071, 100K MWCO) and then dialyzed with 0.9% sodium chloride solution for 24 hours (3 changes, 800 ml each). The dialyzed solution (1.5 mL) was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain the 14 PS-CRM conjugate solution. [Table 55]

[0338] Example 26: Preparation of pneumococcal PS serotype 15B conjugates in eCRM from Table 2 1. Oxidation Purity of 15B type PS: 71% (Antron) Molecular weight: 1185 kDa (repeating unit = 1069.80 g / mol) Reaction procedure 15B type PS (14.6 mg, 13.65 μmol) powder was dissolved using a stirring bar in 7.30 mL of aqueous solution (5.1 mL of water and 2.2 mL of 0.2 M acetate buffer, pH 5.5) in a 50 mL polystyrene sample tube. Once the PS was solubilized, 160 μL of NaIO4 solution (0.59 mg, 2.75 μmol, 0.20 molar equivalent) was added. The reaction tube was wrapped in foil and placed in a water bath, where it was stirred at 24°C. After 3.5 hours, the reaction mixture was dialyzed using one AMICON® Ultra-15 centrifugal filter device (30 kDa MWCO, 15 mL) with six exchanges (15 mL each) with HPLC-grade water to obtain oxidized PS-15B solution. [Table 56]

[0339] 2. DBCO derivatization Reaction procedure To a solution of oxidized PS type 15B (7.56 mg, 7.07 μmol, 1.271 mL), buffer solution (0.640 mL of 0.5 M phosphate buffer, pH 6.0), DMSO (0.063 mL), and DBCO-PEG4-NH2 solution (17 mg, 7.07 μmol, 1 molar equivalent in 221 μL of DMSO) were added. The reaction mixture was stirred at 25°C for 30 minutes, after which 350 μL of sodium borohydride solution (0.90 mg, 2 molar equivalents in 350 μL of water) was added. The reaction mixture was wrapped in aluminum foil and stirred continuously in a water bath set at 25°C for 2 days. On the second day, the reaction was interrupted by adding 163 μL of sodium borohydride solution (0.27 mg, 7.07 μmol, 2 molar equivalents). After stirring for 30 minutes, the reaction mixture was extracted with dichloromethane (3 × 15 mL). The extract was bubbling with N2 for 20 minutes to remove residual dichloromethane, and then transferred to one AMICON® Ultra-15 centrifugal filter device (30 kDa MWCO, 15 mL). Dialysis was performed by three exchanges with 3% DMSO solution (15 mL each), three exchanges with 20% ethanol solution (15 mL each), and three exchanges with HPLC-grade water (15 mL each) to obtain the 15B DBCO derivative. Sucrose solution (68.6 mg in 0.686 mL of water) was added to this solution (1.982 mL, 6.86 mg). This combined solution was divided into two fractions, each of which was lyophilized to obtain a fine white powder. All fractions were lyophilized until dry enough to be used for the conjugation reaction and then stored at 4°C. [Table 57]

[0340] 3. Bonding of PS 15B-DBCO derivative with eCRM PS 15B-DBCO: 3.85 mg (containing 38.5 mg of sucrose) white powder DBCO percentage (%): 5.9% CRM concentration: Solution with a concentration of 6.009 mg / mL PS:CRM (input ratio): 1.5:1 Reaction procedure A 15B DBCO derivative (3.85 mg of white powder containing 38.5 mg of sucrose) was dissolved in 0.9% sodium chloride solution (4.302 mL), pH 7 phosphate buffer (0.220 mL, 0.5 M), and DMSO (0.550 mL). A azide-functionalized eCRM solution (0.467 mL of solution) was added to obtain a PS15B:CRM mass ratio of 1.5:1 (w / w). The reaction mixture was gently mixed in a cyclic stirrer at room temperature (20°C) for 18 hours, followed by a further 2 hours at 37°C. The conjugation reaction was terminated by the addition of sodium azide (0.23 mg, 3.60 μmol). Next, the reaction mixture was diluted with 0.9% sodium chloride solution to a final volume of 7 mL and transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO). The samples were dialyzed in a 0.9% sodium chloride solution for 48 hours (8 exchanges, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 15B PS-CRM conjugate solution. [Table 58]

[0341] Example 27: Preparation of pneumococcal PS serotype 17F conjugates into eCRM from Table 2 1. Oxidation Purity of 17F type PS: 84% (Antron) Molecular weight: 1274 kDa (repeating unit = 1203.00 g / mol) Reaction procedure 17F type PS (28.50 mg, 23.69 μmol) powder was dissolved using a stirring bar in 14.25 mL of aqueous solution (9.925 mL of water and 4.275 mL of 0.2 M acetate buffer, pH 5.5) in a 50 mL polystyrene sample tube. Once the PS was solubilized, 53.8 μL of NaIO4 solution (0.65 mg, 3.03 μmol, 0.128 molar equivalent) was added. The reaction tube was wrapped in foil and placed in a water bath, where it was stirred at 24°C. After 1 hour, the reaction mixture was dialyzed using two AMICON® Ultra-15 centrifugal filter devices (30 kDa MWCO, 15 mL) with five exchanges (15 mL each) with HPLC-grade water to obtain oxidized PS-17F solution. [Table 59]

[0342] 2. DBCO derivatization Reaction procedure To a solution of oxidized PS type 17F (22.0 mg, 18.29 μmol, 2.48 mL), buffer solution (1.31 mL of 0.5 M phosphate buffer, pH 6.0) and DBCO-PEG4-NH2 solution (9.58 mg, 18.29 μmol, 1 molar equivalent in 95.8 μL of DMSO) were added. The reaction mixture was stirred at 25°C for 30 minutes, after which sodium borohydride solution (2.30 mg in 200 μL of water; 36.60 μmol, 2 molar equivalents) was added. The reaction mixture was wrapped in aluminum foil and stirred continuously in a water bath set at 25°C for 2 days. On the second day, the reaction was interrupted by adding sodium borohydride solution (0.48 mg, 18.29 μmol, 1 molar equivalent). After stirring for 30 minutes, the reaction mixture was extracted with dichloromethane (3 × 15 mL). The extract was bubbling with N2 for 20 minutes to remove residual dichloromethane, and then transferred to one AMICON® Ultra-15 centrifugal filter device (30 kDa MWCO, 15 mL). Dialysis was performed by five exchanges (15 mL each) with 20% ethanol solution and three exchanges (15 mL each) with HPLC-grade water to obtain the 17F DBCO derivative. To this solution (3.27 mL, 11.58 mg), sucrose solution (115.8 mg in 1.158 mL of water) was added. This combined solution was divided into three fractions (2 × 5 mg, 1 × 1.58 mg), each of which was lyophilized to obtain a fine white powder. All fractions were lyophilized until dry enough to be required for the conjugation reaction and then stored at 4°C. [Table 60]

[0343] 3. Conjugation of PS 17F-DBCO derivative with eCRM PS 17F-DBCO: 5 mg (containing 50 mg of sucrose) - White powder DBCO percentage (%): 3.1% CRM concentration: Solution with a concentration of 5.996 mg / mL PS:CRM (input ratio): 1.5:1 Reaction procedure A 17F DBCO derivative (5 mg of white powder containing 50 mg of sucrose) was dissolved in 0.9% sodium chloride solution (3.742 mL), pH 7 phosphate buffer (0.200 mL, 0.5 M), and DMSO (0.500 mL). A azide-functionalized eCRM solution (0.558 mL of solution) was added to obtain a PS17F:CRM mass ratio of 1.5:1 (w / w). The reaction mixture was gently mixed with a cyclic stirrer at room temperature (20°C) for 19 hours. The conjugation reaction was terminated by the addition of sodium azide (0.27 mg, 4.16 μmol, 1 molar equivalent). Next, the reaction mixture was diluted with 0.9% sodium chloride solution to a final volume of 8 mL and transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO). The samples were dialyzed in a 0.9% sodium chloride solution for 48 hours (8 exchanges, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain a 17F PS-CRM conjugate solution. [Table 61]

[0344] Example 28: Preparation of Streptococcus pneumoniae PS serotype 18C conjugates into eCRM from Table 2 1. Oxidation Purity of 18C type PS: 72% (Anthron) Molecular weight: 970.76 NaIO4 solution in water (5.41 mg / mL) Reaction procedure 18C type PS (61 mg, 62.84 μmol) powder was dissolved in 30.5 mL of aqueous solution (27.45 mL of water and 3.05 mL of 2 M acetic acid). The solution was then heated at 95°C for 40 minutes, then cooled, and the pH was adjusted to 6.0 by adding NaOH solution (1 N, 5.2 mL). The reaction mixture was dialyzed using an AMICON ultracentrifuge (100 kDa MWCO 6-12 mL) with three exchanges (12 mL each) with HPLC-grade water. The supernatant was transferred to a 50 mL Falcon tube containing 12.4 mL of water. To this solution, 5.15 mL of water, 5.8 mL of 200 mM acetate buffer (pH 5.35), and 153 μL of NaIO4 solution (1.53 mg, 7.175 μmol, 0.15 equivalents) were added. The mixture was stirred at 25°C for 3 hours, and then the oxidized sample was purified using HPLC-grade water in an AMICON ultracentrifuge (6-12 mL of 100 kDa MWCO) with 6 exchanges (12 mL) to obtain oxidized PS-18C solution. [Table 62]

[0345] 2. DBCO derivatization Reaction procedure To an oxidized (assuming a 10% oxidation level) 18C type PS solution (10.0 mg, 10.3 μmol, 1.55 mL of water), buffer solution (0.211 mL of 200 mM phosphate buffer, pH=6.74), DMSO (141 μL), and DBCO-PEG-4-NH2 solution (5.4 mg, 16.17 μmol, 10 equivalents in 54 μL of DMSO) were added, all at 25°C. The reaction mixture was then stirred at 25°C for 30 minutes, after which 130 μL of sodium cyanoborohydroxide solution (1.3 mg, 20.6 μmol, 20 equivalents in 130 μL of water) was added, and the mixture was stirred at 37°C for 2 days. The reaction mixture was diluted with phosphate buffer (500 μL of 200 mM solution, pH=6), and then 80 μL of sodium borohydride solution (0.01 mg / μL, 10 equivalents) was added to water. After stirring for 30 minutes, the reaction mixture was extracted with dichloromethane (2 × 10 mL), followed by ethyl acetic acid (10 mL). The extract was transferred to an AMICON ultracentrifuge filter (6-12 mL of 100 kDa MWCO), and then dialyzed using four exchanges with 20% ethanol in water (12 mL each), followed by three exchanges with water (12 mL each) to obtain the 18C DBCO derivative. To this solution (1.31 mL, 7.0 mg), sucrose solution (70 mg in 0.7 mL of water) was added. The combined solution was divided into two equal parts, each of which was freeze-dried to obtain two samples of white powder. Each sample contained 3.5 mg of 18C DBCO and 35 mg of sucrose for use in the subsequent conjugation reaction. [Table 63]

[0346] 3. Bonding of PS 18C-DBCO derivative with eCRM PS 18C-DBCO: 3.5 mg (containing 35 mg of sucrose) white powder DBCO percentage (%): 5.32% CRM concentration: Solution with a concentration of 2.76 mg / mL

[0347] PS:CRM (input ratio): 1.5:1 Reaction procedure 18C DBCO derivative (3.5 mg of white powder containing 35 mg of sucrose) was dissolved in 0.661 mL of 0.9% sodium chloride solution, 0.07 mL of pH 7 phosphate buffer (0.5 M), and DMSO (0.175 mL). A azide-functionalized eCRM solution (0.844 mL of solution) was added to obtain a PS18C:CRM mass ratio of 1.5:1 (w / w). The reaction mixture was gently mixed and then gently stirred with a cyclic stirrer at room temperature (20°C) for 2 hours. Next, the reaction mixture was diluted with 0.661 mL of 0.9% sodium chloride solution, 0.07 mL of pH 7 phosphate buffer (0.5 M), and DMSO (0.175 mL) to a final concentration of PS-18 of 1 mg / mL, and the mixture was reacted for 18 hours. Sodium azide solution (23 μL, 10 mg / mL in water) was added. After 30 minutes, the combined PS-CRM mixture was transferred to a pre-washed dialysis device (SpectrumLab Float-A-Lyzer G2, Cat. No. G235060, 300K MWCO), and then dialyzed with 0.9% sodium chloride solution for 48 hours (8 changes, 800 ml each). The dialyzed solution was filtered through Millex-GP (0.22 μm, 33 mm polyethersulfone) to obtain an 18C PS-CRM conjugate solution. [Table 64]

[0348] Example 29: Preparation of Streptococcus pneumoniae PS serotype 18C conjugates into eCRM from Table 2 1. Oxidation Molecular weight of the repeating unit of 18C-type PS: 10¹² NaIO4 solution in water (10 mg / mL) Reaction procedure PS-18C (20 mg, 19.76 μmol) powder was dissolved in 3 mL of aqueous solution (10 mM sodium acetate solution, pH 4.5). To this solution, 63.4 μL of NaIO4 solution (0.634 mg, 2.96 μmol, 0.15 equivalents) was added. The mixture was stirred at 23°C for 18 hours, and then the oxidized sample was transferred to a pre-washed dialysis tube (SpectrumLab Float-A-Lyzer G2, Cat. No. G235057, 20K MWCO). Next, the sample was dialyzed with 50 ml of PB buffer at pH 6.8 for 24 hours (four changes, 600 ml each) to obtain the oxidized PS-18C solution. After dialysis, DMSO was added to prepare PS-18C in 10% DMSO containing 50 ml of PB buffer at pH 6.8. [Table 65]

[0349] 2. DBCO derivatization Final PS concentration: 3.75 mg / ml Final buffer concentration: 10% DMSO in 50 mM PB (pH 6.8) Reaction procedure To a solution of type 18C oxidized PS (15 mg, 14.8 μmol, 4.4 mL in 10% DMSO, 50 mM PB, pH 6.8), DBCO-PEG4-NH2 solution (7.76 mg, 14.8 μmol, 10 equivalents in 77.6 μL of DMSO) was added at 25°C. Next, the reaction mixture was stirred at 25°C for 60 minutes, after which sodium cyanoborohydroxide solution (0.93 mg, 14.8 μmol, 10 equivalents i...

Claims

1. A carrier protein comprising at least one T cell activation epitope, exhibiting at least 90% sequence identity with SEQ ID NO: 1, wherein the 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF) non-natural amino acid (nnAA) is replaced with naturally occurring amino acids at residues K34, K213, K245, K265, K386 and K527 of SEQ ID NO:

1.

2. The carrier protein according to claim 1, wherein the carrier protein has at least 95% sequence identity with SEQ ID NO:

1.

3. The carrier protein according to claim 1 or 2, wherein the carrier protein comprises SEQ ID NO:

9.

4. A method for producing a carrier protein according to any one of claims 1 to 3, (a) To provide a nucleic acid that encodes a carrier protein and includes a plurality of repression codons, (b) Creating a reaction mixture by combining a cell-free bacterial extract containing non-natural amino acids, tRNA complementary to repressive codons and capable of being charged with pAMF, and aminoacyl-tRNA synthetase with nucleic acids, (c) Culturing the reaction mixture from (b) under conditions sufficient to selectively incorporate pAMF at the sites corresponding to each repressive codon in the carrier protein. A method that includes [this].

5. The method of claim 4, wherein the culture is carried out at a temperature of approximately 10°C to approximately 30°C, and the resulting carrier protein contains both a soluble fraction and an insoluble fraction, and the ratio of soluble fraction / (soluble fraction + insoluble fraction) is at least 30% (w / w).

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