WO2020010000A1

WO2020010000A1 - Improved methods for the preparation of immunogenic conjugates

Info

Publication number: WO2020010000A1
Application number: PCT/US2019/040143
Authority: WO
Inventors: Jeffery Fairman; Jon Heinrichs; Wei Chan; Olivier MARCQ; Christopher BEHRENS
Original assignee: Sutrovax, Inc.
Priority date: 2018-07-04
Filing date: 2019-07-01
Publication date: 2020-01-09

Abstract

Improved methods are provided for preparing immunoconjugates from a saccharide antigen and a carrier protein in which the saccharide antigen is activated by functionalization with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, and the activated antigen so provided is then covalently conjugated to a carrier protein. The carrier protein comprises a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety. Improved methods for activating saccharide antigens in preparation for covalent conjugation to a carrier protein are also provided.

Description

IMPROVED METHODS FOR THE PREPARATION OF

IMMUNOGENIC CONJUGATES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application priority benefit of provisional U.S. patent application serial number 62/693,978 filed July 4, 2018, the content of which is hereby incorporated by reference in its entirety.

INCORPORATION OF THE ELECTRONIC TEXT FILE SUBMITTED HEREWITH

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: STRO_003_0lWO_SeqList_ST25.txt, date recorded: July 1, 2019, file size ~23 kilobytes).

BACKGROUND OF THE INVENTION

[0003] The immune response to a“weak” saccharide antigen can be amplified by conjugation to a known“strong” carrier polypeptide antigen such as diphtheria toxoid, tetanus toxoid, H.influenzae protein D, or CRM197. International Patent Publication No. WO 2018/126229 (SutroVax, Inc., Foster City, California) discloses methods, compositions, and techniques for the production of conjugate vaccine antigens from a saccharide antigen and a carrier polypeptide that includes a non-natural amino acid (nnAA). Orthogonal attachment chemistry via the nnAA permits conjugation of antigens to the carrier polypeptides to produce immunogenic conjugates which are useful for immunization.

[0004] A two-part method for preparing the conjugates is disclosed in WO 2018/126229. The method involves a first, activation step in which a saccharide antigen is covalently modified with a selected reagent or reagents to provide a plurality of first functional groups on the saccharide, the functional groups serving as a "first chemical handle." The activated saccharide so provided is then covalently conjugated to the carrier polypeptide having a plurality of second functional groups each associated with an nnAA and serving as a "second chemical handle," where the covalent coupling between the saccharide and the polypeptide occurs by reaction of the first chemical handle with the second chemical handle.

[0005] It is an object of the invention to provide variants and improvements of the aforementioned method. The variants and improvements described below can be applied to or combined with any of the methods disclosed in WO 2018/126229 or provisional U.S. Patent Applications Serial Nos. 62/693,978 and 62/693,981, both filed on 4 July 2018. The

aforementioned patent applications are incorporated by reference herein in their entireties.

SUMMARY OF THE INVENTION

[0006] In one embodiment, a method is provided for preparing a conjugate of a saccharide antigen and a carrier protein, where the method comprises:

(a) functionalizing the saccharide antigen with a reactive moiety (i.e., the "first chemical handle") capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety (i.e., the "second chemical handle") on a second reactant, by (i) providing the saccharide as a solution in an aqueous buffer having a pH in the range of 7 to 11; (ii) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the saccharide solution to provide a cyanate-substituted saccharide, and thereafter (iii) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide; and

(b) combining the activated saccharide antigen with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0007] In one aspect of the embodiment, the method further includes (c) recovering the conjugate from the reaction mixture. [0008] In another aspect of the embodiment, the saccharide antigen is subject to a mechanical size reduction step prior to activation, i.e., prior to step (a).

[0009] In a further aspect of the embodiment, the cyanylating reagent comprises cyano-4- dimethylamino pyridinium tetrafluorob orate (CDAP) and the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0010] In another embodiment, a method is provided for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(a) functionalizing the saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety on a second reactant, by (i) providing the saccharide as a solution in an aqueous medium; (ii) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the saccharide solution to provide a cyanate-substituted saccharide, and thereafter (iii) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide; and

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one non-natural amino acid (nnAA) residue bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein. Thus, Arg-l92 or Arg-l93 of SEQ ID NO: 1 can be deleted or can be substituted with a different amino acid. The nnAA residue(s) can be introduced by substitution of an amino acid residue in SEQ ID NO: 1 and/or by insertion.

[0011] In one aspect of the aforementioned embodiment, the method further includes (c) recovering the conjugate from the reaction mixture.

[0012] In another aspect of the embodiment, the saccharide antigen is subject to a mechanical size reduction step prior to activation, i.e., prior to step (a).

[0013] In a further aspect of the embodiment, the cyanylating reagent comprises cyano-4- dimethylamino pyridinium tetrafluorob orate (CDAP) and the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0014] In another embodiment, the invention provides a method for preparing a conjugate of a saccharide antigen and a carrier protein, where the method comprises:

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1 and (ii) includes a nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-2l 1; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp-507; Asp-519; Asn-296; Asn-359; Asn-399;

Asn-48l; Asn-486; Asn-502; Asn-524; Glu-240; Glu-248; Glu-249; Glu-256; Glu-259; Glu-292; Glu-362; Gln-252; Gln-287; Lys-2l2; Lys-2l8; Lys-22l; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-498; Lys-5l6; Lys-522; Lys-534; Arg-377; Arg-407; Arg-455; Arg-460; Arg-462; Arg-472; Arg-493; Ser-l98; Ser-200; Ser-23 l; Ser-233; Ser-239; Ser-26l; Ser-374; Ser-38l; Ser-297; Ser-397; Ser-45l; Ser-475; Ser-494; Ser-495; Ser-496; Ser-50l; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and/or Thr-5l7, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0015] In one aspect of the aforementioned embodiment, the method further includes (c) recovering the conjugate from the reaction mixture.

[0016] In another aspect of the embodiment, the saccharide antigen is subject to a mechanical size reduction step prior to activation, i.e., prior to step (a).

[0017] In a further aspect of the embodiment, the cyanylating reagent comprises cyano-4- dimethylamino pyridinium tetrafluorob orate (CDAP) and the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0018] In another embodiment, the invention provides a method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(a) functionalizing the saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety on a second reactant, by (i) providing the saccharide as a solution in an aqueous medium; (ii) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the saccharide solution to provide a cyanate-substituted saccharide, and thereafter (iii) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide; and (b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues

(numbered according to SEQ ID NO: 1): Asp-2l l; Asp-295; Asp-352; Asp-392; Asp-465; Asp- 467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys 212; Lys 218; Lys 221; Lys 229; Lys 236; Lys 264; Lys 299; Lys 385; Lys 456; Lys 474; Lys 498; Lys 516; Lys 522; Lys 534; Arg 377; Arg 407; Arg 455; Arg 460; Arg 462; Arg 472; Arg 493; Ser 198; Ser 200; Ser 231; Ser 233; Ser 239; Ser 261; Ser 374; Ser 381; Ser 297; Ser 397; Ser 451; Ser 475; Ser 494; Ser 495; Ser 496; Ser 501; Ser 505; Thr 253; Thr 265; Thr 267; Thr 269; Thr 293; Thr 386; Thr 400; Thr 408; Thr-469; and/or Thr 517, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0019] In one aspect of the aforementioned embodiment, the method further includes (c) recovering the conjugate from the reaction mixture.

[0020] In another aspect of the embodiment, the saccharide antigen is subject to a mechanical size reduction step prior to activation, i.e., prior to step (a).

[0021] In a further aspect of the embodiment, the cyanylating reagent comprises cyano-4- dimethylamino pyridinium tetrafluorob orate (CDAP) and the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0022] The invention additionally provides, in another embodiment, a method for

functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous buffer having a pH in the range of 7 to 11; (b) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the buffered saccharide solution to provide a cyanate-substituted saccharide; and thereafter

(c) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen.

[0023] In a further aspect of the embodiment, the cyanylating reagent comprises cyano-4- dimethylamino pyridinium tetrafluorob orate (CDAP) and the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0024] Another embodiment of the invention pertains to a method for activating a saccharide antigen by reaction with periodate followed by reductive amination, and thereafter conjugating the activated saccharide so provided to a carrier. More specifically, in this embodiment, a saccharide antigen is functionalized with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, where the method comprises:

(a) providing the saccharide as a solution in an aqueous medium;

(b) oxidizing the saccharide by adding a periodate reagent to the solution, thereby providing an aldehyde-bearing saccharide;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with 8 to 12 equivalents of sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen; and (f) combining the activated saccharide antigen with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0025] In one aspect of the aforementioned embodiment, the method further includes (g) recovering the conjugate from the reaction mixture.

[0026] In another aspect of the embodiment, the saccharide antigen is subject to a mechanical size reduction step prior to activation, i.e., prior to step (a).

[0027] In a further aspect of the embodiment, the activating reagent comprises a

dibenzylcyclooctyne (DBCO) derivative having the structure (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0028] Another embodiment of the invention also pertains to a method that employs periodate activation in the preparation of saccharide-polypeptide conjugates, where, in this embodiment, the method comprises:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer having a pH in the range of 5.5 to 5.9;

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen; and (f) combining the activated saccharide antigen with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein. Step (a) may be provided as a solution in an aqueous buffer, (e.g., a phosphate buffer), and having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3- 5.7, 5.4-5.6, 5.4-5.9).

[0029] A further embodiment of the invention provides a method for preparing a conjugate of a saccharide antigen and a polypeptide carrier, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen; and

(f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one non-natural amino acid (nnAA) residue bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein. [0030] In a related embodiment, a method for preparing a conjugate of a saccharide and a polypeptide carrier is provided that comprises (a) through (e) in the preceding embodiment, followed by (f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1 and (ii) includes a nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-2l 1; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys 212; Lys 218; Lys 221; Lys 229; Lys 236; Lys 264; Lys 299; Lys 385; Lys 456; Lys 474; Lys 498; Lys 516; Lys 522; Lys 534; Arg 377; Arg 407; Arg 455; Arg 460; Arg 462; Arg 472; Arg 493; Ser 198; Ser 200; Ser 23 l; Ser 233; Ser 239; Ser 26l; Ser 374; Ser 381; Ser 297; Ser 397; Ser 451; Ser 475; Ser 494; Ser 495; Ser 496; Ser 501; Ser 505; Thr 253; Thr 265; Thr 267; Thr 269; Thr 293; Thr 386; Thr 400; Thr 408; Thr-469; and/or Thr 517, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0031] In another related embodiment, a method for preparing a conjugate of a saccharide and a polypeptide carrier is provided that comprises (a) through (e) as in the preceding two embodiments, followed by (f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg- Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-2l 1; Asp-295; Asp-352; Asp-392; Asp- 465; Asp-467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys 212; Lys 218; Lys 221; Lys 229; Lys 236; Lys 264; Lys 299; Lys 385; Lys 456; Lys 474; Lys 498; Lys 516; Lys 522; Lys 534; Arg 377; Arg 407; Arg 455; Arg 460; Arg 462; Arg 472; Arg 493; Ser 198; Ser 200; Ser 231; Ser 233; Ser 239; Ser 261; Ser 374; Ser 381; Ser 297; Ser 397; Ser 451; Ser 475; Ser 494; Ser 495; Ser 496; Ser 501; Ser 505; Thr 253; Thr 265; Thr 267; Thr 269; Thr 293; Thr 386; Thr 400; Thr 408; Thr-469; and/or Thr 517, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0032] In another embodiment of the invention, a method is provided for preparing an activated saccharide antigen that is functionalized for subsequent conjugation to a polypeptide carrier via a click chemistry reaction, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer having a pH in the range of 5.5 to 5.9; and

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with at least two equivalents of an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with 8 to 12 equivalents of sodium

cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate- substituted saccharide, thereby providing an activated saccharide antigen.

[0033] In an additional embodiment, a method is provided for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, where the method is

"standardized" insofar as it is substantially applicable to saccharide antigens across a plurality of serotypes, the method comprising:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 7 to 11;

(b) cyanylating hydroxyl groups on the saccharide antigen with an effective cyanylating amount of CDAP to provide a cyanate-substituted saccharide; and (c) after 3 to 13 minutes, contacting the cyanate-substituted saccharide with 0.25 equivalents to 2.0 equivalents of a dibenzylcyclooctyne (DBCO) derivative having the structure of formula (I) wherein m is zero or 1 and n is an integer in the range of 2 to 12, thereby transferring the DBCO moiety to the cyanate-substituted saccharide.

[0034] In a further additional embodiment, a method is provided for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method being substantially applicable to saccharide antigens across a plurality of serotypes and comprising:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 5.5 to 5.9;

(b) oxidizing the saccharide antigen with an effective oxidizing amount of a periodate reagent, thereby providing an aldehyde-bearing saccharide;

(c) purifying the aldehyde-bearing saccharide; and

(d) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with 8 to 12 equivalents of sodium cyanoborohydride for a time period in the range of 18 to 30 hours, thereby providing an activated saccharide antigen.

[0035] The aforementioned standardized methods are substantially applicable across a plurality of antigen serotypes insofar as most reaction variables can be maintained constant from serotype to serotype, e.g., reaction pH, reaction temperature, reaction time, and the like, while any variables that need to be altered from serotype to serotype are kept to a minimum.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Various details of methods, compositions, and techniques for the production of conjugated antigens are disclosed in PCT/US2017/069129, the complete contents of which have been previously incorporated by reference herein. A. Immunogenic Conjugates:

[0037] The present invention provides, in part, improved methods for the preparation of immunogenic conjugates by activating an antigen and then coupling the activated antigen to a polypeptide carrier as explained herein. Immunogenic conjugates provided herein thus comprise a carrier polypeptide which is covalently linked to an antigen. This linking can convert a T-cell independent immunogen (such as a saccharide) into a T-cell dependent immunogen, thereby enhancing the immune response that is elicited (particularly in children). The conjugates prepared using the present methods contain covalent linkages that are formed between an antigen and a non-natural amino acid (nnAA) residue within the carrier polypeptide. These nnAA residues can provide functional groups which facilitate reactivity with an antigen of interest.

[0038] Typically, a single carrier polypeptide will be linked to multiple antigen molecules.

The antigens can have a single linking group per molecule (e.g. the reducing terminus of a saccharide) for attaching to a carrier polypeptide, or can have multiple linking groups

(e.g. multiple aldehyde or cyanate ester groups). Where an antigen molecule has multiple linking groups this generally leads to the formation of high molecular weight cross-linked or lattice conjugates, involving links between multiple carrier polypeptides via the antigens. Cross-linked conjugates are preferred herein (particularly for pneumococcus), and thus antigens with multiple linking groups are also preferred.

[0039] Covalent linkages are formed between the antigen and a nnAA residue within the carrier polypeptide. Preferably the antigen is not conjugated to a lysine residue in the carrier polypeptide; more preferably, the antigen is not conjugated to a natural amino acid residue in the carrier polypeptide.

[0040] Useful carrier polypeptides contain a T-cell epitope. Various such carrier polypeptides are known in the art, and within approved vaccines it is known to use diphtheria toxoid

(chemically treated toxin from Corynebacterium diphtheriae;‘Dt’), tetanus toxoid (chemically treated tetanospasmin toxin from Clostridium tetani;‘Tt’), protein D from Haemophilus influenzae (‘PD’ or‘HiD’), the outer membrane protein complex of serogroup B meningococcus (‘OMPC’), and the CRM197 mutant C.diphtheriae toxin. [0041] A preferred carrier polypeptide upon which to base the carriers of the present invention is CRM197. CRM197 is well-known in the art (e.g. see Broker et al. 2011 Biologicals 39: 195- 204) and has the following amino acid sequence (SEQ ID NO: 1), where the underlined residue (Glu-52) differs from the natural diphtheria toxin, whereby the substitution of Gly Glu leads to the loss of toxic enzymatic activity in the protein:

GADDWDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNEN

PLSGKAGGWKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRWLSLPFAE

GSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKT

KTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAW

VAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGF

AAYNFVESIINLFQWHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP

LPIAGVLLPTIPGKLDWKSKTHISWGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSE

KIHSNEISSDSIGVLGYQKTVDHTKWSKLSLFFEIKS

[0042] The invention does not use native CRM197. Instead of using CRM197 comprising SEQ ID NO: 1, a modified amino acid sequence is used which contains at least one nnAA.

These modified CRM 197 carrier polypeptides are described in more detail below.

[0043] Aside from CRM 197, other detoxified mutant forms of diphtheria toxin can be used. For instance, a non-toxic K51E/E148K double mutant has also been used as a carrier polypeptide in conjugates (Pecetta et al. 2016 Vaccine 34: 1405-11) and nnAA residues can be incorporated into the sequence of this double mutant in the same way as in CRM197.

[0044] Another carrier polypeptide of interest is PD from H. influenzae, which naturally has the following amino acid sequence (SEQ ID NO: 5):

CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLWIHDHFLDGL

TDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQ

GLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMG

MDLKLVQLIAYTDWKETQEKDPKGYWWYNYDWMFKPGAMAEWKYADGVGPGWYMLWKEESKPDNIVYT

PLVKELAQYNVEVHPYTVRKDALPEFFTDWQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

[0045] Rather than using native PD, a modified amino acid sequence is used which contains at least one nnAA. For instance, one or more Lys residues within SEQ ID NO: 5 can be replaced with a nnAA. There are 36 Lys residues within SEQ ID NO: 5, so several can be replaced by nnAA and then used for conjugation. T-cell epitope prediction and recognition for PD has been reported by Hua et al. (2016) Clin Vaccine Immunol 23: 155-61.

[0046] More generally, any polypeptide including a T-cell epitope can be used as a carrier polypeptide. The T-cell epitope can bind to MHC class II and interact with T-cell receptors on the surface of CD4+ T-cells, thereby enhancing antibody responses against antigens or haptens conjugated thereto ( e.g . see Costantino et al. 2011, Expert Opin Drug Discov 6:1045-66). Micoli et al. (2018) Molecules 23, 1451 reviews various carrier polypeptides and criteria for their selection. Tontini et al. (2016) Vaccine 34:4235-42 discuss pre-clinical studies of 28 carrier polypeptides, including tests of their ability to induce antibodies against saccharide antigens. Poly epitope carrier polypeptides containing multiple broadly-reactive (i.e. immunogenic in the context of most human MHC class II molecules) human CD4+ T-cell epitopes from various pathogen-derived antigens have been designed e.g. the N19 and other polypeptides as disclosed by Falugi et al. (2001) Eur J Immunol 31 :3816-24, Baraldo et al. (2004) Infect Immun 72:4884- 7, and US patents 6,855,321 & 7,867,498. The ability to design these polyepitope carriers demonstrates the ability of those skilled in the art to identify suitable T cell epitopes from diverse sources and also to use them to design effective carrier polypeptides. See also patent application US2016-0101187. T cell epitopes found within known carriers (e.g. Tt, PD,

CRM197) can be used. Various detoxified bacterial toxins have been successfully used as carriers e.g. Tt, Dt, the P. aeruginosa exotoxin, the C.diffwile A & B toxins, etc. Many different carrier polypeptides have been used for pneumococcal saccharides e.g. CRM 197 in Prevnar™, PD, Tt and Dt in Synflorix™, and various peptides in Velasco et al. (1995) Infect Immun 63:961-8. The invention can use any of these numerous carrier polypeptides, modified to include at least one nnAA, to enhance the immunogenicity of antigens of interest.

[0047] Carrier polypeptides to be used with the invention, containing nnAA, can be prepared in general using the techniques disclosed in section 6 (‘Carrier Protein Production Methods’) of PCT/US2017/069129. Preferred carriers contain nnAAs outside of at least one T-cell epitope of the carrier. If the T-cell epitope regions for a carrier are unknown then one can identify the epitopes using standard techniques e.g. see Reece et al. (1993) IJ Immunol 151 :6175-84, Beissbarth et al. (2005) Bioinformatics 21 Suppl 1 : Ϊ29-37, Maciel Jr et al. (2008) Virol

378: 105-17, Fridman et al. (2012) Oncoimmunol 1 :1258-70, etc. (including empirical and/or predictive approaches). It is also possible to confirm that any particular modification of a carrier polypeptide’s sequence does not eliminate the desired T-cell response to a conjugated antigen, such as the saccharides herein. A preferred group of carriers do not contain any modification, including insertion or substitution of a nnAA, within a T-cell epitope. Particularly preferred carriers contain as least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs. Particularly preferred carriers may also have a maximum of 10, 9, 8, 7 or 6 nnAAs. Particularly preferred ranges of nnAAs in a carrier polypeptide are 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 4- 10, 4-9, 4-8, 4-7, and 4-6 nnAAs.

[0048] Various antigens can be included within immunogenic conjugates used herein.

Typically the antigen is a saccharide. The term“saccharide” includes polysaccharides having 50 or more repeat units, and oligosaccharides having fewer than 50 repeating units. Typically, polysaccharides have from about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about 2,000 (sometimes more) repeating units, and optionally from about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 repeating units to about, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 repeating units. Oligosaccharides typically have from about 6, 7, 8, 9, or 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units.

[0049] Useful saccharides for incorporation into immunogenic conjugates include those found in bacteria. These can be non-capsular saccharides (such as an exopolysaccharide e.g. the S. aureus exopolysaccharide) but are preferably bacterial capsular saccharides.

[0050] Bacterial capsular saccharides are high molecular weight saccharides found in the capsule of Gram-positive or Gram-negative bacteria and they can be used as vaccine antigens. Such capsular saccharides are generally prepared from whole cell lysates or culture supernatant of the corresponding bacterium via processes that involve diafiltration, protein removal, ethanol precipitation, nucleic acid removal, and freeze drying. Bacterial saccharides used with the invention can be intact as found in the bacteria, or can be fragments obtained from intact saccharides e.g. obtained by hydrolysis of saccharides purified from the bacteria.

[0051] Saccharide antigens of particular interest include, but are not limited to: Capsular saccharides of S. pneumoniae. Further details of pneumococcal capsular saccharides useful as antigens roe implementing the invention are given below.

Saccharides of Streptococcus pyogenes : The antigen can be a saccharide from

S.pyogenes. In one embodiment the antigen is the capsular saccharide of S. pyogenes, which is composed of hyaluronic acid, a high molecular weight polymer where the repeating unit has the structure:

[ 4)-p-D-GlcUAp-(l 3)-?-D-Glc^NAc-( ] which appears to be invariant between S. pyogenes serotypes. In another embodiment the antigen is a non-capsular saccharide from S. pyogenes, such as the group-A-strep cell wall saccharide, which comprises a backbone of poly-L-rhamnopyranosyl units connected by alternating a-L-(l 3) and a-L-(l 2) linkages, to which N-acetyl-b-O- glucosamine residues are attached at the 3 -position of the rhamnose backbone.

Capsular saccharides of Streptococcus agalactiae: The antigen can be a capsular saccharide from S.agalactiae (Group B Streptococcus or GBS). There are at least 10 GBS serotypes with distinct capsular saccharide repeating units (la, lb, II-IX), but only a few serotypes are commonly responsible for disease. These include serotypes la, lb, II, III, and V, and conjugates of capsular saccharides from these serotypes can be prepared.

Capsular saccharides of Haemophilus influenzae: The antigen can be a capsular saccharide from H. influenzae. There are at least 6 serotypes of H. influenzae with distinct capsular saccharide chemical structures (types a-f). However, only type a and type b are considered“high-virulence” strains, and the preferred type of H. influenzae capsular saccharide for use with the invention is type b (Hib).

Capsular saccharides of Neisseria meningitidis: The antigen can be a capsular saccharide from N.meningitidis. There are at least 13 serogroups of N. meningitidis with distinct capsular saccharide chemical structures (serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z'), but only six (A, B, C, W-135, X, Y) are thought to be life-threatening. The saccharide antigen is usefully derived from any of serogroups A, C, W135, X, or Y. Capsular saccharides of Porphyromonas gingivalis : The antigen can be a capsular saccharide derived from one of the six serotypes Kl, K2, K3, K4, K5 and K6 of

P .gingivalis .

Capsular saccharides of Salmonella typhi: The antigen can be a Vi saccharide. Vi is the capsular saccharide of Salmonella typhi (the typhi serovar of S.enterica). The Vi saccharide is a linear homopolymer of a hexosaminuronic acid, al,4-/V-acetylgalactos- aminouronic acid, which is 60 - 90% acetylated at the C-3 position.

Saccharides of Staphylococcus aureus : The antigen can be a saccharide from S. aureus. The saccharide can be the exopolysaccharide of S.aureus , which is a poly-N- acetylglucosamine (PNAG), or a capsular saccharide of S.aureus , which can be e.g. serotype 5, serotype 8 or serotype 336.

Surface saccharides of Clostridium difficile. The antigen can be a surface glycan from C.difficile , such as PS-I or PS-II.

Glucans: The antigen can be a glucan containing b- l ,3-linkages and/or b-l, 6-linkages. These conjugated glucans can be useful for raising an anti-fungal immune response, for example against Candida albicans.

[0052] Further details of these saccharide antigens can be found in PCT/US2017/069129.

[0053] Antigens often do not intrinsically contain functional groups that are suitable or ideal for conjugation. Thus an antigen might need to be functionalized prior to its conjugation to the nnAA. Further details of such functionalization are given below.

[0054] Preferred antigens for use with the invention are capsular saccharides from

Streptococcus pneumoniae. S.pneumoniae is an encapsulated Gram-positive bacterium that can cause pneumonia, bacteremia, and meningitis. There are at least 90 distinct documented serotypes of S.pneumoniae (see e.g. Kalin, M. Thorax 1998;53: 159-162) which bear capsular saccharides with serotype-specific repeating unit structures. As will be understood by those in the field, it has been proposed that S.pneumoniae serotype 20 is actually made up of two closely related serotypes, the capsular polysaccharides of which are largely cross-protecting (Calix et al. 2012 JBiol Chem 287:27885-94). Thus, as would be further appreciated by those of skill in the art, serotype 20 refers to a saccharide that would have previously been classified in the field as serotype 20, and could therefore structurally be either 20A or 20B (from a strain which would have previously been classified in the field as serotype 20, but could genotypically be either 20A or 20B) as disclosed by Calix et al. For example, the strain used to produce serotype 20 polysaccharide in Pneumovax™ (Merck) is now believed to be serotype 20A. In some instances, 20A may be preferred. In other instances, 20B may be preferred. Prevalence in a target population could be a basis for selecting between these serotypes. Nevertheless, because of strains classified as 20, 20A and 20B are serologically similar, they are largely cross- protective in a vaccine and the choice among strain may not be critical.

[0055] The antigen used with the invention can be a capsular saccharide from any of

S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A,

10B, 10C, 11F, 11 A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17 A, 18F, 18 A, 18B, 18C, 19F, 19A, 19B, 19C, 20, 21, 22F, 22A, 23F, 23 A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33 A, 33B, 33C, 33D, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41 A, 42, 43, 44, 45, 46, 47F, 47A, or 48 (Henrichsen J Clin Microbiol 1995; 33 :2759-2762). However, only a subset of these serotypes are commonly responsible for bacterial infection of clinical significance, so the antigen can be a capsular saccharide from any of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. Serotypes 6C, 7C,

15 A, 15C, 16F, 20A, 20B, 23 A, 23B, 24B, 31, 34, 35B, 35F, 37 and 38 have also become of clinical concern, so the antigen can be a capsular saccharide from one of these S.pneumoniae serotypes.

[0056] Where the invention uses conjugates from different pneumococcal serotypes, it is preferred to include saccharides from at least 14 different S.pneumoniae serotypes (e.g. from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) Where a composition includes 14 or more serotypes, these preferably include the 13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In addition to these 13 S.pneumoniae serotypes a compositions preferably includes one or more of serotypes 2, 8, 9N, 10 A, 11 A, 12F, 15B, 17F, 20 (alternatively, 20 A or 20B), 22F, and/or 33F. Alternatively, in addition to the above 13 serotypes, a composition preferably includes one or more S.pneumoniae serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B,

15C, 16F, 17F, 20, 20A, 20B, 22F, 23 A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F and 38. A useful combination of 15 or more (e.g., 16 or more) serotypes includes each of S.pneumoniae 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 (e.g.21 or more) S.pneumoniae serotypes includes each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. A useful combination of 24 or more serotypes includes each of S.pneumoniae serotypes 1,

2, 3, 4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0057] The structures of common pneumococcal serotype capsular saccharide repeating units are described in Jones etal. (Jones C et al. An Acad Bras Cienc.2005 Jun;77(2):293-324):

Type 1

[ 3)-D-AAT-a-Gal/-(l 4)-a-D-Gal/A(2/30Ac)-(l 3)-a-D-Gal/A-(l ]

Type 2

[ 4)-P-D-Glcp-(l 3)-[a-D-GlcpA-(l 6)-a-D-Glcp-(l 2)]-a-L-Rhap-(l 3)-a-L- Rhap-( 1 3 )P-L-Rhap-( 1 ]

Type 3

[ 3)-p-D-GlcA-(l 4)-p-D-Glc^-(l ]

Type 4

[ 3)-p-D-ManpNAc-(l 3)-a-L-FucpNAc-(l 3)-a-D-GalpNAc-(l 4) -a-D- Galp2, 3 (S)Py-( 1 ]

Type 5

[ 4)-P-D-Glcp-(l 4)-[a-L-PnepNAc-(l 2)-P-D-GlcpA-(l 3)] -a-L-FucpNAc- (l 3)-P-D-Sugp-(l ]

Type 6B

[ 2)-a-D-Galp-(l 3)-a-D-Glcp-(l 3)-a-L-Rhap-(l 4)-D-Rib-ol-(5 P ]

Type 9N

[ 4)-a-D-Glcp A-( 1 3 )-a-D-Glcp-( 1 3 )-p-D-ManpNAc-( 1 4) -p-D-Glcp-( 1 4)-a- D-GlcpNAc-(l ]

Type 9V

[ 4)-a-D-GlcpA(2/3 O Ac)-( 1 3 )-a-D-Galp-( 1 3 )-p-D-ManpN Ac(4/60 Ac)-( 1 4) -b- D-Glcp-(l 4)-a-D-Glcp-( 1 ] Type 12F

[ 4)-[a-D-Galp-(l 3)]a-L-FucpNAc-(l 3)-p-D-GlcNAc-(l 4)-[a-D-Glc-(l 2) -a- D-Glc-(l 3)]-p-D-ManNAcA-( ]

Type 14

[ 4)-p-D-Glcp-(l 6)-[p-D-Galp-(l 4)]-p-D-GlcpNAc-(l 3)-p-D-Galp-(l ]

Type 18C

[ 4)-p-D-Glcp-(l 4)-[a-D-Glcp(60Ac) (l 2)][Gro-(l P 3)]-p-D-Galp-(l 4) -a- D-Glcp-(l 3)-p-L-Rhap-(l ]

Type 19F

[ 4)-p-D-ManpNAc-( 1 4)-a-D-Glcp-( 1 2)-a-L-Rhap-( 1 P ]

Type 23F

[ 4)-p-D-Glcp-(l 4)-[a-L-Rhap-(l 2)]-[Gro-(2 P 3)] -p-D-Galp-(l 4)-p-L- Rhap-(l ]

[0058] A more extensive discussion of the saccharides is found in Geno et al. (2015) Clin. Microbiol. Rev. 28:871-99, in which Table 1 shows the structures for 97 known serotypes. This table also discloses the proportion of saccharide residues which are acetylated when acetylation is not complete.

[0059] The capsular saccharide can be O-acetylated. In some embodiments, the capsular saccharide from serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F comprises a saccharide which has a degree of O-acetylation of between 10-100%, between 20-100%, between 30-100%, between 40-100%, between 50-100%, between 60-100%, between 70-100%, between 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 saccharide can be determined, for example, by proton NMR (see Lemercinier & Jones (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J.

Pharmaceutical and Biomedical Analysis 30: 1233-1247). Normally the saccharide used to prepare a conjugate will retain at least 50% (e.g. 75%, or even 100%) of the O-acetylation levels seen in the starting capsular saccharide purified from a bacterium. [0060] The S.pneumoniae capsular saccharides can be obtained directly from bacteria using isolation procedures known to one of ordinary skill in the art (see for example methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752). As an alternative, they may be obtained from a commercial source ( e.g ., ATCC).

[0061] A pneumococcal capsular saccharide antigen used with the invention can usefully have a molecular weight between lOkDa and 4,000kDa e.g. between 50 kDa and 3,000 kDa, or between 100 kDa and 2,000 kDa. For instance, the molecular weight can be between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 4,000 kDa; between 200 kDa and 3,500 kDa;

between 200 kDa and 3,000 kDa; between 200 kDa and 2,500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500 kDa. Further details and guidance with respect to molecular weights are available in U.S. Serial No. 62/693,978, incorporated by reference earlier herein.

[0062] The capsular saccharide is optionally chemically modified relative to the capsular saccharide found in nature. For example, the saccharide is optionally de-O-acetylated (partially or fully), de-N-acetylated (partially or fully), N-propionated (partially or fully), etc.

De-acetylation optionally occurs before, during or after activation, derivatization, or conjugation, but typically occurs before conjugation.

Non-natural amino acids:

[0063] As mentioned above, conjugates used herein include covalent linkages between an antigen and a functional group within a nnAA residue in the carrier polypeptide. The side chains of nnAA residues can provide reactive functional groups which are useful for conjugating antigens to discrete sites in the carrier polypeptide. [0064] In general terms the nnAA can be any amino acid that can be incorporated into a polypeptide during translation but is not one of the 20 common amino acids. A nnAA can be conveniently incorporated into a polypeptide by converting a tRNA molecule such that its codon incorporates the nnAA rather than the natural cognate amino acid. One technique for achieving this involves using a“suppression codon” i.e. a nucleotide triplet that is introduced into a coding sequence at a desired position and is recognized by a specific tRNA that can recognize a natural stop codon (e.g., an amber, ochre or opal stop codon) but allows translation to continue, with incorporation of the nnAA (thereby suppressing the natural stop codon).

[0065] The nnAA residue can be any of the nnAA residues described herein, or any other that has been identified as compatible with cell-based or cell-free protein synthesis (see, e.g, Schultz et al. Annu Rev Biochem. 2010;79:413-44 particularly pp.418-420; and Chin et al. Annu Rev Biochem. 20l4;83:5.1-5.30, which are hereby incorporated by reference). Ideally the nnAA is not one which arises naturally within a cell by modification of one of the 20 common amino acids (e.g. pyrolysine, selenocysteine, phosphotyrosine, formyl-methionine, etc.).

[0066] Particularly preferred nnAA for use herein are those which can be incorporated during translation (in a cellular or a cell-free system) with a side chain that provides a functional group which is not found in the side chain of any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into polypeptides are known e.g. see Young & Schultz (2010) J Biol Chem 285: 11039-44, Maza et al. (2015) Bioconjugate Chem. 26: 1884-9, and Zimmerman et al. (2014) Bioconjugate Chem. 25:351-61. PCT/US2017/069129 discloses in detail how nnAA residues can be incorporated into carrier polypeptides e.g. using cell-free expression mixtures, orthogonal tRNA/aminoacyl-tRNA synthetase pairs specific for the nnAA, suppression codons, etc. See also U.S. Patent Publication No. US2017/0267637.

[0067] The nnAA can include a chemical group suitable for“click” chemistry reaction with a corresponding group on an antigen of interest. Suitable chemical groups for“click” chemistry include, but are not limited to azido (-N3), alkyne (-CºC-), alkene (-C=C-) and l,2,4,5-tetrazine

phosphine (e.g. -P(Ph)₂) groups. [0068] The nnAA can be any of 2-amino-3-(4-azidophenyl)propanoic acid (para-azido-L- phenylalanine or pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (para-azidomethyl-L- phenylalanine or pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3- (4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 -yl)propanoic acid, or 2-amino-5-azidopentanoic acid.

[0069] The most preferred nnAA for use herein is pAMF:

pAMF provides very favorable reaction kinetics for producing conjugates (e.g. much faster than using pAF when reacting with an alkyne-containing carbohydrate antigen in a SPA AC method).

[0070] The nnAA can be a 2,3-disubstituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl-containing substituent, or an ethynyl-containing substituent at the 3-position. Preferably, the substituent at the 3-position is an azido-containing substituent, particularly an azido-containing substituent comprising a terminal azido group bound to the carbon atom at the 3-position through a linking group. For example, the linking group may comprise an arylene moiety that is optionally substituted and optionally heteroatom-containing. For instance, the linking group may comprise a 5- or 6- membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 non-hydrogen ring substituents.

[0071] The nnAA can have the structure of formula (II): (II):

wherein: Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom; W⁵ is selected from C1-C10 alkylene, -NH-, -O- and -S-; Ql is zero or 1; and W⁶ is selected from azido, l,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl. 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 infra and Ar may be e.g. a pyridine or a pyridazine. In a particularly preferred embodiment, Ql is 1, W⁵ is lower alkylene, and W⁶ is azido.

[0072] The nnAA can be an azido-containing nnAA, such as a nnAA of formula I:

wherein: D is— Ar— W3— or— Wi— Yi— C(O)— Y2— W2— ; each of Wi, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently -NH-, -O-, or -S-; each Yi is independently a single bond, -NH-, or -O-; each Y2 is independently a single

bond, -NH-, -O-, or an N-linked or C-linked pyrrolidinylene;

one of Zi, Z₂, and Z₃ is -N- and the others of

Zi, Z₂, and Z₃ are independently -CH-.

[0073] In other embodiments, the nnAA has formula (III):

(III):

wherein W₄ is C1-C10 alkylene.

[0074] Preparation of azido-containing amino acids according to formulas II and III are found, for example, in Stafford et al. US2014-0066598A1, particularly paragraphs [0331]-[0333], which are incorporated by reference. The process involves substitution of hydroxyl groups for chloride on derivatives of the corresponding aryl amino acids using thionyl chloride, followed by nucleophilic displacement of the chloride with azide. Suitable aryl side-chain containing amino acids are also acquired commercially.

[0075] The nnAA can be a l,2,4,5-tetrazine-containing nnAA. For example, formula IV:

IV:

wherein:

single bond, lower alkylene, or -W1-W2-; one of Wi and W2 is absent or lower alkylene, and the other is -NH-, -0-, or -S-; each one of Zi, Z2, and Z3 is independently -CH- or -N-; and Xi is independently -NH-, -0-, or -S-; and R is lower alkyl.

[0076] Preparation of l,2,4,5-tetrazine-containing amino acids according to formula IV is found, for example, in Yang et al. US2016/0251336, particularly paragraphs [034l]-[0377], which are incorporated by reference. The process involves Negishi coupling of an

amino/carboxyl protected derivative of (R)-2-amino-3-iodopropanoic acid with an aminopyridyl bromide to introduce Ar, followed by reaction with a methylthio-l,2,4,5-tetrazine derivative to introduce the tetrazine moiety into the amino acid.

[0077] The nnAA can be an alkyne-containing nnAA. In one embodiment, this is a propargyl group. A variety of propargyl-containing amino acids, including syntheses thereof, 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. JACS 2009 (13 l):8720-l . Such propargyl-containing amino acids are suitable for incorporation into proteins using cell-based systems. In some embodiments, the propargyl-containing nnAA is selected from the group consisting of horn opropargylgly cine, ethynylphenylalanine, and N6-[(2-propynyloxy)carbonyl]-L-lysine.

[0078] The nnAA used herein are generally a-amino acids with a chiral center at the a-carbon, and they are preferably L-stereoisomers.

[0079] A polypeptide carrier used with the invention includes at least one nnAA residue. It is preferred that a carrier polypeptide should include multiple nnAA e.g. 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues (or sometimes more). Carrier polypeptides with fewer than 10 nnAA residues are preferred. Thus the polypeptide can include 2-9 nnAA residues, and preferably includes 4-6 nnAA residues. [0080] Where a carrier polypeptide includes multiple nnAA residues it is preferred to include only a single species of nnAA ( e.g . the only nnAA in the carrier is pAMF). This permits the same conjugation chemistry to be used simultaneously at each nnAA. 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 species of nnAA in a carrier is preferred. Moreover, where multiple different conjugates are used (e.g. different pneumococcal serotypes) it is sometimes preferred that each conjugate includes the same single species of nnAA. Furthermore, where a composition includes multiple different conjugates (e.g. different pneumococcal serotypes) it is sometimes preferred that each conjugate includes the same carrier polypeptide.

[0081] A nnAA can be incorporated into a carrier polypeptide by substitution or by insertion (or by C-terminal or N-terminal extension). In one embodiment, the nnAA residue(s) are incorporated by substitution. Conveniently, the nnAA can be substituted for a lysine residue in the native polypeptide. For instance, in CRM197 the substitution can occur at one or more of positions K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 in SEQ ID NO: 1 or 2. Substitution of a nnAA (e.g pAMF) at each of K33, K212, K244, K264, K385, and K526 (and in one embodiment at no other positions) is preferred.

[0082] Substitutions to incorporate nnAA are not limited to lysine positions, however, and it is also possible to substitute other amino acids with a nnAA e.g. Phe, Asp, Asn, Glu, Gln, Arg, Ser, and/or Thr.

[0083] The nnAA within a carrier polypeptide is/are ideally surface-accessible residues. This can be assessed using a 3D structure of the polypeptide, or by performing a comprehensive replacement of natural amino acids for nnAAs followed by conjugation tests, to assess the utility of each site.

[0084] To preserve the function of a carrier polypeptide it preferred not to incorporate a nnAA within a T-cell activating epitope of the carrier polypeptide. The use of nnAA allows the selective placement of sites for conjugation and so the T-cell activating epitopes of a carrier polypeptide can be avoided as sites for antigen conjugation. As mentioned above, these epitopes can easily be identified. For example, studies of CRM197 by Raju et al ., Bixler et al, Leonard et a/., and Pillai et al. (e.g. Eur J Immunol. 1995 Dec;25(l2):3207-l4, WO89/06974) have identified various T-cell epitopes e.g. within residues P271-D290, V321-G383, and Q411-1457. Thus it is preferred to avoid introducing nnAA within these regions of SEQ ID NO: 1.

B. Antigen Activation and Conjugation:

1. Overview of Conjugation

[0085] Conjugation involves formation of a covalent linkage between the nnAA and the antigen. This requires a reactive functional group in both the nnAA and the antigen. A nnAA for the carrier polypeptide will generally be chosen because it already has a suitable functional group (e.g. the azido group of pAMF), but antigens often do not intrinsically contain functional groups that are suitable or ideal for conjugation. Thus an antigen normally needs to be functionalized prior to its conjugation to the nnAA.

[0086] Detailed technical information about conjugation can be found in Bioconjugate Techniques (Greg T Hermanson, 3rd edition, 2013). WO2018/126229 discloses in detail how antigens can be functionalized and then conjugated to nnAA. As noted above, useful nnAA include a functional group (e.g. an azido group) that is suitable for a“click” chemistry reaction with a functional group on the antigen. Thus a functionalized antigen ideally includes a group suitable for such“click” reactions.

[0087] In general terms, conjugation thus takes place by a process comprising 3 steps:

(a) activating the antigen; (b) optionally derivatizing the activated antigen (e.g. with a linker or nucleophilic group) to introduce a reactive functional group not normally present in the antigen; and (c) conjugating the antigen to the carrier polypeptide via a group introduced in step (a) or, if present, step (b). In some embodiments step (a) includes a first step of removing a blocking group on the antigen, such that certain functional groups (e.g. hydroxyls, amines, thiols) are more accessible to activation. Sometimes the steps (a)-(c) can occur essentially simultaneously (e.g. where a reactive moiety such as N-hydroxysuccinimide is added to the antigen), but in other embodiments two or more of steps (a)-(c) are discrete, with optional purification between steps. [0088] As noted above, cross-linked conjugates are preferred, so it is also preferred to introduce multiple reactive functional groups per antigen molecule. For instance, multiple aldehyde or cyanate ester groups can be introduced when activating a saccharide molecule.

These groups can then be derivatized e.g. to introduce a reactive cyclooctyne which can then react with azido groups in in the nnAA.

[0089] An antigen can be activated using various chemistry, including but not limited to: periodate oxidation (e.g. to oxidize hydroxyl groups on adjacent carbon atoms to give reactive aldehyde groups), for instance as disclosed in WO2011/110531; cyanylation e.g. using l-cyano- 4-dimethylamino pyridinium tetrafluorob orate (CDAP); hydroxyl activation with

l,l'-carbonyldiimidazole (CDI) followed by nucleophilic addition; or unmasking of an intrinsic aldehyde (e.g. a reducing terminus of a saccharide).

[0090] Periodate oxidation and CDAP cyanylation are two preferred activation techniques. Periodate oxidation has been shown to be useful for activating inter alia pneumococcal serotypes 1, 2, 3, 7F, 8, 9N, and 11 A. CDAP cyanylation has been shown to be useful for activating inter alia pneumococcal serotypes 3, 7F, and 10 A.

[0091] The activated antigen can be conjugated to a nnAA directly, but usually the activated group is derivatized to introduce a functional group that shows better reactivity towards the nnAA’s functional group. For instance, an alkynyl group can be introduced. A bifunctional reagent with an amino group and an alkyne group can react with an aldehyde group that has been introduced into an antigen (e.g. via reductive amination) thereby leaving a pendant alkyne which can react with a nnAA. For instance, bifunctional reagents including amino and DBCO functional groups can be used.

[0092] In one embodiment, the nnAA reacts with an alkynyl group in the antigen (e.g. a propargyl group). An alkyne group in an antigen is ideal for reacting with an azido group in a nnAA e.g. using the reactions known in the art as copper-catalyzed azide-alkyne cycloaddition (CuAAC), ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), or Huisgen azide-alkyne l,3-dipolar cycloaddition. The alkynyl group may have a molecular context that increases its reactivity e.g. it can be within a ring. For instance, the alkylene can be within a cyclooctyne ring (optionally including a heteroatom), such as a diaryl-strained cyclooctyne ring (e.g. DBCO). This reaction can be a [3+2] cycloaddition referred to in the art as strain-promoted azide-alkyne cycloaddition (SPAAC). DIFO- and DBCO-based reagents are readily available for these reactions.

2. Antigen Activation - Periodate Method

[0093] Prior to functionalization of a saccharide antigen to provide an activated antigen capable of conjugation to a carrier polypeptide, the antigen may be subject to a process that reduces molecular weight. The process may be a mechanical sizing step, in which case the polysaccharide is subjected to shearing forces (as may accomplished using a high shear homogenizer or the like), and/or the process may involve treatment with heat or mild acid. Ideally, the molecular weight of the antigen following this initial treatment will approximate, within 25-30%, the native molecular weight of the corresponding serotype.

[0094] One preferred technique for activating saccharide antigens herein involves treatment . with a periodate reagent followed by reductive deamination, with the activated antigen so provided then available for conjugation to an appropriately functionalized polypeptide carrier. Periodate activation is thus a two-step method, with an initial periodate oxidation reaction followed by purification of the oxidation product and then a second reaction involving reductive amination as will be explained infra. Periodate oxidation, as is understood in the field, involves the cleavage of adjacent hydroxyl groups, i.e., hydroxyl groups in the form of a "vicinal" diol in which two adjacent carbon atoms are each substituted with a hydroxyl group. Periodate cleavage of such a diol results in the breakage of the carbon-carbon bond and formation of an aldehyde moiety at each carbon atom, e.g., a -CH2(OH)-CH2(OH)- motif is converted upon periodate oxidation to -C(CO)H at each carbon atom.

[0095] For periodate oxidation of antigens: (a) the antigen is dissolved in a solution, e.g., in water or an aqueous buffer; (b) a source of periodate is added to the antigen from a concentrated stock solution to form an oxidation mixture; (c) the reaction mixture is incubated; and (d) (optional) excess periodate is removed.

[0096] Deionized water or a suitable buffered solution 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) comprises an effective amount of a buffer with a pKa around physiological pH. In some embodiments, the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH, wherein the buffer does not comprise an amine group. Examples of amine-free buffers include, but are not limited to acetate, formate, and phosphate. Step (a) may be provided as a solution in an aqueous buffer, e.g., having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3-5.7, 5.4-5.6, 5.4-5.9).

[0097] The periodate source in step (b) is optionally selected from any periodate source with appropriate stability in aqueous solution. Examples of periodate sources include, but are not limited to, sodium periodate, potassium periodate, tetrabutylammonium (meta)periodate, barium periodate, sodium hydrogen periodate, sodium (para)periodate, and tetraethylammonium

(meta)periodate.

[0098] In some embodiments, the level of periodate addition and reaction conditions are adjusted to convert all available diols on a polysaccharide to aldehydes. For example, large excesses of sodium periodate (>1000c excess with respect to the molar concentration of polysaccharide, or a lOmM solution of sodium periodate) in combination with incubation at room temperature favor total conversion of diols to aldehydes. In general, however, the relative amount of periodate reagent added into the saccharide antigen solution is typically, although not necessarily, in the range of 0.1 equivalents to 0.5 equivalents of periodate, with "equivalents" being relative to individual saccharide units (i.e., saccharide "monomer" units in a

polysaccharide). Reaction conditions for periodate oxidation of a saccharide antigen can vary, but in a preferred embodiment are as follows: a reaction pH in the range of 5 to 7, such as 5 to 6, e.g., 5.4; a reaction temperature in the range of 4 °C to 25 °C; a reaction time in the range of 2 to 24 hours; and polysaccharide-aldehyde (PS-aldehyde) purification carried out using dialysis, size exclusion chromatography (SEC), ultrafiltration/diafiltration (ETF/DF), or the like. The PS- aldehyde is maintained in an aqueous buffer throughout the activation process, and is not "isolated" from aqueous buffer at any point during the activation reactions.

[0099] Following purification of the PS-aldehyde intermediate, the PS-aldehyde is dissolved in an aqueous buffer, typically maintained at a pH in the range of 5 to 7, such as 5 to to 6.7, 5 to 6.5, 5.5 to 6.7, 5.5 to 5.9, or 5.7. A reductive amination reaction is carried out by adding to the PS-aldehyde solution an activating reagent in the form of a reactive moiety coupled to a primary amino group, followed by admixture with sodium cyanoborohydride, for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen. In some embodiments, 2 to 12 equivalents of the

cyanoborohydride are used; in other embodiments, 4 to 12, 6 to 12, or 8 to 12 equivalents are used. The reactive moiety is one that is capable of participating in a click chemistry reaction with a second, bio-orthogonal reactive moiety at nnAA residues on the polypeptide carrier, as will be discussed below.

[0100] One example of a preferred activating reagent is a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12. In one embodiment, m is 1 and n is an integer in the range of 2 to 12, e.g., 2 to 8, 2 to 6, 2 to 4, or 4. In another embodiment, m is zero. The reactive moiety in this case is the alkyne functionality in the eight-membered ring, the reactivity of which is enhanced by the strain imposed by the adjacent phenyl rings. The amount of the DBCO derivative used in the reaction is usually in the range of 1 to 3 equivalents, e.g., at least 2 equivalents, such as 2 equivalents or 3 equivalents, again relative to the individual saccharide units, with the DBCO dissolved in a suitable solvent such as DMSO. The amount of sodium cyanoborohydride employed is typically in the range of 2.0 to 12.0 equivalents, again relative to the saccharide units. In one embodiment, the amount of sodium cyanoborohydride is in the range of in the range of 8.0 to 12.0 equivalents, as this excess can increase the yield of the activation reaction, or "DBCO%," (i.e., the amount of DBCO derivative that reacts with the saccharide relative to the total DBCO derivative employed, as explained earlier herein). The reaction is allowed to proceed, with stirring, for 6 to 48 hours at a reaction temperature typically in the range of 20 °C to 25 °C, with the DBCO-derivatized antigen then purified using any conventional technique, e.g., dialysis, SEC, UF/DF, or the like.

[0101] In one embodiment, a periodate activation method is provided that can be used across a plurality of antigen serotypes to provide a target DBCO%, wherein the only variable that may need to be adjusted for a specific serotype is the relative amount of periodate used per polysaccharide unit, i.e., periodate molar equivalents. "DBCO%" refers to one measure of the yield of the activation reaction and is defined the amount of DBCO derivative that reacts with the saccharide relative to the total DBCO derivative employed. Other variables can be kept constant regardless of the specific serotype undergoing activation, i.e., reaction pH, activation time, reaction temperature, molar equivalents of DBCO, and overall reaction time. These standardized conditions provide a target DBCO%, typically in the range of 3% to 10%, e.g., 3% to 5%, 5% to 10%, 5% to 7%, and the like.

[0102] Representative standardized conditions in this embodiment are as follows:

(a) providing the saccharide antigen as a solution in an aqueous buffer, e.g., a phosphate buffer, having a pH in the range of 5.5 to 5.9;

(b) oxidizing the saccharide antigen with an effective oxidizing amount of a periodate reagent, e.g., sodium periodate, thereby providing an aldehyde-bearing saccharide, where the amount of periodate reagent may vary with antigen serotype;

(c) purifying the aldehyde-bearing saccharide;

(d) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with 8 to 12 equivalents of sodium cyanoborohydride for a time period in the range of 18 to 30 hours, e.g., 18 to 24 hours, or 24 hours, during which the reactive moiety is transferred to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen. Exemplary reaction conditions that are generally useful for all periodate-activatable serotypes (e.g., serotypes 5, 6A, 6B, 7F, 12F, 14, 20. and 23F; see Table 2, infra ) are set forth in Table 1 :

Table 1 :

3. Antigen Activation - CD AP Method

[0103] In another embodiments, a different method is used to activate the saccharide antigen in preparation for conjugation. In this embodiment, the antigen is also functionalized with a reactive moiety capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety on the polypeptide carrier, but functionalization is carried out by cyanylating the antigen with a cyanylating reagent to provide cyanate (-0-CºN) group in place of hydroxyl groups, and thereafter, in a "one pot" reaction, contacting the cyanylated antigen (PS-0-CºN) with an activating reagent as was done in periodate activation. The activating reagent again comprises the reactive moiety coupled to a primary amino group, and may be a DBCO derivative as discussed in the preceding section.

[0104] In one embodiment, the saccharide antigen is provided at the outset in an aqueous buffer, typically having a pH in the range of 7 to 11, e.g., greater than 7 and up to 11, such as 8.5 to 10, 8.5. to 9.5, or 8.5 to 9.0, or 8.9, or 9.0. Starting with the antigen in an alkaline buffer solution obviates the need for pH adjustment during the reactions (i.e., during or after cyanylation and prior to conjugation to a polypeptide carrier). Other preferred reaction conditions are as follows: addition of the DBCO derivative 3 to 13 minutes after contacting the antigen with the cyanylation reagent; and a DBCO: cyanylated PS ratio achieved by using 0.25 equivalents to 3.0 equivalents, e.g., 0.25 to 2.0 equivalents, including 0.25 to 1.5 equivalents, 0.25 to 1.25 equivalents, 1.0 equivalent, and 2.0 equivalents, of DBCO.

[0105] In another embodiment, a CDAP activation method is provided that can be used across a plurality of antigen serotypes to provide a target DBCO%, wherein the only variable that may need to be adjusted for a specific serotype is the relative amount of CDAP used per

polysaccharide unit, i.e., CDAP molar equivalents. Other variables can be kept constant regardless of the specific serotype undergoing activation, i.e., reaction pH, activation time, reaction temperature, and molar equivalents of DBCO. These standardized conditions provide a target DBCO%, typically in the range of 3% to 10%, e.g., 3% to 5%, 5% to 10%, 5% to 7%, and the like.

[0106] Representative standardized conditions in this embodiment are as follows:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 7 to 11, e.g., 8.5 to 10; (b) cyanylating hydroxyl groups on the saccharide antigen with an effective cyanylating amount of CDAP to provide a cyanate-substituted saccharide, where the effective cyanylating amount of CDAP may vary with antigen serotype;

(c) allowing the cyanylation reaction to proceed for 3 to 13 minutes;

(d) thereafter contacting the cyanate-substituted saccharide with 0.25 equivalents to 2.0 equivalents, e.g., 0.25 to 1.5 equivalents, such as 1.0 equivalent, of a dibenzylcyclooctyne (DBCO) derivative having the structure of formula (I) wherein m is zero, or m is 1 and n is an integer in the range of 2 to 12, e.g., 2 to 8, such as 4, thereby transferring the DBCO moiety to the cyanate-substituted saccharide.

[0107] Exemplary activation technique(s) for various pneumococcal serotypes are set forth in Table 2 (where serotype 20 includes 20 A and 20B):

Table 2:

4. Conjugation method

[0108] To form an immunogenic conjugate, the antigen activated as described in the preceding sections is combined with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety that reacts with the reactive moiety on the activated antigen. A solution of the activated antigen is admixed with a selected buffer and preferably a solvent such as DMSO. The nnAA-containing carrier bearing the bio-orthogonal reactive moiety is then added, typically at a ratio of antigenxarrier in the range of about 0.5 w/w to 2.5 w/w. The reaction is allowed to proceed for a reaction time in the range of 4 to 48 hours, with gentle mixing, typically at a reaction temperature in the range of 4 °C to 37 °C, e.g., 20 °C to 25 °C. Unreacted DBCO groups can be quenched by addition of 0.25 to 2.0 molar equivalents of sodium azide, with stirring for on the order of 0.5 to 2 hours. For periodate-activated antigens, unreacted aldehyde groups can be quenched by addition of 0.5 to 2.0 molar equivalents of sodium borohydride and stirring for about 1 to 4 hours. The saccharide antigen-polypeptide carrier conjugates are purified as described above for the activated antigens, e.g., via dialysis, size exclusion chromatography, or UF/DF.

[0109] A conjugate of the invention can have a molecular weight of at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa, or more. In some embodiments, the conjugate has a molecular weight of between about 750 kDa and about 5,000 kDa. In some embodiments, the conjugate has a molecular weight of between about 800 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 850 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 900 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 950 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 1,000 kDa and about 2,800 kDa. The molecular weight of a conjugate is calculated by size exclusion chromatography (SEC) combined with multiangle laser light scattering (MALS).

[0110] Conjugates of the invention include antigen ( e.g ., saccharide) and carrier polypeptide, and the weight ratio of these two components can be used as a parameter to define the conjugate. Higher anti gen: carrier weight ratios for saccharide-carrier conjugates allow for more saccharide antigen to be delivered with a lower amount of carrier polypeptide. For pneumococcal conjugate vaccines, the ratio is typically in the range 0.3-3.0, but this can vary with the serotype and aspects of the conjugation chemistry {Annex 2: Recommendations for the production and control of pneumococcal conjugate vaccines; WHO Technical Report Series, No. 927, 2005). The ratio of the commercial vaccine Prevnar-l3™ is 0.9. For compositions which include conjugates of multiple pneumococcal serotypes {e.g. more than 13 serotypes) the ratio for the complete composition is ideally above 1.0 {i.e. a weight excess of pneumococcal saccharide antigen) and is preferably 1.5 or more {e.g. within the range 1.5-3.0, or preferably 1.5-2.0).

C. Modified CRM197 Carrier Polypeptides:

[0111] As mentioned above, the carrier polypeptides of main interest herein are modified forms of CRM197. Thus preferred carrier polypeptides for use with the invention comprise an amino acid sequence that has at least 80% sequence identity (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) to SEQ ID NO: 1. For instance, the carrier polypeptide can comprise the amino acid sequence SEQ ID NO: 1 except for the presence of up to 10 nnAA, as discussed above.

[0112] SEQ ID NO: 1 includes an Arg-Arg dipeptide sequence at positions 192-193. This sequence can be subject to proteolytic cleavage in some circumstances. If desired, this site can be modified to prevent cleavage and improve yield. Thus in some embodiments a modified CRM197 carrier polypeptide used herein is free from an Arg-Arg dipeptide sequence. For instance, Arg-l92 and/or Arg-l93 of SEQ ID NO: 1 can be deleted or can be substituted with a different amino acid. Thus a preferred carrier polypeptide comprises an amino acid sequence which (i) has at least 80% (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one (e.g. at least 2, and preferably more, as discussed above) nnAA residue.

[0113] One such amino acid sequence is SEQ ID NO: 2, which differs from SEQ ID NO: 1 by having an Arg Asn substitution at position 193:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGY SVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDG ASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSV GSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPEL SELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNT EEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFL HDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGR KIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDH TKVNSKLSLFFEIKS

[0114] Any embodiment described herein, or in WO2018/ 126229, by reference to SEQ ID NO: 1 can be put into effect using SEQ ID NO: 2 instead.

[0115] Thus we provide a carrier polypeptide comprising amino acid sequence SEQ ID NO: 2, wherein SEQ ID NO: 2 has been modified to include from 1-10 (e.g. from 3-9 or from 2-8, or from 2-6, or from 3-6, or from 4-6) nnAA residues. These nnAA residue modifications can be incorporated into SEQ ID NO: 2 as insertions and/or substitutions (e.g. SEQ ID NO: 4, which includes 6 Lys nnAA substitutions). Residue Asn-l93 of SEQ ID NO: 2 is preferably not substituted by a nnAA. This carrier polypeptide can be used to prepare immunogenic conjugates (e.g. of saccharide antigens) via the nnAA residue(s) therein.

[0116] In some embodiments these carrier polypeptides include amino acid sequences upstream and/or downstream of SEQ ID NO: 1 or 2. Thus, for instance, they can include a methionine residue upstream of the N-terminus amino acid residue of SEQ ID NO: 1 or 2. This methionine residue may be formylated. A methionine residue is not present at this position in wild-type CRM197 but it can be included herein for initiating translation (e.g. in a cell-free polypeptide synthesis system) without requiring the whole native leader sequence. In some embodiments a carrier polypeptide includes (i) no amino acids upstream of the N-terminus of SEQ ID NO: 1 or 2, except for an optional methionine, and (ii) no amino acids downstream of the C-terminus of SEQ ID NO: 1 or 2.

[0117] Preferably, at least one Lys residue in SEQ ID NO: 1 or 2 is substituted by a nnAA residue. It is preferred to substitute more than one residue in SEQ ID NO: 1 or 2 with a nnAA and, ideally, only one species of residue in SEQ ID NO: 1 is substituted by a nnAA e.g. only Lys residues are substituted. Where more than one residue in SEQ ID NO: 1 is substituted for a nnAA it is preferred that the same nnAA is used at each position e.g. pAMF at each substitution position. As noted above, in some embodiments residues other than Lys are substituted.

[0118] Carrier polypeptides comprising amino acid sequence SEQ ID NO: 1 or 2 with from 2-9 substitutions by nnAA residues (e.g. Lys— nAA substitutions, preferably Lys pAMF) are preferred, and ideally with from 2-8, 2-6, 3-8, 3-6, 4-9, 4-8, or 4-6 nnAA substitutions e.g. 4, 5 or 6 nnAA residues. This permits more extensive attachment of antigens to the carrier than using a single nnAA, thereby increasing the anti gen: carrier ratio, while avoiding excessive disruption of the native sequence and structure, which can result in insolubility.

[0119] Structural studies of CRM 197 reveal two general 3D regions within SEQ ID NO: 1 or 2: the first region runs from the N-terminus to Asn-373; and the second region runs from

Ser-374 to the C-terminus. These first of these corresponds roughly to the domains known as‘C’ &‘T’ (catalytic and transmembrane), and the second to domain‘R’ (receptor binding). Ideally a carrier polypeptide includes at least one nnAA in the first region and at least one nnAA in the second region e.g. at least 2 nnAA in each region, or at least 3 nnAA in each region. This permits conjugated antigens to be spatially separated when attached to the carrier. A carrier with 3 nnAA in the first region and 3 nnAA in the second region is useful.

[0120] The first region contains 27 Lys residues, and the second region contains 12 Lys residues. Thus one or more (e.g. 3) Lys residues within the N-terminal 374 amino acids and one or more (e.g. 3) Lys residues within the C-terminal 162 amino acids of SEQ ID NO: 1 or 2 can be substituted with a nnAA e.g. within pAMF. [0121] Preferred embodiments of nnAA-containing carriers based on CRM197 have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in which one or more of residues K24, K33, K37, K39, K212, K214, K227, K264, K385, K522 and K526 is/are replaced by a nnAA (such as pAMF). One such sequence is SEQ ID NO:3, in which each X represents a nnAA (preferably the same nnAA, such as pAMF):

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAG YSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGD GASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRS VGSSLSCINLDWDVIRDXTKTKIESLKEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPE LSELXTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHN TEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHXTQPF LHDGYAVSWNTVEDSI IRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNG RKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVD HTKVNSXLSLFFEIKS (SEQ ID NO: 3)

[0122] Another such sequence is SEQ ID NO: 4, in which each X represents a nnAA

(preferably the same nnAA, such as pAMF):

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAG YSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGD GASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNS VGSSLSCINLDWDVIRDXTKTKIESLKEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPE LSELXTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHN TEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHXTQPF LHDGYAVSWNTVEDSI IRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNG RKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVD HTKVNSXLSLFFEIKS (SEQ ID NO: 4)

[0123] SEQ ID NOs: 3 & 4 can be very well-expressed in a cell-free protein synthesis system, while retaining good solubility and providing good immunogenic responses when conjugated to pneumococcal capsular saccharides. SEQ ID NO: 4 lacks the native Arg-Arg dipeptide.

[0124] A polypeptide consisting of SEQ ID NO: 4, in which each X is pAMF, is another preferred carrier polypeptide for use with the invention.

[0125] WO2018/126229 describes several amino acid residues which are suitable for nnAA substitution ( e.g . Lys-24, Lys-33, Lys-37, Lys-39, Lys-2l2, Lys-2l4, Lys-227, Lys-244, Lys- 264, Lys-385, Lys-522, Lys-526, Phe-l2, Phe-53, Phe-l23, Phe-l27, Phe-l40, Phe-l67, Phe- 250, Phe-389, Phe-530, or Phe-531, numbered according to SEQ ID NO: 1 herein). Other residues which can be substituted are: Asp-2l 1; Asp-295; Asp-352; Asp-392; Asp-465; Asp- 467; Asp-507; Asp-5l9; Asn-296; Asn-359; Asn-399; Asn-48l; Asn-486; Asn-502; Asn-524; Glu-240; Glu-248; Glu-249; Glu-256; Glu-259; Glu-292; Glu-362; Gln-252; Gln-287; Lys-2l2; Lys-2l8; Lys-22l; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-498; Lys-5l6; Lys-522; Lys-534; Arg-377; Arg-407; Arg-455; Arg-460; Arg-462; Arg-472; Arg-493; Ser-l98; Ser-200; Ser-23 l; Ser-233; Ser-239; Ser-26l; Ser-374; Ser-38l; Ser-297; Ser-397; Ser-45l; Ser-475; Ser-494; Ser-495; Ser-496; Ser-50l; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and/or Thr-5l7.

[0126] We also provide a polypeptide comprising an amino acid sequence which (i) has at least 80% (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one nnAA residue; and wherein the polypeptide has a N-terminus methionine and/or is in monomeric form.

[0127] These CRMl97-derived carrier polypeptides can be used in the same manner for conjugation as CRM197 has been used in the prior art (e.g. see Broker el al. 2011 supra , WO2015/117093, etc.), but with the improvement of permitting site-specific conjugation via the nnAA residue(s). They will generally be used in monomeric monomeric form, rather than being associated with other CRM197 or CRMl97-derived subunits to form polypeptide multimers. Similarly, they will generally include at least one disulfide bridge e.g. between Cys-l86 & Cys-20l (numbered according to SEQ ID NO: 1) and, optionally, between Cys-46l & Cys-47l.

D. General:

[0128] The term“comprising” encompasses“including” as well as“consisting,” e.g., a composition“comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.

[0129] The term“about” in relation to a numerical value x is optional and means, for example, c+10%. [0130] The word“substantially” does not exclude“completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0131] The term“sequence identity” in the context of two amino acid sequences refers to two sequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm (e.g., BLASTP). The percent identity is determined over the full-length reference sequence disclosed herein, such as the reference sequence set forth in SEQ ID NO: 1 or 2. The method for calculating the sequence identity as provided herein is the BLASTP program having its defaults set at a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g, Henikoff & Henikoff,

1989, Proc Natl Acad Sci USA 89: 10915). See e.g., the BLAST alignment tool available on the WWW at blast.ncbi.nlm.nih.gov/Blast.cgi or elsewhere.

[0132] The term“lower alkyl” as used herein, and unless otherwise specified, refers to a saturated straight or branched hydrocarbon having one to six carbon atoms, i.e., Ci to C6 alkyl. In certain embodiments, the lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted moieties. See also US-2014/0066598. The term“lower alkylene” refers to an alkylene radical of a lower alkyl.

[0133] Unless defined otherwise, all technical and scientific terms used herein have the commonly understood meaning. Practitioners are particularly directed to Green & Sambrook (eds ) Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), and Ausubel, F. M., et al, Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012), and Plotkin, S.A., Orenstein, W.A., & Offit, P.A., Vaccines, 6th ed, Elsevier, London (2013).

[0134] Methods for cell-free synthesis are described in Spirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. Methods for incorporation of non-natural amino acids into proteins using cell-free synthesis are described in Shimizu et al. (2006) FEBS Journal, 273, 4133-4140 and also in Chong (2014) Curr Protoc Mol Biol. 108: 16.30.1-11. ENUMERATED EMBODIMENTS

[0135] Some embodiments of this disclosure relate to Embodiment I, as follows:

[0136] Embodiment 1-1. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(a) functionalizing the saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety on a second reactant, by (i) providing the saccharide as a solution in an aqueous buffer having a pH in the range of 7 to 11; (ii) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the saccharide solution to provide a cyanate-substituted saccharide, and thereafter (iii) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide; and

[0137] Embodiment 1-2. The method of embodiment 1-1, wherein step (iii) is carried out about 3 to about 13 minutes after step (ii).

[0138] Embodiment 1-3. The method of embodiment 1-2, wherein step (iii) is carried out about 5 minutes after step (ii).

[0139] Embodiment 1-4. The method of any one of embodiments 1-1 through 1-3, wherein (a) comprises a one-pot reaction without addition of a pH-adjusting agent during or subsequent to step (ii).

[0140] Embodiment 1-5. The method of any one of embodiments 1-1 through 1-4, wherein the cyanylating reagent comprises cyano-4-dimethylamino pyridinium tetrafluorob orate (CDAP). [0141] Embodiment 1-6. The method of embodiment 1-5, wherein the reactive moiety comprises an alkyne functionality.

[0142] Embodiment 1-7. The method of embodiment 1-6, wherein the alkyne functionality comprises a chemically constrained alkyne.

[0143] Embodiment 1-8. The method of embodiment 1-7, wherein the reactive moiety comprises a diarylcyclooctyne group.

[0144] Embodiment 1-9. The method of embodiment 1-8, wherein the diarylcyclooctyne group is a dibenzylcyclooctyne group.

[0145] Embodiment I- 10. The method of embodiment 1-9, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0146] Embodiment 1-11. The method of embodiment 1-10, wherein m is 1 and n is an integer in the range of 2 to 8.

[0147] Embodiment 1-12. The method of embodiment 1-11, wherein n is 4.

[0148] Embodiment 1-13. The method of embodiment I- 10, wherein m is zero.

[0149] Embodiment 1-14. The method of embodiment 1-10, wherein step (iii) is carried out using 0.25 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide. [0150] Embodiment 1-15. The method of embodiment 1-14, wherein step (iii) is carried out using 0.25 equivalents to 1.5 equivalents of the DBCO derivative.

[0151] Embodiment 1-16. The method of embodiment 1-15, wherein step (iii) is carried out using 1.0 equivalents of the DBCO derivative.

[0152] Embodiment 1-17. The method of embodiment 1-15 or 1-16, wherein the CDAP is added to the saccharide solution in an amount effective to achieve a target yield for step (iii) in the range of 5% to 10%, wherein the target yield is the percentage of the DBCO derivative that reacts with the cyanylated saccharide.

[0153] Embodiment 1-18. The method of embodiment 1-17, wherein step (i) is carried out using 0.5 equivalents to 5.0 equivalents CDAP, relative to the saccharide.

[0154] Embodiment 1-19. The method of embodiment 1-18, wherein the antigen is a bacterial capsular saccharide.

[0155] Embodiment 1-20. The method of embodiment 1-19, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0156] Embodiment 1-21. The method of embodiment 1-20, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0157] Embodiment 1-22. The method of embodiment 1-21, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A,

6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0158] Embodiment 1-23. The method of embodiment 1-1, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

[0159] Embodiment 1-24. The method of embodiment 1-1, further including (c) recovering the conjugate. [0160] Embodiment 1-25. The method of embodiment 1-1, wherein the polypeptide comprises at least two nnAA residues.

[0161] Embodiment 1-26. The method of embodiment 1-25, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0162] Embodiment 1-27. The method of embodiment 1-1, wherein the at least one T-cell activating epitope is from a native carrier protein selected from the group consisting of

Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D, and CRM 197.

[0163] Embodiment 1-28. The method of embodiment 1-1, wherein the at least one nnAA is substituted for a lysine in the native carrier protein.

[0164] Embodiment 1-29. The method of embodiment 1-1, wherein the polypeptide has at least 80% sequence identity to SEQ ID NO: l.

[0165] Embodiment 1-30. The method of embodiment 1-29, wherein the at least one nnAA is substituted for a lysine in SEQ ID NO: 1.

[0166] Embodiment 1-31. The method of embodiment 1-30, wherein the at least one nnAA is substituted for herein at least one nnAA is substituted for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 of SEQ ID NO: l.

[0167] Embodiment 1-32. The method of embodiment 1-31, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0168] Embodiment 1-33. The method of embodiment 1-32, wherein the at least one 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)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof. [0169] Embodiment 1-34. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one non-natural amino acid (nnAA) residue bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0170] Embodiment 1-35. The method of embodiment 1-34, further including (c) recovering the conjugate.

[0171] Embodiment 1-36. The method of embodiment 1-34 or 1-35, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0172] Embodiment 1-37. The method of any one of embodiments 1-34 to 1-36, wherein the polypeptide comprises at least two nnAA residues.

[0173] Embodiment 1-38. The method of embodiment 1-37, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0174] Embodiment 1-39. The method of embodiment 1-38, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent. [0175] Embodiment 1-40. The method of embodiment 1-39, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0176] Embodiment 1-41. The method of embodiment 1-34, wherein the antigen is a bacterial capsular saccharide.

[0177] Embodiment 1-42. The method of embodiment 1-41, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0178] Embodiment 1-43. The method of embodiment 1-41, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0179] Embodiment 1-44. The method of embodiment 1-43, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A,

[0180] Embodiment 1-45. A conjugate vaccine prepared according to the method of any one of embodiments 1-34 through 1-44.

[0181] Embodiment 1-46. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

[0182] Embodiment 1-47. The method of embodiment 1-46, further including (c) recovering the conjugate.

[0183] Embodiment 1-48. The method of embodiment 1-46 or 1-47, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0184] Embodiment 1-49. The method of any one of embodiments 1-46 to 1-48, wherein the polypeptide comprises at least two nnAA residues.

[0185] Embodiment 1-50. The method of embodiment 1-49, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0186] Embodiment 1-51. The method of embodiment 1-50, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent. [0187] Embodiment 1-52. The method of embodiment 1-51, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0188] Embodiment 1-53. The method of embodiment 1-46, wherein the antigen is a bacterial capsular saccharide.

[0189] Embodiment 1-54. The method of embodiment 1-53, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0190] Embodiment 1-55. The method of embodiment 1-54, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0191] Embodiment 1-56. The method of embodiment 1-55, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A,

[0192] Embodiment 1-57. A conjugate vaccine prepared according to the method of any one of embodiments 1-46 through 1-56.

[0193] Embodiment 1-58. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues

[0194] Embodiment 1-59. The method of embodiment 1-58, further including (c) recovering the conjugate.

[0195] Embodiment 1-60. The method of embodiment 1-58 or 1-59, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0196] Embodiment 1-61. The method of any one of embodiments 1-58 to 1-60, wherein the polypeptide comprises at least two nnAA residues.

[0197] Embodiment 1-62. The method of embodiment 1-61, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0198] Embodiment 1-63. The method of embodiment 1-62, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent. [0199] Embodiment 1-64. The method of embodiment 1-63, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0200] Embodiment 1-65. The method of embodiment 1-58, wherein the antigen is a bacterial capsular saccharide.

[0201] Embodiment 1-66. The method of embodiment 1-65, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0202] Embodiment 1-67. The method of embodiment 1-66, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0203] Embodiment 1-68. The method of embodiment 1-67, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A,

[0204] Embodiment 1-69. A conjugate vaccine prepared according to the method of any one of embodiments 1-58 through 1-68.

[0205] Embodiment 1-70. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous buffer having a pH in the range of 7 to 11;

(b) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the buffered saccharide solution to provide a cyanate-substituted saccharide; and thereafter (c) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide.

[0206] Embodiment 1-71. The method of embodiment 1-70, wherein step (c) is carried out about 3 to about 13 minutes after step (b).

[0207] Embodiment 1-72. The method of embodiment 1-71, wherein step (c) is carried out about 5 minutes after step (b).

[0208] Embodiment 1-73. The method of any one of embodiments 1-70 through 1-72, comprising a one-pot reaction without addition of a pH-adjusting agent during or subsequent to step (b).

[0209] Embodiment 1-74. The method of any one of embodiments 1-70 through 1-73, wherein the cyanylating reagent comprises cyano-4-dimethylamino pyridinium tetrafluorob orate (CDAP).

[0210] Embodiment 1-75. The method of embodiment 1-74, wherein the reactive moiety comprises an alkyne functionality.

[0211] Embodiment 1-76. The method of embodiment 1-75, wherein the alkyne functionality comprises a chemically constrained alkyne.

[0212] Embodiment 1-77. The method of embodiment 1-76, wherein the reactive moiety comprises a diarylcyclooctyne group.

[0213] Embodiment 1-78. The method of embodiment 1-77, wherein the diarylcyclooctyne group is a dibenzylcyclooctyne group.

[0214] Embodiment 1-79. The method of embodiment 1-78, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0215] Embodiment 1-80. The method of embodiment 1-79, wherein m is 1 and n is an integer in the range of 2 to 8.

[0216] Embodiment 1-81. The method of embodiment 1-80, wherein n is 4.

[0217] Embodiment 1-82. The method of embodiment 1-79, wherein m is zero.

[0218] Embodiment 1-83. The method of embodiment 1-79, wherein step (c) is carried out using 0.25 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0219] Embodiment 1-84. The method of embodiment 1-83, wherein step (c) is carried out using 0.25 equivalents to 1.5 equivalents of the DBCO derivative.

[0220] Embodiment 1-85. The method of embodiment 1-84, wherein step (c) is carried out using 1.0 equivalents of the DBCO derivative.

[0221] Embodiment 1-86. The method of embodiment 1-84 or 1-85, wherein the CDAP is added to the saccharide solution in an amount effective to achieve a target yield for step (c) in the range of 5% to 10%, wherein the target yield is the percentage of the DBCO derivative that reacts with the cyanylated saccharide.

[0222] Embodiment 1-87. The method of embodiment 1-86, wherein step (a) is carried out using 0.5 equivalents to 5.0 equivalents CDAP, relative to the saccharide.

[0223] Embodiment 1-88. The method of embodiment 1-70, wherein the saccharide comprises an antigen. [0224] Embodiment 1-89. The method of embodiment 1-88, wherein the antigen is a bacterial capsular saccharide.

[0225] Embodiment 1-90. The method of embodiment 1-89, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0226] Embodiment 1-91. The method of embodiment 1-90, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A,

[0227] Embodiment 1-92. The method of embodiment 1-70, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

[0228] Embodiment 1-93. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

[0229] Embodiment 1-94. The method of embodiment 1-93, wherein after step (c) and prior to step (d), the aldehyde-bearing saccharide is dissolved in an aqueous buffer having a pH in the range of 5.5 to 6.9.

[0230] Embodiment 1-95. The method of embodiment 1-94, wherein the aqueous buffer has a pH of 5.7.

[0231] Embodiment 1-96. The method of any one of embodiments 1-93, 1-94, and 1-95, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0232] Embodiment 1-97. The method of embodiment 1-96, wherein m is 1 and n is an integer in the range of 2 to 8.

[0233] Embodiment 1-98. The method of embodiment 1-97, wherein n is 4.

[0234] Embodiment 1-99. The method of embodiment 1-96, wherein m is zero. [0235] Embodiment I- 100. The method of any one of embodiments 1-96 through 1-99, wherein step (e) is carried out using 1 equivalent to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0236] Embodiment 1-101. The method of embodiment 1-100, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0237] Embodiment 1-102. The method of embodiment 1-101, wherein step (e) is carried out for 24 hours.

[0238] Embodiment 1-103. The method of embodiment 1-93, wherein the antigen is a bacterial capsular saccharide.

[0239] Embodiment 1-104. The method of embodiment 1-103, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0240] Embodiment 1-105. The method of embodiment 1-104, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0241] Embodiment 1-106. The method of embodiment 1-105, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3,

4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0242] Embodiment 1-107. The method of embodiment 1-93, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

[0243] Embodiment 1-108. The method of embodiment 1-93, further including (c) recovering the conjugate.

[0244] Embodiment 1-109. The method of embodiment 1-93, wherein the polypeptide comprises at least two nnAA residues. [0245] Embodiment 1-110. The method of embodiment 1-109, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0246] Embodiment 1-111. The method of embodiment 1-93, wherein the at least one T-cell activating epitope is from a native carrier protein selected from the group consisting of

[0247] Embodiment 1-112. The method of embodiment 1-93, wherein the at least one nnAA is substituted for a lysine in the native carrier protein.

[0248] Embodiment 1-113. The method of embodiment 1-93, wherein the polypeptide has at least 80% sequence identity to SEQ ID NO: l.

[0249] Embodiment 1-114. The method of embodiment 1-113, wherein the at least one nnAA is substituted for a lysine in SEQ ID NO: 1.

[0250] Embodiment 1-115. The method of embodiment 1-114, wherein the at least one nnAA is substituted for herein at least one nnAA is substituted for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 of SEQ ID NO: l.

[0251] Embodiment 1-116. The method of embodiment 1-115, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0252] Embodiment 1-117. The method of embodiment 1-116, wherein the at least one 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)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0253] Embodiment 1-118. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising: (a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(f) combining the activated saccharide antigen with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0254] Embodiment 1-119. The method of embodiment 1-118, wherein the aqueous buffer has a pH of 5.7.

[0255] Embodiment 1-120. The method of embodiment 1-118, wherein after step (c) and prior to step (d), the aldehyde-bearing saccharide is dissolved in an aqueous buffer having a pH in the range of 5.5 to 6.9.

[0256] Embodiment 1-121. The method of any one of embodiments I- 118, 1- 119, and 1-120, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0257] Embodiment 1-122. The method of embodiment 1-121, wherein m is 1 and n is an integer in the range of 2 to 8.

[0258] Embodiment 1-123. The method of embodiment 1-122, wherein n is 4.

[0259] Embodiment 1-124. The method of embodiment 1-121, wherein m is zero.

[0260] Embodiment 1-125. The method of any one of embodiments 1-121 through 1-124, wherein step (e) is carried out using 1 equivalent to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0261] Embodiment 1-126. The method of embodiment 1-125, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0262] Embodiment 1-127. The method of embodiment 1-126, wherein step (e) is carried out for 24 hours.

[0263] Embodiment 1-128. The method of embodiment 1-118, wherein the antigen is a bacterial capsular saccharide.

[0264] Embodiment 1-129. The method of embodiment 1-128, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali. [0265] Embodiment 1-130. The method of embodiment 1-129, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0266] Embodiment 1-131. The method of embodiment 1-130, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0267] Embodiment 1-132. The method of embodiment 1-118, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

[0268] Embodiment 1-133. The method of embodiment 1-118, further including (c) recovering the conjugate.

[0269] Embodiment 1-134. The method of embodiment 1-118, wherein the polypeptide comprises at least two nnAA residues.

[0270] Embodiment 1-135. The method of embodiment 1-134, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0271] Embodiment 1-136. The method of embodiment 1-118, wherein the at least one T-cell activating epitope is from a native carrier protein selected from the group consisting of

[0272] Embodiment 1-137. The method of embodiment 1-118, wherein the at least one nnAA is substituted for a lysine in the native carrier protein.

[0273] Embodiment 1-138. The method of embodiment 1-118, wherein the polypeptide has at least 80% sequence identity to SEQ ID NO: l .

[0274] Embodiment 1-139. The method of embodiment 1-138, wherein the at least one nnAA is substituted for a lysine in SEQ ID NO: 1. [0275] Embodiment 1-140. The method of embodiment 1-139, wherein the at least one nnAA is substituted for herein at least one nnAA is substituted for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 of SEQ ID NO: l.

[0276] Embodiment 1-141. The method of embodiment 1-140, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0277] Embodiment 1-142. The method of embodiment 1-141, wherein the at least one 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)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0278] Embodiment 1-143. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one non-natural amino acid (nnAA) residue bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

[0279] Embodiment 1-144. The method of embodiment 1-143, wherein the aqueous buffer has a pH in the range of 5.5 to 5.9.

[0280] Embodiment 1-145. The method of embodiment 1-143 or 1-144, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0281] Embodiment 1-146. The method of embodiment 1-145, wherein m is 1 and n is an integer in the range of 2 to 8.

[0282] Embodiment 1-147. The method of embodiment 1-146, wherein n is 4.

[0283] Embodiment 1-148. The method of embodiment 1-145, wherein m is zero.

[0284] Embodiment 1-149. The method of any one of embodiments 1-145 through 1-148, wherein step (e) is carried out using 1 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0285] Embodiment 1-150. The method of embodiment 1-149, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide. [0286] Embodiment 1-151. The method of embodiment 1-150, wherein step (e) is carried out for 24 hours.

[0287] Embodiment 1-152. The method of embodiment 1-143, further including (g) recovering the conjugate.

[0288] Embodiment 1-153. The method of embodiment 1-143, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0289] Embodiment 1-154. The method of any one of embodiment 1-143, wherein the polypeptide comprises at least two nnAA residues.

[0290] Embodiment 1-155. The method of embodiment 1-154, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0291] Embodiment 1-156. The method of embodiment 1-154, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0292] Embodiment 1-157. The method of embodiment 1-156, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0293] Embodiment 1-158. The method of embodiment 1-143, wherein the antigen is a bacterial capsular saccharide.

[0294] Embodiment 1-159. The method of embodiment 1-158, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0295] Embodiment 1-160. The method of embodiment 1-159, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae. [0296] Embodiment 1-161. The method of embodiment 1-160, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3,

4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F,

24F, 31, and 33F.

[0297] Embodiment 1-162. A conjugate vaccine prepared according to the method of any one of embodiments 1-143 through 1-161.

[0298] Embodiment 1-163. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1 and (ii) includes a nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-2l 1; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp-507; Asp-519; Asn-296; Asn-359; Asn-399;

[0299] Embodiment 1-164. The method of embodiment 1-163, wherein the aqueous buffer has a pH in the range of 5.5 to 5.9.

[0300] Embodiment 1-165. The method of embodiment 1-163 or 1-164, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0301] Embodiment 1-166. The method of embodiment 1-165, wherein m is 1 and n is an integer in the range of 2 to 8.

[0302] Embodiment 1-167. The method of embodiment 1-166, wherein n is 4.

[0303] Embodiment 1-168. The method of embodiment 1-165, wherein m is zero.

[0304] Embodiment 1-169. The method of any one of embodiments 1-165 through 1-168, wherein step (e) is carried out using 1 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide. [0305] Embodiment 1-170. The method of embodiment 1-169, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0306] Embodiment 1-171. The method of embodiment 1-170, wherein step (e) is carried out for 24 hours.

[0307] Embodiment 1-172. The method of embodiment 1-163, further including (g) recovering the conjugate.

[0308] Embodiment 1-173. The method of embodiment 1-163, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0309] Embodiment 1-174. The method of any one of embodiment 1-163, wherein the polypeptide comprises at least two nnAA residues.

[0310] Embodiment 1-175. The method of embodiment 1-174, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0311] Embodiment 1-176. The method of embodiment 1-175, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0312] Embodiment 1-177. The method of embodiment 1-176, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0313] Embodiment 1-178. The method of embodiment 1-163, wherein the antigen is a bacterial capsular saccharide.

[0314] Embodiment 1-179. The method of embodiment 1-178, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali. 10315] Embodiment 1-180. The method of embodiment 1-179, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0316] Embodiment 1-181. The method of embodiment 1-180, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0317] Embodiment 1-182. A conjugate vaccine prepared according to the method of any one of embodiments 1-163 through 1-181.

[0318] Embodiment 1-183. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues

[0319] Embodiment 1-184. The method of embodiment 1-183, wherein the aqueous buffer has a pH in the range of 5.5 to 5.9.

[0320] Embodiment 1-185. The method of embodiment 1-183 or 1-184, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0321] Embodiment 1-186. The method of embodiment 1-185, wherein m is 1 and n is an integer in the range of 2 to 8.

[0322] Embodiment 1-187. The method of embodiment 1-186, wherein n is 4.

[0323] Embodiment 1-188. The method of embodiment 1-185, wherein m is zero. [0324] Embodiment 1-189. The method of any one of embodiments 1-185 through 1-188, wherein step (e) is carried out using 1 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0325] Embodiment 1-190. The method of embodiment 1-189, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

[0326] Embodiment 1-191. The method of embodiment 1-190, wherein step (e) is carried out for 24 hours.

[0327] Embodiment 1-192. The method of embodiment 1-183, further including (g) recovering the conjugate.

[0328] Embodiment 1-193. The method of embodiment 1-183, further including, prior to (a), subjecting the saccharide antigen to mechanical size reduction.

[0329] Embodiment 1-194. The method of any one of embodiment 1-183, wherein the polypeptide comprises at least two nnAA residues.

[0330] Embodiment 1-195. The method of embodiment 1-194, wherein the polypeptide comprises 4 to 9 nnAA residues.

[0331] Embodiment 1-196. The method of embodiment 1-195, wherein the at least one nnAA is a 2,3-disubstituted propanoic acid bearing an azido-containing substituent.

[0332] Embodiment 1-197. The method of embodiment 1-196, wherein the at least one 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)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.

[0333] Embodiment 1-198. The method of embodiment 1-183, wherein the antigen is a bacterial capsular saccharide. [0334] Embodiment 1-199. The method of embodiment 1-198, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0335] Embodiment 1-200. The method of embodiment 1-199, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0336] Embodiment 1-201. The method of embodiment 1-200, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6 A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0337] Embodiment 1-202. A conjugate vaccine prepared according to the method of any one of embodiments 1-183 through 1-201.

[0338] Embodiment 1-203. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio- orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate- substituted saccharide, thereby providing an activated saccharide antigen. [0339] Embodiment 1-204. The method of embodiment 1-203, wherein the aqueous buffer has a pH of 5.7.

[0340] Embodiment 1-205. The method of embodiment 1-203 or 1-204, wherein the activating reagent comprises a dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

[0341] Embodiment 1-206. The method of embodiment 1-205, wherein m is 1 and n is an integer in the range of 2 to 8.

[0342] Embodiment 1-207. The method of embodiment 1-206, wherein n is 4.

[0343] Embodiment 1-208. The method of embodiment 1-205, wherein m is zero.

[0344] Embodiment 1-209. The method of any one of embodiments 1-205 through 1-208, wherein step (e) is carried out using 3 equivalents of the DBCO derivative, relative to the saccharide.

[0345] Embodiment 1-210. The method of embodiment 1-209, wherein step (e) is carried out for 24 hours.

[0346] Embodiment 1-211. The method of embodiment 1-203, wherein the antigen is a bacterial capsular saccharide.

[0347] Embodiment 1-212. The method of embodiment 1-211, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

[0348] Embodiment 1-213. The method of embodiment 1-212, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

[0349] Embodiment 1-214. The method of embodiment 1-213, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3,

24F, 31, and 33F.

[0350] Embodiment 1-215. The method of embodiment 1-203, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

[0351] Embodiment 1-216. A method for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method substantially applicable to saccharide antigens across a plurality of serotypes and comprising:

(b) cyanylating hydroxyl groups on the saccharide antigen with an effective cyanylating amount of CDAP to provide a cyanate-substituted saccharide;

(c) after 3 to 13 minutes, contacting the cyanate-substituted saccharide with 0.25 equivalents to 2.0 equivalents of a dibenzylcyclooctyne (DBCO) derivative having the structure

(I) (I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12, thereby transferring the DBCO moiety to the cyanate-substituted saccharide, wherein the effective cyanylating amount of CDAP is selected to correspond to a particular serotype.

[0352] Embodiment 1-217. The method of embodiment 1-216, wherein the aqueous buffer has a pH in the range of 8.75 to 9.5.

[0353] Embodiment 1-218. The method of embodiment 1-216 or 1-217, wherein step (c) is carried out 5 minutes after step (b).

[0354] Embodiment 1-219. The method of any one of embodiments 1-216 through 1-218, wherein in step (c), the cyanate-substituted saccharide is contacted with 1 equivalent of the DBCO derivative.

[0355] Embodiment 1-220. A method for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method being substantially applicable to saccharide antigens across a plurality of serotypes and comprising:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 5,5 to 5.9;

(b) oxidizing the saccharide antigen with an effective oxidizing amount of a periodate reagent, thereby providing an aldehyde-bearing saccharide; (c) purifying the aldehyde-bearing saccharide;

[0356] Embodiment 1-221. The method of embodiment 1-220, wherein the time period is 24 hours.

EXAMPLES

[0357] The invention is illustrated in the following examples. The materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. The examples are carried out using well known and routine techniques to those of skill in the art, except where otherwise described in detail.

Examples from WO2018/126229:

[0358] The examples of WO2018/ 126229 describe in full detail the synthesis of single-site eCRM moieties (e.g. Kl 1TAG). These were expressed in a cell-free protein synthesis (CFPS) extract, and pAMF was incorporated in place of natural Lys.

[0359] Variants of CRM containing multiple nnAA per polypeptide were also expressed, with various different numbers of Lys pAMF substitutions per protein. In general it was found that higher numbers of substitutions gave carriers which led to higher MW conjugates but the carriers had lower solubility. Carriers with six pAMF residues generally provided both good solubility (»50mg/mL) and immunogenicity. The high solubility was surprising because replacement of charged Lys residues in the native sequence with hydrophobic pAMF residues increased the hydrophobicity of CRM197, which is a protein whose hydrophobicity has already been reported to affect its solubility. Thus it was shown that it is possible to maintain the same attachment sites which have been used in known CRM 197 conjugates (namely Lys residues) without causing insolubility when the charged residues are lost.

[0360] A particularly useful set of 6 Lys pAMF substitutions was seen using K34, K213, K245, K265, K386 and K527 (numbered according to SEQ ID NO: 3). This combination of sites for pAMF substitution is surprisingly effective, in particular because individual substitutions at positions K245 and K527 led to relatively poor levels of expression.

[0361] This set of six substitutions can be combined with disruption of the Arg-Arg dipeptide at residues 192-193 of SEQ ID NO: 1 (RR RN) to provide SEQ ID NO: 4, where each X is pAMF.

[0362] The examples of WO2018/126229 further describe general protocols for saccharide activation with sodium meta-periodate, for periodate-oxidized polysaccharide derivatization with DBCO, for saccharide activation with CDAP, and for conjugation of saccharide-DBCO with eCRM.

General protocols for antigen activation:

[0363] The following protocol was used to activate saccharide antigens using periodate oxidation followed by reductive amination:

[0364] To a 50 mM pH 5.4 acetate buffered polysaccharide solution was added (0.1-0.5) molar equivalents (to polysaccharide repeating unit) of sodium periodate with vigorous stirring. The reaction was stirred for (2-24) hours at (4-25) °C. The oxidized polysaccharide was then purified via dialysis, size exclusion chromatography, or UF/DF.

[0365] To a 100 mM pH (5.7-6.7) phosphate buffered oxidized polysaccharide solution was added (1-3) molar equivalents of dibenzocyclooctyne-PEG4-amine linker (dissolved in DMSO). The final concentration of DMSO in the reaction was (5-20)% v/v. Then (2-12) molar equivalents of sodium cyanoborohydride was added and the reaction was stirred for (6-48) hours at (20-25) °C. The dibenzocyclooctyne-derivatized polysaccharide was then purified via dialysis, size exclusion chromatography, or UF/DF. [0366] For activation of saccharide antigens via CDAP, the following protocol was used:

[0367] To a 100 mM pH (8-10) borate buffered polysaccharide solution was added (0.5-5) molar equivalents (to polysaccharide repeating unit) of l-cyano-4-dimethylaminopyridinium tetrafluorob orate (CDAP; from 100 mg/mL solution in acetonitrile) with vigorous stirring. (3- 13) min after addition of CDAP, (0.5-2.5) molar equivalents of dibenzocyclooctyne-PEG4- amine linker (dissolved in DMSO) was added. The final concentration of DMSO in the reaction was (5-10)% v/v. After 1 h of further reaction, the reaction was quenched by addition of glycine. The dibenzocyclooctyne-derivatized polysaccharide was then purified via dialysis, size exclusion chromatography, or UF/DF. All steps were performed at (20-25) °C.

General protocol for the conjugation reaction:

[0368] To a solution of dibenzocyclooctyne-derivatized polysaccharide was added potassium phosphate pH 7.5 buffer, DMSO for a final concentration of (0-15)% v/v, and sodium chloride for a final concentration of (0-300) mM. The carrier protein used for conjugation was the polypeptide having SEQ ID NO: 4 where each X is pAMF, which was then added for a final ratio of polysaccharide to carrier of (0.5-2.5) w/w. The solution was gently mixed for (4-48) hours typically at (20-25) °C but as low as 4 °C and as high as 37 °C. ETnreacted

dibenzocyclooctyne groups were quenched by addition of (0.25-2) molar equivalents of sodium azide and stirring for (0.5-2) hours. For serotypes prepared with periodate chemistry, unreacted aldehyde groups were quenched by addition of (0.5-2) molar equivalents of sodium borohydride and stirring for (1-4) hours. For serotypes prepared with CDAP chemistry, no sodium

borohydride quench was performed. The polysaccharide-carrier conjugate was then purified via dialysis, size exclusion chromatography, or EIF/DF.

Example 1

Periodate Activation of Pneumococcal Polysaccharide Serotypes 7F and 14:

[0369] Table 1 provides information regarding the activation conditions and product obtained upon activation of Pneumococcal Polysaccharide Serotypes 7F and 14 with sodium periodate (protocol as described above unless modified as indicated in the table): Table 1 :

Example 2

CHAP Activation of Pneumococcal Serotypes 3, 4, 17F, 22F, 6B, and 9N:

[0370] Table 2 provides information regarding the conditions and product obtained upon CDAP activation of the Pneumococcal serotypes activated. The CDAP protocol set forth above was followed unless modified as indicated in the table. Table 2:

[0371] Sizing for the activated serotypes is given in Table 3, for both periodate and CDAP:

Table 3:

Example 3

Conjugation of Activated Antigens to the Polypeptide Carrier:

[0372] The general conjugation protocol set forth above is used to prepare conjugates of activated antigens to a polypeptide carrier. The factors varied during conjugate preparation are identified in Table 4, with additional detail regarding the experiments set forth in Table 5:

Table 4:

Table 5:

Example 4

Pneumococcal PS Serotype 5 Sizing, Activation, and Conjugation:

PS5 Sizing and activation:

[0373] Serotype 5 polysaccharide (PS5) at 3 mg/mL was reduced in size via high pressure homogenization using 20,000 psi for 20 passes. The sized polysaccharide was analyzed via anthrone assay to determine the concentration. [0374] To the sized PS5 (42.2 mL, 2.275 mg/mL) was added 2.286 mL water and 2.4 mL acetate buffer (1.0 M, pH 5.4). The solution was stirred at 250 rpm and 1.116 mL sodium periodate solution (5 mg/mL in water) was added. After 3 h of reaction time at room

temperature, the solution was purified by tangential flow filtration against 10 diavolumes of water. The concentration of the oxidized polysaccharide PS5-OX was measured by anthrone assay.

[0375] To PS5-OX (17 mL, 4.322 mg/mL) was added 2.190 mL water and 2.449 mL sodium phosphate buffer (1.0 M, pH 5.1). The solution was stirred and then DBCO-PEG₄-amine was added (2.449 mL, 34.1 mg/mL in DMSO). After the solution was stirred to homogeneity, sodium cyanoborohydride was added (0.402 mL, 100 mg/mL in water). After 19 h of stirring at room temperature, the solution was purified by tangential flow filtration. The concentration of the activated polysaccharide PS5-DBCO was measured by anthrone assay and the concentration of DBCO-PEG₄-amine measured by absorbance at 309 nm.

PS-5 Conjugation:

[0376] To 3.377 mL of water was added 0.108 mL potassium phosphate (0.5 M, pH 7.5),

0.343 mL NaCl (5M), and 0.857 mL of DMSO. The mixture was stirred to homogeneity, then 0.870 mL of serotype 5 activated polysaccharide (PS5-DBCO, 2.3 mg/mL) was added. After mixing to homogeneity eCRM (0.160 mL, 5.2 mg/mL) was added and the reaction was stirred at 80 rpm for 15 h. The unreacted DBCO was quenched by addition of sodium azide (0.057 mL, 10 mg/mL in water) followed by stirring for 2 h. The resulting quenched conjugate was purified via dialysis, then analyzed to determine total saccharide concentration, total protein concentration, molecular weight, and free saccharide content.

Example 5

Method for Periodate Activation of Pneumococcal PS Serotype 6B:

[0377] To 6B sized polysaccharide (47.3 mL, 2.73 mg/mL) was added 9.019 mL water and 7.106 mL acetate buffer (1.0 M, pH 5.4). The solution was stirred at 250 rpm and 1.173 mL sodium periodate solution (5 mg/mL in water) was added. After 3 h of reaction time at room temperature, the solution was purified by tangential flow filtration against 10 diavolumes of water. The concentration of the oxidized polysaccharide PS6B-OX was measured by anthrone assay.

[0378] To the 6B oxidized polysaccharide (31.3 mL, 3.75 mg/mL) was added 3.908 mL sodium phosphate buffer (1.0 M, pH 5.1). The solution was stirred and then DBCO-PEG₄-amine was added (3.908 mL, 44.5 mg/mL in DMSO). After solution was stirred to homogeneity, sodium cyanoborohydride was added (0.556 mL, 150 mg/mL in water). After 16.5 h of stirring at room temperature, the solution was purified by tangential flow filtration. The concentration of the activated polysaccharide (PS6B-DBCO) was measured by anthrone assay and the concentration of DBCO-PEG₄-amine measured by absorbance at 309 nm.

Example 6

Method for Periodate Activation of Pneumococcal PS Serotype 23 F:

[0379] To 23F sized polysaccharide (54.0 mL, 2.517 mg/mL) was added 5.016 mL water and 7.475 mL acetate buffer (1.0 M, pH 5.4). The solution was stirred at 250 rpm and 1.467 mL sodium periodate solution (5 mg/mL in water) was added. After 3 h of reaction time at room temperature, the solution was purified by tangential flow filtration against 10 diavolumes of water. The concentration of the oxidized polysaccharide was measured by anthrone assay.

[0380] To 23F oxidized polysaccharide (25.5 mL, 4.726 mg/mL) was added 4.626 mL water and 4.017 mL sodium phosphate buffer (1.0 M, pH 5.1). The solution was stirred and then DBCO-PEG₄-amine was added (4.017 mL, 39.6 mg/mL in DMSO). After solution was stirred to homogeneity, sodium cyanoborohydride was added (2.008 mL, 38.056 mg/mL in water). After 18 h of stirring at room temperature, the solution was purified by tangential flow filtration. The concentration of the activated polysaccharide was measured by anthrone assay and the concentration of DBCO-PEG₄-amine measured by absorbance at 309 nm. SEQUENCE LISTING

SEQ ID NO: 1 (native CRM197)

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKE FYSTDNKYDAAGYSVDNE NPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPF AEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIR DKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAA WAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKI TAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVA FHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

SEQ ID NO: 2 (CRM197 with Arg-Asn substitution)

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKE FYSTDNKYDAAGYSVDNE NPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPF AEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIR DKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAA WAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDS IIRTGFQGESGHDIKI TAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVA FHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

SEQ ID NO: 3 (CRM197 with 6 preferred nnAA sites and N-terminus Met)

MGADDVVDSSKS FVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLP FAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVI RDXTKTKIESLKEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPELSELXTVTGTNPVFAGANYA AWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGE LVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHXTQPFLHDGYAVSWNTVEDSI IRTGFQGESGHDIK ITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVT FCRPKSPVYVGNGVHANLHV AFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSXLSLFFEIKS

SEQ ID NO: 4 (CRM197 with Arg-Asn subsf, 6 preferred nnAA sites, and N-terminus Met)

MGADDVVDSSKS FVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLP FAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVI RDXTKTKIESLKEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPELSELXTVTGTNPVFAGANYA AWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGE LVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHXTQPFLHDGYAVSWNTVEDSI IRTGFQGESGHDIK ITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHV AFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSXLSLFFEIKS

SEQ ID NO: 5 (H.influenzae protein D)

CSSHSSNMANTQMKSDKI IIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDG LTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEF IQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLP QMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDN IVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

Claims

CLAIMS:

1. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

2. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(b) cyanylating hydroxyl groups on the saccharide by adding a cyanylating reagent to the buffered saccharide solution to provide a cyanate-substituted saccharide; and thereafter

(c) contacting the cyanate-substituted saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, under conditions effective to transfer the reactive moiety to the cyanate-substituted saccharide.

3. The method of claim 2, wherein the saccharide comprises an antigen.

4. The method of claim 1, wherein step (iii) is carried out about 3 to about 13 minutes after step (ii) or the method of claim 2 or 3, wherein step (c) is carried out about 3 to about 13 minutes after step (b).

5. The method of claim 4, wherein step (iii) is carried out about 5 minutes after step (ii), or wherein step (c) is carried out about 5 minutes after step (b).

6. The method of any one of claims 1 through 5, wherein (a) comprises a one-pot reaction without addition of a pH-adjusting agent during or subsequent to step (ii) or during or subsequent to step (b).

7. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

8. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 11 and (ii) includes a nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-211; Asp- 295; Asp-352; Asp-392; Asp-465; Asp-467; Asp-507; Asp-519; Asn-296; Asn-359; Asn-399; Asn-48l; Asn-486; Asn-502; Asn-524; Glu-240; Glu-248; Glu-249; Glu-256; Glu-259; Glu-292; Glu-362; Gln-252; Gln-287; Lys-2l2; Lys-2l8; Lys-22l; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-498; Lys-5l6; Lys-522; Lys-534; Arg-377; Arg-407; Arg-455; Arg-460; Arg-462; Arg-472; Arg-493; Ser-l98; Ser-200; Ser-23 l; Ser-233; Ser-239; Ser-26l; Ser-374; Ser-38l; Ser-297; Ser-397; Ser-45l; Ser-475; Ser-494; Ser-495; Ser-496; Ser-50l; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and/or Thr-5l7, such that a click chemistry reaction between the reactive moiety and the bio- orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

9. A method for preparing a conjugate of a saccharide antigen and a carrier protein, comprising:

(b) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: l; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues

10. The method of any one of claims 1 through 10, wherein the cyanylating reagent comprises cyano-4-dimethylamino pyridinium tetrafluorob orate (CDAP).

11. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(b) oxidizing the saccharide by adding a periodate reagent to the solution, thereby providing an aldehyde-bearing saccharide; (c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with 8 to 12 equivalents of sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen; and

12. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(e) in a reductive amination reaction, contacting the aldehyde-bearing saccharide with an activating reagent comprising the reactive moiety coupled to a primary amino group, followed by admixture with sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen; and (f) combining the activated saccharide antigen with a carrier protein comprising a polypeptide having at least one T-cell activating epitope and at least one non-natural amino acid (nnAA) bearing the bio-orthogonal reactive moiety, such that a click chemistry reaction between the reactive moiety and the bio-orthogonal reactive moiety results in a conjugate of the saccharide antigen and the carrier protein.

13. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

14. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

15. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

(d) dissolving the aldehyde-bearing saccharide in an aqueous buffer;

(f) combining the activated saccharide antigen with a carrier protein comprising a modified CRM197 carrier polypeptide comprising: an amino acid sequence which (i) has at least 90% sequence identity to SEQ ID NO: l; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes a nnAA substitution at one or more of the following amino acid residues

16. The method of any one of claims 13-15, wherein the aqueous buffer has a pH in the range of 5.5 to 5.9.

17. The method of any one of claims 1 or 8-16, further including recovering the conjugate.

18. A method for functionalizing a saccharide with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method comprising:

(a) providing the saccharide as a solution in an aqueous medium;

(c) purifying the aldehyde-bearing saccharide;

19. The method of claim 18, wherein the aqueous buffer has a pH of 5.7.

20. The method of any one of claims 1-19, wherein the reactive moiety comprises an alkyne functionality.

21. The method of claim 20, wherein the alkyne functionality comprises a chemically constrained alkyne.

22. The method of any one of claims 1-21, wherein the reactive moiety comprises a diarylcyclooctyne group.

23. The method of claim 22, wherein the diarylcyclooctyne group is a

dibenzylcyclooctyne group.

24. The method of claim 23, wherein the activating reagent comprises a

dibenzylcyclooctyne (DBCO) derivative having the structure (I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12.

25. The method of claim 24, wherein m is 1 and n is an integer in the range of 2 to 8.

26. The method of claim 25, wherein n is 4.

27. The method of any one of claims 24-26, wherein step (e) is carried out using 1 equivalent to 3 equivalents of the DBCO derivative, relative to the saccharide.

28. The method of any one of claims 24-26, wherein step (e) is carried out using 2 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

29. The method of claim 28, wherein step (e) is carried out for 24 hours.

30. The method of any of claims 24-26 or 29, wherein step (e) is carried out using 3 equivalents of the DBCO derivative, relative to the saccharide.

31. The method of any of claims 24-26, wherein the cyanate-substituted saccharide is contacted with 0.25 equivalents to 3 equivalents of the DBCO derivative, relative to the saccharide.

32. The method of any of claims 24-26, wherein the cyanate-substituted saccharide is contacted with 0.25 equivalents to 1.5 equivalents of the DBCO derivative, relative to the saccharide.

33. The method of any of claims 24-26, wherein the cyanate-substituted saccharide is contacted with 1.0 equivalents of the DBCO derivative, relative to the saccharide.

34. The method of 10, wherein the CDAP is added to the saccharide solution in an amount effective to achieve a target yield for step (iii) in the range of 5% to 10%, wherein the target yield is the percentage of the DBCO derivative that reacts with the cyanylated saccharide or wherein the CDAP is added to the saccharide solution in an amount effective to achieve a target yield for step (c) in the range of 5% to 10%, wherein the target yield is the percentage of the DBCO derivative that reacts with the cyanylated saccharide.

35. The method of any of claims 1-34, wherein the antigen is a bacterial capsular saccharide.

36. The method of claim 35, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae , Neisseria meningitidis , Haemophilus influenzae , Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivali.

37. The method of claim 36, wherein the capsular saccharide is from a bacterium selected from Streptococcus pneumoniae.

38. The method of claim 37, wherein the antigen is a capsular saccharide of an S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 9N,

10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

39. The method of claim 38, wherein the antigen of serotype 20 is serotype 20B.

40. The method any of claims 1-39, further including, prior to (a), subjecting the saccharide to mechanical size reduction.

41. The method of claim 20-40, further including recovering the conjugate.

42. The method of any of claims 1, 4-17, or 20-41, wherein the polypeptide comprises at least two nnAA residues.

43. The method of claim 42, wherein the polypeptide comprises 4 to 9 nnAA residues.

44. The method of any one of claims 1, 4-6, 10-12, 17, 20-43, wherein the at least one T-cell activating epitope is from a native carrier protein selected from the group consisting of Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D, and CRM197.

45. The method any one of claims 1-45, wherein the at least one nnAA is substituted for a lysine in the native carrier protein.

46. The method of claim 1, 4-6, 10-12, 17, or 20-45, wherein the polypeptide has at least 80% sequence identity to SEQ ID NO: l.

47. The method of claim 46, wherein the at least one nnAA is substituted for a lysine in SEQ ID NO: 1.

48. The method of claim 47, wherein the at least one nnAA is substituted for herein at least one nnAA is substituted for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 of SEQ ID NO:l.

49. The method any one of claims 1, 4-6, 10-12, 17, or 20-48, wherein the at least one nnAA is a 2, 3 -di substituted propanoic acid bearing an azido-containing substituent.

50. The method of claim 49, wherein the at least one 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)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 -yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, and any combination thereof.

51. A conjugate vaccine prepared according to the method of any one of claims 1, 4- 6, 10-12, 17, or 20-50.

52. A method for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method substantially applicable to saccharide antigens across a plurality of serotypes and comprising:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 7 to 11 ;

(I)

(I):

wherein m is zero or 1 and n is an integer in the range of 2 to 12, thereby transferring the DBCO moiety to the cyanate-substituted saccharide,

wherein the effective cyanylating amount of CDAP is selected to correspond to a particular serotype.

53. The method of claim 52, wherein the aqueous buffer has a pH in the range of 8.75 to 9.5.

54. The method of claim 52 or claim 53, wherein step (c) is carried out 5 minutes after step (b).

55. The method of any one of claims 52 through 54, wherein in step (c), the cyanate- substituted saccharide is contacted with 1 equivalent of the DBCO derivative.

56. A method for functionalizing a saccharide antigen with a reactive moiety capable of participating in a click chemistry reaction with a second reactant comprising a bio-orthogonal reactive moiety, the method being substantially applicable to saccharide antigens across a plurality of serotypes and comprising:

(a) providing the saccharide antigen as a solution in an aqueous buffer having a pH in the range of 5.1 to 5.9;

(c) purifying the aldehyde-bearing saccharide;

57. The method of claim 56, wherein the time period is 24 hours.

58. The method of any one of claims 52-57, further including, prior to (a), subjecting the saccharide to mechanical size reduction.