Improvements in immunogenic conjugates

Abstract

The present application discloses various improvements regarding immunogenic conjugates comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Patent Application Serial No. 62 / 693,981, filed July 4, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Merging of electronic text files submitted with this document

[0004] The contents of the text file submitted electronically with this article are incorporated herein by reference in their entirety: a computer-readable copy of the sequence list (filename: STRO_005_01WO_SeqList_ST25.txt, date of record: July 1, 2019, file size: approximately 23 kilobytes). Background Technology

[0005] An immune response to a “weak” glycoantigen can be enhanced by conjugation to a known “strong” carrier peptide antigen, such as diphtheria toxoid, tetanus toxoid, Haemophilus influenzae (H. influenzae) protein D, or CRM197. WO 2018 / 126229 (SutroVax, Inc., Foster City, California) discloses methods, compositions, and techniques for generating conjugated vaccine antigens using carrier peptides containing non-natural amino acids (nnAAs). Orthogonal linking chemistry of nnAAs allows the antigen to conjugate with the carrier peptide to produce an immunogenic conjugate that can be used for immunization.

[0006] The object of this invention is to provide variations and improvements of such methods, compositions, and techniques. The variations and improvements described below can be applied to or combined with any of the methods, compositions, or techniques disclosed in WO 2018 / 126229 or U.S. Provisional Patent Application Serial Nos. 62 / 693,978 and 62 / 693,981 (both filed July 4, 2018). The foregoing patent applications are incorporated herein by reference in their entirety. Summary of the Invention

[0007] In one embodiment, a sterile container (e.g., a vial) is provided containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. The container may contain a unit dose of the pharmaceutical composition. A sterile glass container is preferred.

[0008] In another embodiment, a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.) is provided, the delivery device containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide. The delivery device may contain a unit dose of the pharmaceutical composition. The delivery device may be used to administer the pharmaceutical composition to a mammalian subject.

[0009] In another embodiment, an airtight container is provided containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. Suitable containers for sealing include, for example, vials. When airtight, the contents are preferably sterile.

[0010] In another embodiment, a syringe is provided containing 0.25-0.75 mL (e.g., 0.3-0.75 mL, preferably 0.5 mL) of a pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0011] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) the aluminum salt adjuvant is aluminum hydroxide or aluminum phosphate adjuvant; and (iii) the volume of the pharmaceutical composition is 0.25-0.75 mL (e.g., 0.3-0.75 mL, preferably 0.5 mL).

[0012] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of aluminum ions in the composition is <300 μg / mL (e.g., between 100 and 300 μg / mL). Ideally, the concentration of aluminum ions is ≤1.7 mg / mL. The conjugates within the composition can be adsorbed onto the aluminum phosphate adjuvant.

[0013] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) the carrier polypeptide does not include SEQ ID NO:3; and (iii) the concentration of aluminum ions in the composition is <2.5 mg / mL. Ideally, the concentration of aluminum ions is ≤1.7 mg / mL. The conjugates within the composition can be adsorbed onto the aluminum phosphate adjuvant.

[0014] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the volume of the pharmaceutical composition is 0.25-1.25 mL (e.g., 0.3-0.7 mL, preferably 0.5 mL). This composition may contain an aluminum phosphate adjuvant, and the conjugates within the composition may be adsorbed onto the aluminum phosphate adjuvant.

[0015] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and a preservative, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0016] In another embodiment, a preservative-free pharmaceutical composition is provided, the preservative-free pharmaceutical composition comprising two or more different immunogenic conjugates, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0017] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide; and (ii) the composition has a permeability of 200-400 mOsm / kg.

[0018] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and at least one excipient, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80. The pharmaceutical composition may also contain an aluminum salt adjuvant. This composition may contain both sodium chloride and polysorbate 80 as excipients.

[0019] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total amount of the carrier polypeptide in the n immunogenic conjugates is less than or equal to 3 n μg per dose of the pharmaceutical composition.

[0020] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) in the pharmaceutical composition, the total concentration of the carrier polypeptide in the n immunogenic conjugates is less than or equal to 6 n μg / mL.

[0021] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total amount of the glycoantigen in the n immunogenic conjugates is less than or equal to 3 n μg per dose of the pharmaceutical composition.

[0022] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) in the pharmaceutical composition, the total concentration of the glycoantigen in the n immunogenic conjugates is less than or equal to 6 n μg / mL.

[0023] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the average amount of the carrier polypeptide in each conjugate is 1-4 μg per dose of the pharmaceutical composition.

[0024] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) in the pharmaceutical composition, the average concentration of the carrier polypeptide of each conjugate is 2-8 μg / mL.

[0025] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the average amount of the glycoantigen in each conjugate is 1-4 μg per dose of the pharmaceutical composition;

[0026] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) in the pharmaceutical composition, the average concentration of the glycoantigen in each conjugate is 2-8 μg / mL.

[0027] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the composition does not contain one or more unconjugated forms of the carrier polypeptide or (iv) the composition contains one or more unconjugated forms of the carrier polypeptide, wherein the mass of the one or more unconjugated forms of the carrier polypeptide in the composition is less than 10% of the mass of the carrier polypeptide in the n immunogenic conjugates.

[0028] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic conjugates, wherein: (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the composition does not contain an unconjugated form of the glycoantigen or (iv) the composition contains at least one of the unconjugated forms of the glycoantigen, wherein the total mass of the unconjugated forms of the glycoantigen in the composition is less than 40% (e.g., ≤30%, ≤20%, or ≤10%) of the total mass of the glycoantigen in the n immunogenic conjugates.

[0029] In another embodiment, a pharmaceutical composition is provided comprising 14 or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the total amount of the carrier polypeptide per dose is <40 μg.

[0030] In another embodiment, a pharmaceutical composition is provided comprising 14 or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of the carrier polypeptide per dose is ≤80 μg / mL.

[0031] In another embodiment, a method for preparing multiple unit doses of a pharmaceutical composition is provided, wherein (i) the pharmaceutical composition comprises an immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide, and (ii) the method includes the steps of preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into multiple individual containers. This method is ideally performed aseptically. The individual containers can be sealed after the unit doses have been packaged into them. The individual containers are ideally syringes.

[0032] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the composition is lyophilized.

[0033] In another embodiment, a method for preparing a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, and (ii) the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and the method comprises one of the following steps: (A) adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant, and then mixing the adsorbed conjugates together; (B) sequentially adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant; or (C) preparing a mixture of two or more of the immunogenic conjugates (e.g., all of the immunogenic conjugates) and combining this mixture with the aluminum salt adjuvant. The adjuvant may be an aluminum phosphate adjuvant.

[0034] In another embodiment, a modified CRM197 carrier polypeptide is provided, the modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity with SEQ ID NO:1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue. Thus, for example, Arg-192 and / or Arg-193 of SEQ ID NO:1 may be omitted or may be substituted with different amino acids. One or more of the nnAA residues may be introduced by substitution of amino acid residues in SEQ ID NO:1 and / or by insertion. The modified CRM197 carrier polypeptide can be used to prepare (e.g., glycoantigens) immunogenic conjugates via one or more of the nnAA residues in the carrier polypeptide.

[0035] In another embodiment, a modified CRM197 carrier polypeptide is provided, the modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity with SEQ ID NO:1 and (ii) contains an nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO:1): Asn-211; Asn-295; Asn-352; Asn-392; Asn-465; Asn-467; Asn-507; Asn-519; Asn-296; Asn-359; Asn-399; Asn-481; Asn-486; Asn-502; Asn-524; Glu-2 40;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. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., glycoantigens) via one or more of the nnAA residues in the carrier polypeptide.

[0036] In another embodiment, a modified CRM197 carrier polypeptide is provided, the modified CRM197 carrier polypeptide comprising: an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO:1; (ii) lacking an Arg-Arg dipeptide sequence; and (iii) containing an nnAA substitution at one or more of the following amino acid residues (as 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-481; Asn-486; Asn-502; Asn-524; Glu-24 0;Glu-248;Glu-249;Glu-256;Glu-259;Glu-292;Glu-362;Gln-252;Gln-287;Lys-212;L Lys-218; Lys-221; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-4 98; 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. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., glycoantigens) via one or more of the nnAA residues in the carrier polypeptide.

[0037] In another embodiment, an immunogenic conjugate is provided, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein (i) the carrier polypeptide comprises the amino acid sequence SEQ ID NO:4; and (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:4. A pharmaceutical composition is also provided, the pharmaceutical composition comprising two or more different immunogenic conjugates, each of the two or more different immunogenic conjugates comprising a carrier polypeptide and a glycoantigen, wherein (i) the carrier polypeptide in each conjugate comprises the amino acid sequence SEQ ID NO:4; and (ii) the glycoantigen in each conjugate is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:4.

[0038] In another embodiment, a syringe is provided containing a pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a pneumococcal glycoantigen, wherein the syringe is a non-silicone syringe. The pharmaceutical composition in the non-silicone syringe ideally has 13 or more different pneumococcal conjugates, and the carrier polypeptide optionally comprises nnAA, but may instead be, for example, CRM197. Further details of the non-silicone syringe are given below. Attached Figure Description

[0039] Figure 1 The present invention provides each of the 32 indicated serotypes in the 32-valent vaccine as described in the examples relative to the polysaccharide / alum formulation and Prevnar-13. TM The geometric mean titer. Detailed Implementation

[0040] International Patent Publication No. WO2018 / 126229 discloses various details of methods, compositions and techniques for generating conjugated antigens, the entire contents of which are incorporated herein by reference.

[0041] Immunogenic conjugates

[0042] This invention generally relates to immunogenic conjugates. These conjugates comprise a carrier polypeptide covalently linked to an antigen. This linkage can convert T-cell-independent immunogens (such as sugars) into T-cell-dependent immunogens, thereby enhancing the induced immune response (especially in children). The conjugates used herein contain covalent bonds formed between the antigen and non-natural amino acid ('nnAA') residues within the carrier polypeptide. These nnAA residues can provide functional groups that promote reactivity with the antigen of interest.

[0043] A single carrier polypeptide is typically linked to multiple antigen molecules. The antigen may have a single linker group (e.g., a reducing end of a sugar) per molecule for linking to the carrier polypeptide, or it may have multiple linker groups (e.g., multiple aldehyde or cyanate groups). In the case of antigen molecules having multiple linker groups, this typically results in the formation of high molecular weight crosslinks or lattice conjugates, involving the linking of the antigen between multiple carrier polypeptides. Crosslinked conjugates are preferred herein (particularly for pneumococci), and therefore antigens having multiple linker groups are also preferred.

[0044] A covalent bond is formed between the antigen and the nnAA residues in the carrier polypeptide. Preferably, the antigen does not conjugate with lysine residues in the carrier polypeptide; more preferably, the antigen does not conjugate with native amino acid residues in the carrier polypeptide.

[0045] Useful carrier peptides contain T-cell epitopes. Various such carrier peptides are known in the art and are known to be used in approved vaccines. These include diphtheria toxoid (a chemically treated toxin from Corynebacterium diphtheriae; 'Dt'), tetanus toxoid (a chemically treated tetanus toxin from Clostridium tetani; 'Tt'), protein D (“PD” or “HiD”) from Haemophilus influenzae, the outer membrane protein complex ('OMPC') of group B meningococci, and CRM197 mutant diphtheria toxin.

[0046] The preferred carrier polypeptide upon which the carrier of the present invention is based is CRM197. CRM197 is well known in the art (e.g., see...). (2011 Biologicals et al. 39:195-204) and has the following amino acid sequence (SEQ ID NO:1), wherein the underlined residue (Glu-52) is different from the natural diphtheria toxin, and thus the substitution of Gly→Glu leads to the loss of toxic enzyme activity in the protein:

[0047] GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK EFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGK RGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSET ADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGES GHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0048] This invention does not use natural CRM197. Instead, it uses a modified amino acid sequence containing at least one nnAA, rather than CRM197 including SEQ ID NO:1. These modified CRM197 carrier peptides are described in more detail below.

[0049] Besides CRM197, other detoxified mutant forms of diphtheria toxin can be used. For example, the nontoxic K51E / E148K double mutant has also been used as a carrier peptide 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 manner as in CRM197.

[0050] Another carrier polypeptide of interest is PD from Haemophilus influenzae, which naturally has the following amino acid sequence (SEQ ID NO:5):

[0051] CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

[0052] Instead of using native PD, a modified amino acid sequence containing at least one nnAA is used. For example, one or more Lys residues in SEQ ID NO:5 can be replaced with nnAA. SEQ ID NO:5 contains 36 Lys residues, so several residues can be replaced with nnAA and then used for conjugation. T-cell epitope prediction and recognition of PD have been reported by Hua et al. (2016) in *Clinical Vaccine Immunol* 23:155-61.

[0053] More generally, any peptide containing a T-cell epitope can be used as a carrier peptide. T-cell epitopes 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 to them (e.g., see Costantino et al. 2011, Expert Opinion on Drug Discovery 6:1045-66). Micoli et al. (2018) in *Molecules* 23, 1451 reviewed various carrier peptides and their selection criteria. Tontini et al. (2016) in *Vaccines* 34:4235-42 discussed preclinical studies of 28 carrier peptides, including tests of their ability to induce antibodies against glycoantigens. Multiepitope vector peptides containing multiple broadly reactive (i.e., immunogenic in most MHC class II molecules) human CD4+ T cell epitopes derived from various pathogen-derived antigens have been designed, such as N19, and other peptides disclosed in Falugi et al. (2001), *Eur J Immunol* 31:3816-24, Baraldo et al. (2004), *Infect Immun* 72:4884-7, and U.S. Patents 6,855,321 and 7,867,498. The ability to design these multiepitope vectors demonstrates the ability of those skilled in the art to identify suitable T cell epitopes from diverse sources and to use them to design effective vector peptides. See also patent application US2016-0101187. T cell epitopes present within known vectors (e.g., Tt, PD, CRM197) can be used. Various detoxified bacterial toxins have been successfully used as carriers, such as Tt, Dt, Pseudomonas aeruginosa exotoxin, and Clostridium difficile A and B toxins. Many different carrier peptides have been used for pneumococcal sugars, such as Prevnar. TM CRM197 and Synflorix TM The present invention utilizes PD, Tt, and Dt, as well as various epitopes described in Velasco et al. (1995), *Infectious Diseases and Immunity*, 63:961-8. Any of these many carrier polypeptides modified to contain at least one nnAA can be used to enhance the immunogenicity of the antigen of interest.

[0054] The nnAA-containing carrier polypeptides used in conjunction with this invention can generally be prepared using the techniques disclosed in Section 6 (“Carrier Protein Production Methods”) of WO2018 / 126229. Preferred carriers contain nnAA in addition to at least one T-cell epitope of the carrier. If the T-cell epitope region of the carrier is unknown, the epitope can be identified using standard techniques, such as Reece et al. (1993) *International Journal of Immunology* 151:6175-84, Beissbarth et al. (2005) *Bioinformatics* 21 Supplement 1:129-37, Maciel Jr et al. (2008) *Virol* 378:105-17, Fridman et al. (2012) *Oncoimmunol* 1:1258-70, etc. (including empirical and / or predictive methods). It can also be confirmed that any specific modification to the sequence of the carrier polypeptide does not eliminate the desired T-cell response to conjugated antigens (such as sugars in this text). A preferred group of vectors does not contain any modifications within the T-cell epitopes, including the insertion or substitution of nnAAs. Particularly preferred vectors contain at least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs. Particularly preferred vectors may also have up to 10, 9, 8, 7, or 6 nnAAs. A particularly preferred range for nnAAs in the carrier polypeptide is 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.

[0055] The immunogenic conjugates used in this article may contain a variety of antigens. Antigens are typically sugars. The term "sugar" includes polysaccharides having 50 or more repeating units and oligosaccharides having fewer than 50 repeating units. Polysaccharides typically have about 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 repeating units up to about 2,000 (sometimes more) repeating units, and optionally about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1,000 repeating units up to about 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800 or 1,900 repeating units. Oligosaccharides typically have about 6, 7, 8, 9 or 10 repeating units to about 15, 20, 25, 30 or 35 to about 40 or 45 repeating units.

[0056] Sugars that can be incorporated into immunogenic conjugates include sugars present in bacteria. These sugars can be non-capsular sugars (such as extracellular polysaccharides, for example, Staphylococcus aureus extracellular polysaccharides), but are preferably bacterial capsular sugars.

[0057] Bacterial capsular sugars are high-molecular-weight sugars present in the capsules of Gram-positive or Gram-negative bacteria, and can be used as vaccine antigens. These capsular sugars are typically prepared from whole-cell lysates or culture supernatants of the corresponding bacteria by methods involving percolation, protein removal, ethanol precipitation, nucleic acid removal, and freeze-drying. Bacterial sugars used in conjunction with this invention can be whole sugars as present in bacteria or fragments obtained from whole sugars, for example, by hydrolysis of sugars purified from bacteria.

[0058] The carbohydrate antigens of particular interest include, but are not limited to:

[0059] - Capsular sugars of Streptococcus pneumoniae: Further details are given below regarding pneumococcal capsular sugars that can be used as antigens for carrying out the present invention.

[0060] - Sugars of Streptococcus pyogenes The antigen may be a sugar derived from Streptococcus pyogenes. In one embodiment, the antigen is a capsular sugar of Streptococcus pyogenes, the capsular sugar being composed of the high molecular weight polymer hyaluronic acid, wherein the repeating unit has the following structure:

[0061] [→4)-β-D-GlcUAp-(1→3)-β-D-GlcpNAc-(→]

[0062] The structure appears to be invariant across Streptococcus pyogenes serotypes. In another embodiment, the antigen is a non-capsulated sugar derived from Streptococcus pyogenes, such as a group A stroma cell wall sugar comprising a backbone of poly-L-pyranoside units linked by alternating α-L-(1→3) and α-L-(1→2) bonds, with an N-acetyl-β-D-glucosamine residue linked to the bond at position 3 of the rhamnose backbone.

[0063] - Capsular sugars of Streptococcus agalactiae The antigen can be a capsular sugar derived from agalactiae (group B streptococci or GBS). At least 10 GBS serotypes have different capsular sugar repeat units (Ia, Ib, II–IX), but only a few serotypes are commonly the cause of the disease. These serotypes include serotypes Ia, Ib, II, III, and V, and conjugates derived from the capsular sugars of these serotypes can be prepared.

[0064] - Capsule of Haemophilus influenzaeThe antigen can be a capsular sugar derived from Haemophilus influenzae. At least six Haemophilus influenzae serotypes have different capsular sugar chemical structures (af type). However, only types a and b are considered "highly virulent" strains, and the preferred type of Haemophilus influenzae capsular sugar used in conjunction with this invention is type b (Hib).

[0065] - Capsule of Neisseria meningitidis The antigen can be a capsular sugar derived from Neisseria meningitidis. At least 13 Neisseria meningitidis serogroups have different capsular sugar 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, and Y) are considered life-threatening. The sugar antigen is usefully derived from any one of serogroups A, C, W135, X, or Y.

[0066] - Capsular sugars of Porphyromonas gingivalis The antigen can be a capsular sugar derived from one of the six serotypes of Porphyromonas gingivalis, namely K1, K2, K3, K4, K5, and K6.

[0067] - Capsular sugar of Salmonella typhi The antigen can be Vi sugar. Vi is the capsular sugar of Salmonella Typhi (the typhoid sera variant of Salmonella enterica). Vi sugar is a linear homopolymer of aminohexuronic acid α1,4-N-acetylgalactose-aminouronic acid, which is 60-90% acetylated at the C-3 position.

[0068] - Sugars of Staphylococcus aureus The antigen can be a sugar derived from Staphylococcus aureus. The sugar can be an extracellular polysaccharide of Staphylococcus aureus or a capsular sugar of Staphylococcus aureus, wherein the extracellular polysaccharide is poly-N-acetylglucosamine (PNAG), and the capsular sugar can be, for example, serotype 5, serotype 8, or serotype 336.

[0069] - surface sugars of Clostridium difficile The antigen can be a surface glycan from Clostridium difficile, such as PS-I or PS-II.

[0070] - beta-glucan Antigens can be dextrans containing β-1,3-bonds and / or β-1,6-bonds. These conjugated dextrans can be used to generate antifungal immune responses, such as against Candida albicans.

[0071] Further details about these glycoantigens can be found in WO2018 / 126229.

[0072] Antigens typically do not inherently contain suitable or ideal functional groups for conjugation. Therefore, antigens may require functionalization before conjugation with nnAA. Further details of such functionalization are given below.

[0073] pneumococcal capsula

[0074] The preferred antigen used in conjunction with this invention is a capsular sugar derived from *Streptococcus pneumoniae*. *Streptococcus pneumoniae* is an encapsulated Gram-positive bacterium that can cause pneumonia, bacteremia, and meningitis. At least 90 documented different *Streptococcus pneumoniae* serotypes (see, for example, Kalin, M. *Thorax* 1998; 53:159-162) carry capsular sugars with serotype-specific repeating unit structures. As those skilled in the art will understand, it has been proposed that *Streptococcus pneumoniae* serotype 20 is actually composed of two closely related serotypes whose capsular polysaccharides are largely cross-protected (Calix et al. 2012 *Journal of Biol Chem* 287:27885-94). Therefore, as those skilled in the art will further understand, serotype 20 refers to sugars previously classified as serotype 20 in the art and thus structurally possible as 20A or 20B (from strains previously classified as serotype 20 in the art, but genotypeably possible as 20A or 20B), as disclosed by Calix et al. For example, it is now considered that sugars used to produce Pneumovax TM The strain of serotype 20 polysaccharide in Merck is serotype 20A. In some cases, 20A may be preferred. In other cases, 20B may be preferred. The prevalence in the target population may be the basis for selection among these serotypes. Nevertheless, since strains classified as 20, 20A, and 20B are serologically similar, these strains largely offer cross-protection in vaccines, and selection between strains may not be important.

[0075] The antigen used in conjunction with this invention may be a capsular sugar derived from any of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19 A, 19B, 19C, 20, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A or 48 (Henrichsen, *Journal of Clinical Microbiology*, 1995; 33:2759-2762). However, only subsets of these serotypes are often the cause of clinically significant bacterial infections, therefore the antigen can be a capsular sugar from any of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. Serotypes 6C, 7C, 15A, 15C, 16F, 20A, 20B, 23A, 23B, 24B, 31, 34, 35B, 35F, 37, and 38 have also raised clinical concerns, therefore the antigen can be a capsular sugar from one of these Streptococcus pneumoniae serotypes.

[0076] When the present invention uses conjugates derived from different pneumococcal serotypes, it preferably contains sugars derived from at least 14 different Streptococcus pneumoniae serotypes (e.g., from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more). When the composition contains 14 or more serotypes, these serotypes preferably include 13 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In addition to these 13 Streptococcus pneumoniae serotypes, the composition preferably contains one or more of serotypes: 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20 (or alternatively, 20A or 20B), 22F, and / or 33F. Alternatively, in addition to the 13 serotypes mentioned above, the composition preferably comprises one or more Streptococcus pneumoniae serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38. Useful combinations of 15 or more (e.g., 16 or more) serotypes include Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6... Each of the following Streptococcus pneumoniae serotypes is included: A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and may also include serotype 8. Useful combinations of 20 or more (e.g., 21 or more) Streptococcus pneumoniae serotypes include each of the following Streptococcus pneumoniae serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. Useful combinations of 24 or more serotypes include each of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0077] The structures of capsular sugar repeat units of common pneumococcal serotypes are described by Jones et al. (Jones C et al., *An Acad Bras Ciênc.*, June 2005; 77(2):293-324):

[0078] Type 1

[0079] [→3)-D-AAT-α-Galp-(1→4)-α-D-GalpA(2 / 3OAc)-(1→3)-α-D-GalpA-(1→]

[0080] Type 2

[0081] [→4)-β-D-Glcp-(1→3)-[α-D-GlcpA-(1→6)-α-D-Glcp-(1→2)]-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)β-L-Rhap-(1→]

[0082] Background3

[0083] [→3)-β-D-GlcA-(1→4)-β-D-Glcp-(1→]

[0084] Settings4

[0085] [→3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp2,3(S)Py-(1→]

[0086] Section5

[0087] [→4)-β-D-Glcp-(1→4)-[α-L-PnepNAc-(1→2)-β-D-GlcpA-(1→3)]-α-L-FucpNAc-(1→3)-β-D-Sugp-(1→]

[0088] Section 6B

[0089] [→2)-α-D-Galp-(1→3)-α-D-Glcp-(1→3)-α-L-Rhap-(1→4)-D-Rib-ol-(5→P→]

[0090] Background 9N

[0091] [→4)-α-D-GlcpA-(1→3)-α-D-Glcp-(1→3)-β-D-ManpNAc-(1→4)-β-D-Glcp-(1→4)-α-D-GlcpNAc-(1→]

[0092] Rating 9V

[0093] [→4)-α-D-GlcpA(2 / 3OAc)-(1→3)-α-D-Galp-(1→3)-β-D-ManpNAc(4 / 6OAc)-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→]

[0094] Range 12F

[0095] [→4)-[α-D-Galp-(1→3)]α-L-FucpNAc-(1→3)-β-D-GlcNAc-(1→4)-[α-D-Glc-(1→2)-α-D-Glc-(1→3)]-β-D-ManNAcA-(→]

[0096] Type 14

[0097] [→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→]

[0098] Type 18C

[0099] [→4)-β-D-Glcp-(1→4)-[α-D-Glcp(6OAc)(1→2)][Gro-(1→P→3)]-β-D-Galp-(1→4)-α-D-Glcp-(1→3)-β-L-Rhap-(1→]

[0100] Type 19F

[0101] [→4)-β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap-(1→P→]

[0102] Type 23F

[0103] [→4)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→]

[0104] A more extensive discussion of sugars can be found in Geno et al. (2015) in *Clin. Microbiol. Rev.* 28:871-99, where Table 1 shows the structures for 97 known serotypes. This table also discloses the proportion of acetylated sugar residues when acetylation is incomplete.

[0105] Capsular sugars can be O-acetylated. In some embodiments, capsular sugars from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F include sugars with O-acetylation levels between 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 75% and 100%, 80% and 100%, 90% and 100%, 50% and 90%, 60% and 90%, 70% and 90%, or 80% and 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 sugar can be determined by proton NMR (see, for example, Lemercinier and Jones (1996), Carbohydrate Research, 296:83-96; Jones et al. (2002), Journal of Pharmaceutical and Biomedical Analysis, 30:1233-1247). The sugar used to prepare the conjugate will typically retain at least 50% (e.g., 75% or even 100%) of the O-acetylation level observed in the starting capsular sugar purified from bacteria.

[0106] The pneumococcal capsular sugar can be obtained directly from bacteria using isolation methods known to those skilled in the art (see, for example, methods disclosed in: U.S. Patent Application Publications Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498, and WO 2008 / 118752). Alternatively, the pneumococcal capsular sugar can be obtained from commercial sources (e.g., ATCC).

[0107] The molecular weight of the pneumococcal capsular glycoantigen used in conjunction with the present invention can be usefully between 10 kDa and 4,000 kDa, for example between 50 kDa and 3,000 kDa or between 100 kDa and 2,000 kDa. For example, 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; 200 kDa... Between Da 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 regarding molecular weights are available previously cited in U.S. Serial No. 62 / 693,978, which is incorporated herein by reference.

[0108] Capsular sugars are optionally chemically modified relative to naturally occurring capsular sugars. For example, the sugars are optionally deacetylated (partially or completely), deacetylated (partially or completely), N-propionic acid-modified (partially or completely), etc. Deacetylation optionally occurs before, during, or after activation, derivatization, or conjugation, but usually occurs before conjugation.

[0109] Some embodiments of the present invention involve the use of two or more different conjugates. Regarding pneumococcal capsular sugar conjugates, this means (when a single type of carrier polypeptide is used for each conjugate) that each 'different' conjugate has sugars derived from different pneumococcal serotypes.

[0110] Multivalent conjugates

[0111] Preferred compositions of the present invention involve, for example, the use of two or more different conjugates within a single pharmaceutical composition. These examples are also referred to as multivalent. When any two conjugates are described as 'different' or provide different valences in a 'multivalent' composition, this refers to the difference between the combination of carrier polypeptide and antigen in the two conjugates. For example, when a single type of modified CRM197 (e.g., SEQ ID NO:4) is conjugated with a capsular sugar from a single serotype of pneumococcus, the reaction product will contain many different types of molecules (different molecular weights, different bond patterns within each molecule, etc.), but is considered herein as a single conjugate. Those skilled in the art are familiar with this heterogeneity at the molecular level and similarly define individual conjugates of vaccines by antigen-carrier combinations of specific conjugates, where other properties (such as molecular weight) are averages within the conjugate composition. Two 'different' conjugates have different carrier polypeptides (i.e., different amino acid sequences) and / or different antigens (i.e., different antigenic structures).

[0112] For example, capsular sugar antigens can be purified from two different serotypes of Streptococcus pneumoniae. These two different capsular sugars can be conjugated separately to a carrier polypeptide (which may be the same or different) to provide two different conjugates. Therefore, regarding bacterial capsular sugar conjugates, the difference between two 'different' conjugates will generally be that one contains a capsular sugar from a first serotype or serogroup of the bacterial species, while the other contains a capsular sugar from a second serotype or serogroup of the said bacterial species, for example, capsular sugars from different Streptococcus pneumoniae serotypes or from different Neisseria meningitidis serogroups. If the two conjugates contain capsular sugars different from antigens from multiple bacterial species, for example, a Hib sugar conjugate and a meningococcal sugar conjugate, then the two conjugates are also 'different'.

[0113] The preferred multivalent compositions of the present invention comprise n different immunogenic glycoconjugates, wherein the glycoantigen of each of the n immunogenic conjugates is different from the glycoantigen of the other n-1 immunogenic conjugates. For example, if the composition contains antigens from a single bacterial species, there may be capsular sugars from n different serotypes or n different serogroups of said species.

[0114] This nomenclature associated with 'distinct' conjugates is used in the field of conjugate vaccines. For example, Glesby et al. (2015) in the *Journal of Infectious Diseases* 212:18-27 mention Prevnar. TMThe PCV13 vaccine contains '13 different conjugates' because it contains glycoantigens from 13 different pneumococcal serotypes, each conjugated to CRM197. Similarly, EP-A-2932979 refers to 'an immunogenic composition comprising 13 different polysaccharide-protein conjugates'.

[0115] Therefore, PCV7 Prevnar TM The vaccine contains 7 different conjugates, PCV13 Prevnar TM The vaccine has 13 different conjugates, Menveo TM The vaccine has four different conjugates, Menactra TM The vaccine has four different conjugates, Nimenrix TM The vaccine has four different conjugates, Menitorix TM The vaccine contains two different conjugates, Menhibrix TM The vaccine has three different conjugates, Synflorix TM The vaccine has 10 different conjugates, etc.

[0116] The multivalent compositions of pneumococcal conjugates preferably contain more than 13 different conjugates, such as 14, 15, 20, 21, 24, 25 or more. Suitable selection of serotypes for these >13-valent compositions has been discussed above.

[0117] For high-valent vaccines (e.g., those with more than 13 different conjugates), it is sometimes preferable to use more than one carrier polypeptide to reduce the possibility of carrier inhibition (e.g., see WO98 / 51339 and WO2011 / 110241). For example, in a multivalent vaccine comprising n different conjugates, a first carrier polypeptide is conjugated to ny different antigens (e.g., capsular sugars from different bacterial serotypes or serogroups), and a second polypeptide carrier is conjugated to the remaining y antigens. Similarly, three, four, or more carriers can be used, with n antigens distributed among these carriers. When more than one carrier is used, at least the first carrier is a carrier polypeptide containing nnAA according to the invention. In a preferred embodiment, at least the first and second carriers are carrier polypeptides containing nnAA according to the invention.

[0118] Non-natural amino acids

[0119] As mentioned above, the conjugates used in this paper contain covalent bonds between functional groups within nnAA residues of the antigen and the carrier polypeptide. The side chains of the nnAA residues can provide reactive functional groups that can be used to conjugate the antigen to discrete sites in the carrier polypeptide.

[0120] Generally, nnAA can be any amino acid that can be incorporated into a polypeptide during translation, but it is not one of the 20 common amino acids. nnAA can be conveniently incorporated into a polypeptide by transforming tRNA molecules so that their codons incorporate nnAA instead of the natural homologous amino acid. One technique for achieving this involves using a "suppression codon," a nucleotide triplet, which is introduced into the desired position of the coding sequence and is recognized by a specific tRNA that can recognize natural stop codons (e.g., amber, ochre, or opal stop codons) but allows translation to continue, in which nnAA is incorporated (thus suppressing the natural stop codon).

[0121] The nnAA residue can be any of the nnAA residues described herein or any other residue identified as compatible with cell-based or cell-free protein synthesis (see, for example, Schultz et al., Annals of Biochemistry 2010; 79:413-44, specifically pp. 418-420; and Chin et al., Annals of Biochemistry 2014; 83:5.1-5.30, which are incorporated herein by reference). Ideally, nnAA is not naturally produced in cells through modification with one of the 20 common amino acids (e.g., pyrrolidone, selenocysteine, phosphotyrosine, formyl-methionine, etc.).

[0122] The particularly preferred nnAA used herein is an nnAA with a side chain that can be incorporated during translation (in a cellular or cell-free system), the side chain providing a functional group not present in the side chains of any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into peptides are known, for example, see Young and Schultz (2010) *Journal of Biochemistry* 285:11039-44, Maza et al. (2015) *Bioconjugate Chemistry* 26:1884-9, and Zimmerman et al. (2014) *Bioconjugate Chemistry* 25:351-61. WO2018 / 126229 discloses in detail how nnAA residues can be incorporated into carrier peptides, for example, using cell-free expression mixtures, orthogonal tRNA / aminoacyl-tRNA synthetase pairs specific for nnAA, repressive codons, etc. See also U.S. Patent Application US-2017 / 0267637.

[0123] nnAA may contain chemical groups suitable for a "click" chemistry reaction with a corresponding group on the antigen of interest. Chemical groups suitable for "click" chemistry include, but are not limited to, azide (-N3), acetylene (-C≡C-), alkene (-C=C-), and 1,2,4,5-tetraazine. And phosphine (e.g., -P(Ph)2) groups.

[0124] nnAA can be any of the following: 2-amino-3-(4-azidophenyl)propionic acid (p-azido-L-phenylalanine or pAF), 2-amino-3-(4-(azidomethyl)phenyl)propionic acid (p-azidomethyl-L-phenylalanine or pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propionic acid, or 2-amino-5-azidopentanoic acid.

[0125] The most preferred nnAA used in this paper is pAMF:

[0126]

[0127] pAMF provides very favorable reaction kinetics for the generation of conjugates (e.g., much faster than pAF when reacting with alkyne-containing carbohydrate antigens using the SPAAC method).

[0128] nnAA can be a 2,3-disubstituted propionic acid carrying: an amino substituent at the 2-position; and an azide-containing substituent, a 1,2,4,5-tetraazine-containing substituent, or an ethynyl-containing substituent at the 3-position. Preferably, the substituent at the 3-position is an azide-containing substituent, particularly an azide-containing substituent including a terminal azide group linked to the carbon atom at the 3-position by a linking group. For example, the linking group can include an arylene moiety that is optionally substituted and optionally contains heteroatoms. For example, the linking group can include a 5- or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 non-hydrogen ring substituents.

[0129] nnAA can have the structure of formula XII:

[0130]

[0131] Wherein: Ar includes a 5- or 6-membered aromatic ring optionally containing at least one heteroatom; W 5 Selected from C1-C 10 Alkylene, -NH-, -O-, and -S-; Q1 is zero or 1; and W 6Ar is selected from azide, optionally substituted with a lower alkyl C-, 1,2,4,5-tetraazine, and ethynyl. In some embodiments, Ar does not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene (i.e., Ar is -C6H4-). In other embodiments, Ar contains a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described below, and Ar can be, for example, pyridine or pyridazine. In a particularly preferred embodiment, Q1 is 1, W 5 It is a lower alkylene group, and W 6 It is an azide group.

[0132] nnAA can be nnAA containing an azide group, such as nnAA in Formula I:

[0133]

[0134] Wherein: D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-; each of W1, W2, and W3 is independently a single bond or a lower alkylene group; each X1 is independently -NH-, -O-, or -S-; each Y1 is independently a single bond, -NH-, or –O-; each Y2 is independently a single bond, -NH-, -O-, or N-linked or C-linked pyrroleylene group; Ar is... Furthermore, one of Z1, Z2, and Z3 is -N-, and the other terms in Z1, Z2, and Z3 are independently -CH-.

[0135] In other embodiments, nnAA has Equation II:

[0136]

[0137] Where W4 is C1-C 10 Alkylene.

[0138] The preparation of azide-containing amino acids according to Formulas I and II can be found, for example, in Stafford et al., US2014-0066598A1, specifically in paragraphs

[0331] -

[0333] , which is incorporated herein by reference. The method involves replacing the hydroxyl group of the chloride on a derivative of the corresponding aryl amino acid with a thionyl chloride, followed by a nucleophilic substitution of the chloride with an azide. Suitable aryl side-chain-containing amino acids can also be commercially obtained.

[0139] nnAA can be nnAA containing 1,2,4,5-tetraazine. For example, Formula III:

[0140]

[0141] Where: Ar is V is a single bond, a lower alkylene group, or -W1-W2-; one of W1 and W2 is absent or a lower alkylene group, while the other is -NH-, -O-, or -S-; each of Z1, Z2, and Z3 is independently -CH- or -N-; and X1 is independently -NH-, -O-, or -S-; and R is a lower alkyl group.

[0142] The preparation of amino acids containing 1,2,4,5-tetraazine according to Formula III can be found, for example, in Yang et al., US2016 / 0251336, specifically in paragraphs

[0341] -

[0377] , which is incorporated herein by reference. The method involves coupling an amino / carboxyl protected derivative of (R)-2-amino-3-iodopropionic acid with brominated aminopyridine to introduce Ar, followed by reaction with a methylthio-1,2,4,5-tetraazine derivative to introduce the tetraazine moiety into the amino acid.

[0143] nnAA can be an alkyne-containing nnAA. In one embodiment, this is propargyl. Various propargyl-containing amino acids (including their synthesis) can be found in the following literature: 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(131):8720-1. 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 homopropargylglycine, ethynylphenylalanine, and N6-[(2-propynyloxy)carbonyl]-L-lysine.

[0144] The nnAA used in this article is typically an α-amino acid with a chiral center at the α-carbon, and is preferably an L-stereoisomer.

[0145] The polypeptide carrier used in conjunction with the present invention comprises at least one nnAA residue. Preferably, the carrier polypeptide should contain a plurality of nnAA residues, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues (or sometimes more). Carrier polypeptides having fewer than 10 nnAA residues are preferred. Thus, the polypeptide may contain 2-9 nnAA residues and preferably 4-6 nnAA residues.

[0146] When the carrier polypeptide contains multiple nnAA residues, it is preferable to contain only a single species of nnAA (e.g., the only nnAA in the carrier is pAMF). This allows the same conjugation chemistry to be used simultaneously at each nnAA. If it is desired to link 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 the carrier is preferred. Furthermore, in the case of using multiple different conjugates (e.g., different pneumococcal serotypes), it is sometimes preferable that each conjugate contains the same single species of nnAA. Additionally, in the case where the composition contains multiple different conjugates (e.g., different pneumococcal serotypes), it is sometimes preferable that each conjugate contains the same carrier polypeptide.

[0147] nnAA can be incorporated into the carrier polypeptide by substitution or by insertion (or by C-terminal or N-terminal extension). In one embodiment, one or more nnAA residues are incorporated by substitution. Conveniently, nnAA can replace lysine residues in the native polypeptide. For example, in CRM197, substitution can occur at one or more of the following positions in SEQ ID NO:1 or 2: K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522, and K526. Preferably, nnAA (e.g., pAMF) is substituted at each of K33, K212, K244, K264, K385, and K526 (and in one embodiment, not at the other positions).

[0148] However, the substitutions used for incorporating nnAA are not limited to the lysine position, and other amino acids such as Phe, Asp, Asn, Glu, Gln, Arg, Ser and / or Thr can also be substituted with nnAA.

[0149] The nnAAs within the carrier peptide are ideally surface-accessible residues. This can be assessed using the 3D structure of the peptide or by performing a complete substitution of native amino acids with nnAAs followed by a conjugation test to evaluate the utility of each site.

[0150] To maintain the functionality of the carrier peptide, it is preferable not to incorporate nnAA into the T-cell activation epitopes of the carrier peptide. The use of nnAA allows for selective placement of conjugation sites and thus avoids using the T-cell activation epitopes of the carrier peptide as sites for antigen conjugation. As mentioned above, these epitopes are readily identifiable. For example, studies of CRM197 by Raju et al., Bixler et al., Leonard et al., and Pillai et al. (e.g., *European Journal of Immunology*, December 1995; 25(12):3207-14, WO89 / 06974) have identified various T-cell epitopes, such as those within residues P271-D290, V321-G383, and Q411-I457. Therefore, it is preferable to avoid introducing nnAA into these regions of SEQ ID NO:1.

[0151] Adhesion

[0152] Conjugation involves forming a covalent bond between an nnAA residue and an antigen. This requires reactive functional groups in both the nnAA and the antigen. The nnAA of a carrier polypeptide is often chosen because it already possesses suitable functional groups (e.g., the azide group in pAMF), but antigens typically do not inherently contain suitable or ideal functional groups for conjugation. Therefore, the antigen may need to be functionalized before conjugation with the nnAA.

[0153] Detailed technical information on conjugation can be found in *Bioconjugate Techniques* (Greg T Hermanson, 3rd edition, 2013). WO2018 / 126229 discloses in detail how antigens are functionalized and then conjugated with nnAAs. As noted above, useful nnAAs contain functional groups (e.g., azide groups) suitable for "click" chemical reactions with functional groups on antigens. Therefore, functionalized antigens ideally contain groups suitable for such "click" reactions.

[0154] Generally, conjugation is therefore performed by a method comprising the following three steps: (a) activating the antigen; (b) optionally derivatizing the activated antigen (e.g., with a linker or nucleophile) to introduce reactive functional groups not normally present in the antigen; and (c) conjugating the antigen to a carrier polypeptide by means of the group introduced in step (a) or (if present) step (b). In some embodiments, step (a) includes a first step of removing blocking groups on the antigen to make certain functional groups (e.g., hydroxyl, amine, thiol) more readily activated. Sometimes, steps (a)-(c) can occur substantially simultaneously (e.g., in the case of adding a reactive moiety such as N-hydroxysuccinimide to the antigen), but in other embodiments, two or more steps in steps (a)-(c) are discrete, with purification optionally occurring between steps.

[0155] As noted above, cross-linked conjugates are preferred, and therefore, the introduction of multiple reactive functional groups into each antigen molecule is also preferred. For example, multiple aldehyde or cyanate groups can be introduced when activating a sugar molecule. These groups can then be derivatized, for example, to introduce reactive cyclooctyne that can then react with the azide group in nnAA.

[0156] Various chemically activated antigens can be used, including but not limited to: periodate oxidation (e.g., for oxidizing hydroxyl groups on adjacent carbon atoms to produce reactive aldehydes), such as those disclosed in WO2011 / 110531; cyanohydrination, for example, using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP); activation with a 1,1'-carbonyldiimidazole (CDI) hydroxyl group followed by nucleophilic addition; or exposure of the intrinsic aldehyde (e.g., the reducing end of a sugar).

[0157] Periodate oxidation and CDAP cyanation are two preferred activation techniques. Periodate oxidation has been shown to be particularly effective for activating pneumococcal serotypes 1, 2, 3, 7F, 8, 9N, and 11A. CDAP cyanation has been shown to be particularly effective for activating pneumococcal serotypes 3, 7F, and 10A.

[0158] Activated antigens can be directly conjugated to nnAA, but the activated group is usually derivatized to introduce a functional group that exhibits better reactivity with nnAA. For example, an alkynyl group can be introduced. Bifunctional reagents having both amino and alkynyl groups can react with the aldehyde group already introduced into the antigen (e.g., by reductive amination), thereby leaving a side alkyne that can react with nnAA. For example, bifunctional reagents containing both amino and DBCO functional groups can be used.

[0159] In one embodiment, nnAA reacts with an alkynyl group (e.g., propargyl) in the antigen. The alkynyl group in the antigen is ideally suited for reaction with the alkynyl group in nnAA using, for example, copper-catalyzed azido-alkynyl cycloaddition (CuAAC), ruthenium-catalyzed azido-alkynyl cycloaddition (RuAAC), or Huisgen azido-alkynyl 1,3-dipolar cycloaddition. The alkynyl group may have a molecular environment that increases its reactivity; for example, it may be intracyclic. For example, an alkylene group may be within a cyclooctyne ring (optionally containing heteroatoms) such as a diaryl-strained cyclooctyne ring (e.g., DBCO). This reaction can be a cycloaddition [3+2] known in the art as a strain-promoted azido-alkynyl cycloaddition reaction (SPAAC). For these reactions, DIFO- and DBCO-based reagents are readily available.

[0160] Useful alkyne rings in the SPAAC reaction include difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne. These can be obtained, for example, using any of the following reagents, utilizing side functional groups for attachment to the activated antigen (e.g., side amino groups for attachment to aldehydes or cyanates):

[0161] DBCO-PEGn-NH2

[0162] DBCO-PEGn-NH2

[0163]

[0164] DBCO carboxylic acid

[0165] DBCO-NH2 DIFO carboxylic acid

[0166] The value of 'n' in 'PEGn' represents the number of ethylene oxide repeating units. The value of n is in the range of 1-20, for example, in the range of 2-18, 3-16, or 4-14. Therefore, n can be any one of, for example, 4, 5, 11, 12, or 13.

[0167] Other click chemistry reactions that can be used to conjugate antigens with nnAA include, but are not limited to, tetrazine-olefin linkages and Staudinger linkages between phosphine and azides.

[0168] The molecular weight of the conjugates of the present invention can be at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa or higher. In some embodiments, the molecular weight of the conjugate is between about 750 kDa and about 5,000 kDa. In some embodiments, the molecular weight of the conjugate is between about 800 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 850 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 900 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 950 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 1,000 kDa and about 2,800 kDa. The molecular weight of the conjugate is calculated by size exclusion chromatography (SEC) combined with multi-angle laser scattering (MALS).

[0169] The conjugates of this invention comprise an antigen (e.g., a sugar) and a carrier polypeptide, and the weight ratio of these two components can be used as a parameter defining the conjugate. A higher antigen-carrier weight ratio in the sugar-carrier conjugate allows for the delivery of a greater amount of sugar antigen with a smaller amount of carrier polypeptide. For pneumococcal conjugate vaccines, this ratio is typically in the range of 0.3–3.0, but this can vary depending on 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). Commercial vaccine Prevnar-13 TM The ratio is 0.9. For compositions containing conjugates of multiple pneumococcal serotypes (e.g., more than 13 serotypes), the ratio of the whole composition is ideally higher than 1.0 (i.e., an excess of pneumococcal glycoantigen by weight) and preferably 1.5 or higher (e.g., in the range of 1.5-3.0 or preferably 1.5-2.0).

[0170] Modified CRM197 carrier peptide

[0171] As mentioned above, the carrier polypeptide of primary interest herein is a modified form of CRM197. Therefore, preferred carrier polypeptides for use with this invention comprise an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1 (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%). For example, in addition to the presence of up to 10 nnAAs, the carrier polypeptide may comprise the amino acid sequence SEQ ID NO:1, as discussed above.

[0172] SEQ ID NO:1 contains the Arg-Arg dipeptide sequence at positions 192-193. In some cases, this sequence is susceptible to proteolytic cleavage. If desired, this site can be modified to prevent cleavage and improve yield. Therefore, in some embodiments, the modified CRM197 carrier polypeptide used herein does not contain the Arg-Arg dipeptide sequence. For example, Arg-192 and / or Arg-193 of SEQ ID NO:1 may be omitted or substituted with different amino acids. Therefore, preferred carrier polypeptides comprise an amino acid sequence that: (i) has at least 80% (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%) sequence identity with SEQ ID NO:1; (ii) does not contain the Arg-Arg dipeptide sequence; and (iii) contains at least one (e.g., at least two, and preferably more, as discussed above) nnAA residues.

[0173] One such amino acid sequence is SEQ ID NO:2, which differs from SEQ ID NO:1 in that it has an Arg→Asn substitution at position 193:

[0174] GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVR NSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0175] Any embodiment described herein or in WO2018 / 126229 by reference to SEQ ID NO:1 may instead be effective using SEQ ID NO:2.

[0176] Therefore, a carrier polypeptide comprising the amino acid sequence SEQ ID NO:2 is provided, wherein SEQ ID NO:2 has been modified to contain 1-10 (e.g., 3-9, 2-8, 2-6, 3-6, or 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 contains 6 Lys→nnAA substitutions). The residue Asn-193 of SEQ ID NO:2 is preferably not substituted with nnAA. This carrier polypeptide can be used to prepare (e.g., glycoantigens) immunogenic conjugates via one or more nnAA residues in the carrier polypeptide.

[0177] In some embodiments, these carrier peptides comprise the amino acid sequence upstream and / or downstream of SEQ ID NO:1 or 2. Thus, for example, these carrier peptides may comprise a methionine residue upstream of the N-terminal 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 may be included here to initiate translation (e.g., in a cell-free peptide synthesis system) without requiring the entire native leader sequence. In some embodiments, the carrier peptide does not comprise (i) the amino acid upstream of the N-terminus of SEQ ID NO:1 or 2, except optionally methionine, and (ii) the amino acid downstream of the C-terminus of SEQ ID NO:1 or 2.

[0178] Preferably, at least one Lys residue in SEQ ID NO:1 or 2 is substituted with an nnAA residue. It is preferable to substitute more than one residue in SEQ ID NO:1 or 2 with nnAA, and ideally, residues of only one species in SEQ ID NO:1 are substituted with nnAA, for example, only the Lys residue is substituted. When more than one residue in SEQ ID NO:1 is substituted with nnAA, it is preferable to use the same nnAA at each position, for example, pAMF at each substitution position. As noted above, in some embodiments, residues other than Lys are substituted.

[0179] Vector polypeptides, including those with 2-9 nnAA residue substitutions (e.g., Lys→nnAA substitution, preferably Lys→pAMF) and ideally having 2-8, 2-6, 3-8, 3-6, 4-9, 4-8, or 4-6 nnAA substitutions, such as amino acid sequences of 4, 5, or 6 nnAA residues, are preferred. This allows for more extensive binding of the antigen to the vector compared to using a single nnAA, thereby increasing the antigen:carrier ratio while avoiding excessive destruction of the native sequence and structure that could lead to insolubility.

[0180] Structural studies of CRM197 revealed two general 3D regions within SEQ ID NO:1 or 2: a first region extending from the N-terminus to Asn-373; and a second region extending from Ser-374 to the C-terminus. The first region roughly corresponds to the 'C' and 'T' (catalytic and transmembrane) domains, and the second region corresponds to the 'R' (receptor binding) domain. Ideally, the carrier polypeptide contains at least one nnAA in the first region and at least one nnAA in the second region, for example, at least two nnAAs in each region or at least three nnAAs in each region. This allows the conjugated antigen to spatially separate upon attachment to the carrier. Carriers having three nnAAs in both the first and second regions are useful.

[0181] The first region contains 27 Lys residues, and the second region contains 12 Lys residues. Therefore, 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 nnAA, for example, within pAMF.

[0182] Preferred embodiments of the CRM197-based nnAA-containing vector have the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2, in which one or more of the following residues are replaced by nnAA (such as pAMF): K24, K33, K37, K39, K212, K214, K227, K264, K385, K522, and K526. One such sequence is SEQ ID NO:3, where each X represents an nnAA (preferably the same nnAA, such as pAMF):

[0183] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO:3)

[0184] Another such sequence is SEQ ID NO:4, where each X represents nnAA (preferably the same nnAA, such as pAMF):

[0185] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ XGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO:4)

[0186] SEQ ID NO:3 and 4 can be adequately expressed in cell-free protein synthesis systems while maintaining good solubility and providing a good immunogenic response upon conjugation with pneumococcal capsular sugars. SEQ ID NO:4 lacks the native Arg-Arg dipeptide.

[0187] The polypeptide consisting of SEQ ID NO:4 in which each X is pAMF is another preferred carrier polypeptide for use with the present invention.

[0188] WO2018 / 126229 describes several amino acid residues suitable for nnAA substitution (e.g., Lys-24, Lys-33, Lys-37, Lys-39, Lys-212, Lys-214, Lys-227, Lys-244, Lys-264, Lys-385, Lys-522, Lys-526, Phe-12, Phe-53, Phe-123, Phe-127, Phe-140, Phe-167, Phe-250, Phe-389, Phe-530 or Phe-531, as numbered according to SEQ ID NO:1 herein). Other residues that can be substituted are: Asp-211; 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;L ys-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-4 93;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;T hr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and / or Thr-517.

[0189] Also provided are polypeptides comprising an amino acid sequence, said amino acid sequence: (i) having at least 80% (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%) sequence identity with SEQ ID NO:1; (ii) being free of an Arg-Arg dipeptide sequence; and (iii) containing at least one nnAA residue; and wherein the polypeptide has an N-terminal methionine and / or is in monomeric form.

[0190] These CRM197-derived carrier peptides can be used for conjugation in the same manner as CRM197 has been used in existing technologies (e.g., see [link]). (e.g., 2011, ibid., WO2015 / 117093, etc.), but the improvement lies in allowing site-specific conjugation via one or more nnAA residues. These carrier peptides will generally be used in monomeric form rather than associated with other CRM197 or CRM197-derived subunits to form peptide multimers. Similarly, these carrier peptides will generally contain at least one disulfide bridge, for example, between Cys-186 and Cys-201 (numbered according to SEQ ID NO:1) and optionally between Cys-461 and Cys-471.

[0191] Immunogenic conjugates are also provided, comprising any of these various carrier polypeptides conjugated to a glycoantigen via at least one of its nnAA residues. The carrier polypeptides are particularly useful for conjugating pneumococcal capsular sugars via one or more nnAA residues in the carrier polypeptide. Immunogenic conjugates prepared in this manner can be combined to form multivalent compositions as discussed elsewhere herein.

[0192] Therefore, immunogenic conjugates comprising a carrier polypeptide and a glycoantigen are provided, wherein (i) the carrier polypeptide has an amino acid sequence SEQ ID NO:4, for example, wherein each X is pAMF; and (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:4. Multivalent pharmaceutical compositions comprising two or more of these immunogenic conjugates are also provided.

[0193] Therefore, a pharmaceutical composition comprising a variety of different conjugates (e.g., different pneumococcal serotypes) is provided, wherein each conjugate comprises a carrier polypeptide having an amino acid sequence SEQ ID NO:4.

[0194] Immunogenic conjugates comprising a carrier polypeptide and a glycoantigen are also provided, wherein (i) the carrier polypeptide has the amino acid sequence SEQ ID NO:4; (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:4; and (iii) the glycoantigen is a capsular sugar from any of the following pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. These individual conjugates can be combined to prepare the multivalent pharmaceutical compositions of the present invention.

[0195] Also provided are polynucleotides encoding the vector polypeptides described herein. In another embodiment, this disclosure provides an expression vector comprising a polynucleotide. In yet another embodiment, this disclosure provides a host cell comprising an expression vector.

[0196] adjuvant

[0197] The pharmaceutical compositions of the present invention may contain aluminum salt adjuvants. Adjuvants can enhance the immunogenicity of conjugates within the pharmaceutical composition. Conjugates within the composition can be adsorbed onto the aluminum salt adjuvant.

[0198] Useful aluminum salt adjuvants include, but are not limited to, aluminum hydroxide adjuvants and aluminum phosphate adjuvants. These adjuvants are described, for example, in Chapters 8 and 9, Vaccine Design… (1995) Powell and Newman, eds. ISBN: 030644867X. Plenum Corporation.

[0199] The adjuvant commonly referred to as "aluminum hydroxide" is typically an aluminum hydroxide salt, which is usually at least partially crystalline. Aluminum hydroxide, which can be represented by the formula AlO(OH), can be detected by infrared (IR) spectroscopy, particularly at 1070 cm⁻¹. –1 The adsorption band at 3090-3100 cm⁻¹ –1 The presence of a strong shoulder distinguishes it from other aluminum compounds such as Al(OH)3 (Powell and Newman, Chapter 9). The crystallinity of aluminum hydroxide adjuvants is reflected by the half-maximum diffraction band width (WHH), where poorly crystallized particles show greater line broadening due to their smaller crystallite size. Surface area increases with increasing WHH, and adjuvants with higher WHH values ​​have been considered to have greater antigen adsorption capacity. For example, aluminum hydroxide adjuvants with needle-like particles of about 2 nm in diameter are often in fibrous form (e.g., as seen in transmission electron microscopy). The pI of aluminum hydroxide adjuvants is typically about 11, meaning that the adjuvant itself has a positive surface charge at physiological pH. Adsorption capacity of aluminum hydroxide adjuvants at pH 7.4 has been reported to be between [value missing] mg Al. +++ Between 1.8 and 2.6 mg of protein.

[0200] The adjuvant commonly referred to as "aluminum phosphate" is typically aluminum hydroxyphosphate, which usually also contains a small amount of sulfate (i.e., aluminum hydroxyphosphate sulfate). This adjuvant can be obtained by precipitation, and the reaction conditions and concentration during precipitation affect the degree to which the phosphate ester substitutes for the hydroxyl group in the salt. The PO4 / Al molar ratio of the hydroxyphosphate is typically between 0.3 and 1.2. The hydroxyphosphate can be distinguished from strictly AlPO4 by the presence of hydroxyl groups. For example, 3164 cm⁻¹ -1The IR spectral bands at a point (e.g., when heated to 200 °C) indicate the presence of structural hydroxyl groups (Powell and Newman, Chapter 9).

[0201] PO4 / Al of aluminum phosphate adjuvant 3+ The molar ratio will typically be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably between 0.95 and 0.1. Aluminum phosphate will generally be amorphous, especially for hydroxyphosphate. A typical adjuvant is 0.6 mg Al. 3+ Aluminum hydroxyphosphate is contained in ml as an amorphous form with a PO4 / Al molar ratio between 0.84 and 0.92. Aluminum phosphate is typically in particulate form (e.g., a sheet-like morphology as seen in transmission electron microscopy, where primary particles range in size from 50 nm). After any antigen adsorption, the typical diameter of the particles ranges from 0.5 to 20 μm (e.g., about 5 to 10 μm). Aluminum phosphate adjuvant has been reported to adsorb between 0.7 and 1.5 mg of protein per mg Al+++ at pH 7.4.

[0202] The point of zero charge (PZC) of aluminum phosphate is inversely proportional to the degree to which the hydroxyl groups of the phosphate ester are substituted, and this degree of substitution can vary depending on the reaction conditions and the concentration of the reactants used to prepare the salt by precipitation. The PZC is also altered by changing the concentration of free phosphate ions in the solution (more phosphate ester = more acidic PZC) or by adding a buffer such as histidine buffer (making the PZC more basic). The PZC of aluminum phosphate used according to the invention is typically between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example, about 5.7.

[0203] The concentration of aluminum ions in the composition for administration to patients is preferably less than 2.5 mg / ml, for example, ≤2 mg / ml, ≤1 mg / ml, etc. The preferred maximum concentration is ≤1.7 mg / mL. The Al in the composition of the present invention... +++ The dosage range can be 0.3-1 mg / ml or 0.3-0.5 mg / ml. The maximum dose is preferably 0.85 mg / ml.

[0204] In solution, both aluminum phosphate adjuvant and aluminum hydroxide adjuvant tend to form stable porous aggregates with a diameter of 1-10 μm. The composition may contain a mixture of both aluminum hydroxide adjuvant and aluminum phosphate adjuvant.

[0205] In compositions comprising multiple conjugates, each of which is adsorbed onto an aluminum salt adjuvant, each conjugate may be adsorbed onto the aluminum salt individually and then mixed, or the conjugates may be added sequentially to the aluminum salt, thereby forming a mixed conjugate composition. Both methods of mixing can also be used.

[0206] Excipients for pharmaceutical compositions

[0207] The pharmaceutical compositions of the present invention will generally contain one or more pharmaceutically acceptable excipients. A full discussion of such excipients can be found in the Handbook of Pharmaceutical Excipients (edited by Rowe et al.), 6th edition, 2009.

[0208] The pharmaceutical composition is preferably in an aqueous form, especially when applied, but it may also be in a dry form (e.g., as a lyophilized product), which can be converted into an aqueous form for application.

[0209] The pharmaceutical composition may contain a buffer or a pH adjuster. The buffer may be selected from the group consisting of phosphate buffers, acetate buffers, histidine buffers, citrate buffers, succinate buffers, Tris buffers, HEPES buffers, etc. The buffer salt is typically contained in the range of 5-20 mM.

[0210] Pharmaceutical compositions may contain physiological salts such as sodium salts, for example, to control stress. Sodium chloride (NaCl) is typical, and it may be present at concentrations of 1-20 mg / ml, for example, 10+2 mg / ml or 9 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium dehydrated phosphate, magnesium chloride, calcium chloride, etc. Other useful salts may have sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions.

[0211] The pharmaceutical composition may contain organic acids, such as acetic acid or succinic acid. This can be part of a buffer system.

[0212] The pharmaceutical composition may contain sugar alcohols, such as mannitol or sorbitol. The pharmaceutical composition may contain sugars, such as sucrose or glucose.

[0213] The pharmaceutical composition may contain a surfactant. Suitable surfactants include, but are not limited to, polysorbate 20, polysorbate 80, and sodium dodecyl sulfate (SDS). In some embodiments, the surfactant is present at a concentration of 0.0003% to 0.3% (w / w), for example, 0.01% to 0.03%. Polysorbate 80 is a preferred surfactant.

[0214] Pharmaceutical compositions may contain preservatives such as thimerosal or 2-phenoxyethanol. Preferably, the composition should be substantially free of (e.g., <10 μg / ml) mercury-free materials, such as thimerosal-free. Mercury-free compositions are more preferred. The inclusion of preservatives can be particularly useful when the composition contains aluminum salt adjuvants, as their insolubility means the composition is typically a suspension with a turbid appearance that can mask the growth of contaminating bacteria. Preservatives are also particularly useful if the composition is intended for use more than once, for example, in multi-dose vials. However, pharmaceutical compositions may generally be preservative-free.

[0215] The permeability of the pharmaceutical composition can be from 200 mOsm / kg to 400 mOsm / kg, for example, 240-360 mOsm / kg or 290-310 mOsm / kg.

[0216] The pH of the pharmaceutical composition is typically 5.0 to 9.5, for example, 5.0 to 8.0 or 6.0 to 8.0.

[0217] The pharmaceutical composition is preferably pyrogen-free, for example, containing <1 EU (endotoxin unit, standard measure) per dose and preferably <0.1 EU per dose.

[0218] The permeability of the pharmaceutical composition can be 200-400 mOsm / kg, for example, 240-360 mOsm / kg or 280-320 mOsm / kg.

[0219] The pharmaceutical composition is preferably gluten-free.

[0220] The pharmaceutical composition is suitable for administration to animal (and especially human) patients and therefore comprises both human and veterinary uses.

[0221] The pharmaceutical composition can be prepared in unit dose form. In some embodiments, the volume of a unit dose can be 0.1-1.0 ml, for example about 0.25 ml or preferably about 0.5 ml. Such a volume is ideal for human injection.

[0222] Conjugate Level

[0223] Pharmaceutical compositions may comprise multiple immunogenic conjugates. Currently licensed meningococcal conjugate vaccines comprise capsular sugars from four different serotypes, and licensed pneumococcal conjugate vaccines comprise capsular sugars from seven, ten, or thirteen different serotypes. Therefore, the compositions of the present invention may comprise, for example, three to fifty different conjugates (e.g., 14, 15, 20, 21, 24, 25, or more). For example, each of these conjugates may comprise capsular sugars from different serotypes or serogroups of the same bacterial species (e.g., multiple meningococcal serogroups or multiple pneumococcal serotypes).

[0224] When a pharmaceutical composition comprises n different immunogenic conjugates, the total amount of the carrier peptide in these n conjugates can be less than or equal to 3 n μg per dose. In other words, the average amount of the carrier peptide in each conjugate is less than 3 μg. The total amount can be, for example, n-2.5 n μg per dose.

[0225] When a pharmaceutical composition comprises n different immunogenic conjugates, the total amount of glycoantigen in these n conjugates can be less than or equal to 4.4n μg per dose. In other words, the average amount of sugar in each conjugate is less than 4.4 μg. The total amount can be, for example, 0.4n-4.4n μg per dose, for example, 1.1n-2.2n.

[0226] When a pharmaceutical composition comprises n different immunogenic conjugates, the total concentration of the carrier peptides of these n conjugates can be less than or equal to 6 n μg / mL. In other words, the average concentration of the carrier peptide for each conjugate is less than 6 μg / mL. The total concentration can be, for example, n-4 n μg / mL.

[0227] When a pharmaceutical composition comprises n different immunogenic conjugates, the total concentration of the glycoantigens of these n conjugates can be less than or equal to 8.8 n μg / mL. In other words, the average concentration of the sugar in each conjugate is less than 8.8 μg / mL. The total concentration can be, for example, 0.8n-8.8n μg / mL, for example, 2.2n-4.4n μg / mL.

[0228] In some embodiments, the total amount of conjugated carrier peptide in a unit dose of the multivalent pharmaceutical composition of the present invention may be 4-128 μg, for example, 8-64 μg or 16-48 μg. The concentration of the conjugated carrier peptide in the multivalent pharmaceutical composition of the present invention may be 8-256 μg / mL, for example, 16-128 μg / mL or 32-96 μg / mL.

[0229] In some embodiments, the total amount of conjugated glycoantigen in a unit dose of the multivalent pharmaceutical composition of the present invention may be 10-120 μg, for example, 20-90 μg or 30-60 μg. The concentration of conjugated glycoantigen in the multivalent pharmaceutical composition of the present invention may be 20-240 μg / mL, for example, 40-180 μg / mL or 60-120 μg / mL.

[0230] Unconjugated components

[0231] As noted above, the pharmaceutical composition may contain a variety of immunogenic conjugates, for example, 3 to 50 different conjugates (e.g., 14, 15, 20, 21, 24, 25 or more). For example, each of these conjugates may contain capsular sugars from different serotypes or serogroups of the same bacterial species.

[0232] In some embodiments, the composition does not contain one or more unconjugated carrier polypeptides of the conjugate. In other embodiments, a low level of one or more unconjugated carrier polypeptides is present, provided that the mass of one or more unconjugated carrier polypeptides in the composition is less than 10% (e.g., <5% or <2%) of the mass of one or more carrier polypeptides in the n immunogenic conjugates of the composition as a whole.

[0233] In some embodiments, the composition does not contain the unconjugated form of sugars in the conjugates. In other embodiments, a low level of unconjugated sugars is present, provided that the mass of unconjugated sugars in the composition is less than 10% (e.g., <5% or <2%) of the total mass of sugars in the n immunogenic conjugates of the composition.

[0234] Containers, delivery devices, etc.

[0235] Pharmaceutical compositions containing immunogenic conjugates can be packaged in sterile containers, delivery devices, etc. Sterility can be maintained by using airtight, airtight containers. Suitable containers include, but are not limited to, vials, syringes, nebulizers, sprayers, inhalers, skin patches, etc. Vials and syringes are preferred.

[0236] The vials typically contain an immunogenic composition. The vials are preferably made of plastic or, more preferably, glass. The vials are sealed after filling, and the seal can be broken during use. Preferably, the vials are sterilized before the composition is added and then sealed. To avoid problems for patients sensitive to latex, the vials can be sealed with latex-free stoppers, and preferably, all packaging materials are latex-free. Ideally, the vials contain a single unit dose of the composition, but sometimes more than one dose ('multi-dose' vials), such as 10 doses, may be contained. The vials are preferably made of colorless glass.

[0237] The vial may have a cap (e.g., a Luer lock) adapted to allow a syringe to be inserted into the cap for transferring material between the vial and the syringe (in both directions). After the syringe is removed from the vial, a needle can then be attached, and the composition can be administered to the subject. The cap is preferably located inside a seal or cap, such that the seal or cap must be removed before access to the cap is possible. The vial may have a cap that allows for aseptic removal of the vial's contents, particularly for multi-dose vials.

[0238] The delivery device may contain a composition prepared for administration to a subject. The composition may be transferred into the delivery device at the time of use (e.g., from a vial), or the composition may be placed into the delivery device during the manufacturing stage (e.g., in the form of a pre-filled syringe).

[0239] The syringe used in conjunction with this invention can be made of glass or plastic (e.g., of a cyclic olefin polymer or cyclic olefin copolymer). The syringe (particularly a glass syringe) can be a siliconized syringe. Non-siliconized syringes can also be used, such as i-Coating from Terumo. TM The system (available in its PLAJEX) TM (Obtained in a syringe) or using Daikyo CZ containing ethylene-tetrafluoroethylene (ETFE) copolymer. TM Syringe or use TriboGlide with perfluoropolyether (PFPE). TM Syringe. A carbon film can be used instead of siliconized (e.g., see JP2001190665). Silicone-free syringes are also disclosed in JP2011212183. Non-silicone syringes containing a plunger stopper, as disclosed in EP-A-0375778, can be used, i.e., wherein the stopper has a thermoplastic elastomer layer at least partially covered by a thermoplastic resin layer with a low dynamic coefficient of friction.

[0240] When the syringe contains a composition, it may have a needle attached thereto for injecting the syringe contents into a subject or container. A pre-attached needle may be supplied with the syringe. If the needle is not attached, a separate needle may be supplied with the syringe for assembly and use, or the needle may be supplied separately. Such needles should be sterile during use and may be sheathed. Safety needles may be used. 1-inch 23, 1-inch 25, and 5 / 8-inch 25 needles are typical. 1 / 2-1 1 / 2 inch 22-25 gauge needle. If the syringe and needle are packaged separately, the needle is preferably fitted with a butyl rubber shield.

[0241] Syringes may be equipped with peel-off labels, which may be printed with the batch number and expiration date of the contents to facilitate record keeping. The plunger in the syringe may have a stopper to prevent accidental removal of the plunger during aspiration. Syringes may have latex rubber caps and / or plungers, but latex-free rubbers may be used, such as latex-free chlorobutyl rubber or latex-free isoprene bromobutyl rubber. Syringes will typically have a tip cap for sealing the tip before connecting the needle, and the tip cap is preferably made of butyl rubber, such as latex-free isoprene bromobutyl rubber. Useful syringes are, for example, those marketed under the trade name "Tip-Lok". TM Syringes for sale.

[0242] Containers may be labeled to indicate a half-dose volume, for example, to facilitate delivery to children. For instance, a syringe containing a 0.5 ml dose may have a label indicating a 0.25 ml volume. The syringe itself may have a volume larger than the dose; for example, a 1 ml syringe may be used to hold a 0.5 ml dose of the pharmaceutical composition. Disposable or pre-filled syringes typically contain a single dose of vaccine.

[0243] When using glass containers (e.g., syringes or vials), the glass containers are preferably made of borosilicate glass rather than soda-lime glass.

[0244] Containers may be packaged (e.g., in the same box) with leaflets containing vaccine details such as administration instructions, details of the antigens contained in the vaccine, etc. The instructions may also contain warnings, such as ensuring the availability of epinephrine solution in case of an allergic reaction after vaccination. Multiple containers may be packaged together, for example, in the same box.

[0245] Pharmaceutical compositions can be presented in unit dose form, where each container (e.g., each syringe or each vial) has a single dose. Instead of manufacturing each unit dose individually, a large volume composition is prepared and unit doses are extracted and individually packaged into their containers. Thus, for example, multiple unit doses are extracted from the large volume composition and each unit dose is placed in a separate container, such as a syringe or vial.

[0246] Produce an immune response

[0247] Immunogenic conjugates can be administered to mammalian subjects to elicit a protective immune response against antigens in the conjugate. The immunogenic conjugates are administered in the form of a pharmaceutical composition. The composition may contain multiple immunogenic conjugates as described elsewhere herein, thus enabling the simultaneous elicit of protective immune responses against numerous antigens.

[0248] Therefore, a method is provided to induce a protective antibody response against one or more antigens in a mammalian subject by administering a conjugate of one or more antigens to the subject.

[0249] Conjugates, as disclosed herein, are also provided for use in inducing protective antibody responses.

[0250] The use of conjugates, as disclosed herein, in the manufacture of drugs for inducing protective antibody responses is also provided.

[0251] Also provided are (i) a method for inducing a protective antibody response against one or more antigens of a mammalian subject by administering the multivalent composition of the invention to the subject; (ii) the multivalent composition of the invention for use in inducing a protective antibody response; and (iii) the use of the various conjugates disclosed herein in the manufacture of multivalent pharmaceutical compositions for inducing a protective antibody response against a variety of antigens.

[0252] The ability to elicit a protective immune response means that the conjugate can be used, for example, to provide active immunization to prevent invasive diseases caused by Streptococcus pneumoniae, to prevent otitis media caused by Streptococcus pneumoniae, to prevent pneumonia caused by Streptococcus pneumoniae, and to provide active immunization to prevent invasive diseases in subjects at risk of exposure to Neisseria meningitidis.

[0253] Pharmaceutical compositions can be prepared in various forms. For example, the composition can be prepared as an injectable preparation, such as a liquid solution or suspension. Injectable preparations for intramuscular administration are typical. An injection volume of about 0.5 ml is preferred for humans. Therefore, a preferred unit dose volume is about 0.5 ml. Intramuscular injection is typical for example, into the anterolateral thigh of infants or into the deltoid muscle of the upper arm in young children and adults.

[0254] Conjugates are typically administered according to a multi-dose regimen. Multiple doses may be used in primary immunization regimens and / or booster immunization regimens. Administration of more than one dose (usually two doses) is particularly useful for patients with an immune blank. Multiple doses are typically administered at intervals of at least one week (e.g., approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, etc.).

[0255] Overview

[0256] The term "comprising" encompasses both "including" and "consisting of," for example, a composition "comprising" X may consist of only X or may contain additional substances, such as X+Y.

[0257] The term "approximately" related to the numerical value x is optional and means, for example, x. + 10%.

[0258] The term "substantially" does not exclude "completely," for example, a composition that is "substantially free of" Y can be completely free of Y. Where necessary, the term "substantially" can be omitted from the definition of this invention.

[0259] In the context of two amino acid sequences, the term "sequence identity" refers to two sequences that are identical or have the same specific percentage of amino acid residues when compared and aligned against the largest correspondence within a comparison window, as measured using a sequence comparison algorithm (e.g., BLASTP). The identity percentage is determined on a reference sequence such as SEQ ID NO: 1 or 2, which is disclosed herein as a full-length reference sequence. The method for calculating sequence identity, as provided herein, is the BLASTP procedure, which is defaulted to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad SciUSA) 89:10915). See, for example, BLAST alignment tools available at blast.ncbi.nlm.nih.gov / Blast.cgi or elsewhere.

[0260] As used herein and unless otherwise stated, the term "lower alkyl" refers to a saturated straight-chain or branched hydrocarbon having one to six carbon atoms, i.e., a C1 to C6 alkyl group. In some embodiments, the lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted portions. See also US-2014 / 0066598. The term "lower alkylene" refers to an alkylene of a lower alkyl group.

[0261] Unless otherwise defined, all technical and scientific terms used herein have their commonly understood meanings. Practitioners shall refer in particular to Green and Sambrook (eds.), *Molecular Cloning: A Laboratory Manual*, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012); Ausubel, FM et al., *Current Protocols in Molecular Biology* (Supplement 99), John Wiley & Sons, New York (2012); and Plotkin, SA, Orenstein, WA, and Offit, PA, *Vaccines*, 6th ed., Elsevier, London (2013).

[0262] Methods for cell-free synthesis are described in Spirin and Swartz (2008), Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. Methods for incorporating non-natural amino acids into proteins using cell-free synthesis are described in Shimizu et al. (2006), FEBS Journal, 273, 4133-4140, and Chong (2014), Laboratory Manual of Molecular Biology, 108:16.30.1-11.

[0263] In some embodiments, the present invention does not cover compositions in which SEQ ID NO:3 is used as a carrier polypeptide for conjugates of each of the following 24 pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F (as illustrated by example in WO2018 / 126229). More generally, in some embodiments, the present invention does not cover compositions in which SEQ ID NO:3 is used as a carrier polypeptide for each conjugate in a multivalent composition.

[0264] Listed Examples

[0265] Example I-1. A sterile container containing a pharmaceutical composition comprising an immunogenic conjugate, said immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein said glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0266] Example 1-2. An airtight container containing a pharmaceutical composition comprising an immunogenic conjugate, said immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein said glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. Suitable containers for airtight sealing include, for example, vials. When airtight, the contents are preferably sterile.

[0267] Example I-3. The container according to Example I-1 or I-2 is a sterile glass container such as a vial.

[0268] Example 1-4. A delivery device comprising a pharmaceutical composition including an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0269] Example I-5. The container according to Example I-1 or I-2 or the delivery device according to Example I-4, wherein the container or the delivery device is a syringe.

[0270] Example 1-6. A pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the aluminum salt adjuvant is aluminum hydroxide or aluminum phosphate adjuvant.

[0271] Example 1-7. A pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of aluminum ions in the composition is <2.5 mg / mL.

[0272] Example 1-8. A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the volume of the pharmaceutical composition is 0.25-1.25 mL.

[0273] Examples I-9. A pharmaceutical composition comprising two or more different immunogenic conjugates and a preservative, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0274] Example I-10. A preservative-free pharmaceutical composition comprising two or more different immunogenic conjugates, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0275] Example I-11. A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the composition has a permeability of 200-400 mOsm / kg.

[0276] Example 1-12. A pharmaceutical composition comprising two or more different immunogenic conjugates and at least one excipient, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80.

[0277] Example I-13. A pharmaceutical composition comprising n different immunogenic conjugates, wherein:

[0278] (i) Each of the n immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide;

[0279] (ii) n is an integer from 3 to 50; and

[0280] (iii) The total amount of carrier polypeptide in the n immunogenic conjugates is less than or equal to 3nμg per dose;

[0281] (iv) The total concentration of the carrier polypeptide in the n immunogenic conjugates is less than or equal to 6 n μg / ml;

[0282] (v) The total amount of glycoantigen in the n immunogenic conjugates is less than or equal to 3 n μg per dose;

[0283] (vi) The total concentration of glycoantigens in the n immunogenic conjugates is less than or equal to 6 n μg / mL;

[0284] (vii) The average amount of the carrier polypeptide for each conjugate is 1-4 μg per dose;

[0285] (viii) The average concentration of the carrier polypeptide for each conjugate was 2-8 μg / mL;

[0286] (ix) The average amount of glycoantigen for each conjugate is 1-4 μg per dose;

[0287] (x) The average concentration of the glycoantigen for each conjugate is 2-8 μg / mL;

[0288] (xi) The composition does not contain one or more unconjugated forms of the carrier polypeptide;

[0289] (xii) The composition contains one or more unconjugated forms of the carrier polypeptide, wherein the mass of the one or more unconjugated forms of the carrier polypeptide in the composition is less than 10% of the mass of the carrier polypeptide in the n immunogenic conjugates;

[0290] (xiii) The composition does not contain the unconjugated form of the glycoantigen; and / or

[0291] (xiv) The composition contains at least one of the unconjugated forms of the glycoantigens, wherein the total mass of the unconjugated forms of the glycoantigens in the composition is less than 10% of the total mass of the glycoantigens in the n immunogenic conjugates.

[0292] Example 1-14. A method for preparing a plurality of unit doses of a pharmaceutical composition, wherein (i) the pharmaceutical composition comprises an immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide, and (ii) the method comprises the steps of preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into a plurality of individual containers.

[0293] Example I-15. A method for preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, and (ii) the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and the method comprises (A) adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant and then mixing the individually adsorbed conjugates together, or (B) sequentially adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant.

[0294] Example I-16. A carrier polypeptide comprising an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO:1; (ii) lacking an Arg-Arg dipeptide sequence; and (iii) containing at least one nnAA residue.

[0295] Example I-17. A carrier polypeptide comprising an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO:1 and (ii) containing nnAA substitutions 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 481; Asn 486; Asn 502; Asn 524; Glu240; 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;Ser496;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.

[0296] Example I-18. The carrier polypeptide according to Example I-16 or I-17, wherein Arg-193 of SEQ ID NO:1 is replaced with different amino acids such as Asn.

[0297] Example I-19. An immunogenic conjugate comprising a carrier polypeptide according to Example I-16, I-17, or I-18, wherein the carrier polypeptide is conjugated to an antigen via an nnAA residue in the carrier polypeptide.

[0298] Example I-20. An immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein (i) the carrier polypeptide comprises the amino acid sequence SEQ ID NO:4; and (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:4.

[0299] Example 1-21. A pharmaceutical composition comprising two or more different immunogenic conjugates according to Example 1-20.

[0300] Examples I-22. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises 4 to 9 nnAA residues.

[0301] Examples I-23. A container, apparatus, composition, method, polypeptide or conjugate according to any of the foregoing examples, wherein at least one nnAA replaces a lysine residue in the natural sequence of the carrier polypeptide.

[0302] Examples I-24. Containers, devices, compositions, methods, polypeptides or conjugates according to any of the foregoing examples, wherein the carrier polypeptide has at least 90% sequence identity with SEQ ID NO:1.

[0303] Examples I-25. Containers, devices, compositions, methods, polypeptides or conjugates according to Examples I-24, wherein at least one nnAA replaces K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and / or K526 in SEQ ID NO:1 or 2.

[0304] Examples I-26. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises the amino acid sequence SEQ ID NO:14.

[0305] Examples I-27. Containers, devices, compositions, methods, polypeptides or conjugates according to any of the foregoing examples, wherein the nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propionic acid.

[0306] Examples I-28. Containers, devices, compositions, methods, polypeptides, or conjugates according to any of the foregoing examples, wherein the antigen has an alkynyl group conjugated to the nnAA via an azide group.

[0307] Examples I-29. A container, apparatus, composition, method, polypeptide, or conjugate according to any of the foregoing examples, wherein the antigen is a bacterial capsular sugar; for example, a capsular sugar from bacteria selected from the group consisting of: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis.

[0308] Examples I-30. Containers, devices, compositions, methods, polypeptides, or conjugates according to any of the foregoing examples, wherein the antigen is a capsular sugar of a Streptococcus pneumoniae serotype selected from the group consisting of: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0309] Example 1-31. A container, apparatus, composition, method, polypeptide or conjugate according to any of the foregoing examples, wherein the ratio (w / w) of sugar to carrier polypeptide in one or more of the conjugates is greater than 1.

[0310] Examples 1-32. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises three or more nnAA residues, and the conjugate has a molecular weight of at least 500 kDa.

[0311] Examples I-33. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the molecular weight of the conjugate is between 900 kDa and 5 MDa.

[0312] Example I-34. A container, device, composition, or method according to any one of Examples I-1 to I-15 or Examples I-21 to I-33, wherein the pharmaceutical composition comprises:

[0313] A conjugate of capsular sugars from two or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0314] A conjugate of capsular sugars from 14 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0315] A conjugate of capsular sugars from 15 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0316] A conjugate of capsular sugars from 20 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0317] A conjugate of capsular sugars from 21 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0318] A conjugate of capsular sugars from 24 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0319] A conjugate of capsular sugars from 25 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F;

[0320] A conjugate of capsular sugars from four or more different serogroups of meningococci, wherein the different serogroups of meningococci are selected from the group consisting of: A, C, W135, X, and Y; or

[0321] A conjugate of capsular sugars from two or more different serotypes of Porphyromonas gingivalis, wherein the different serotypes of Porphyromonas gingivalis are selected from the group consisting of the following serotypes: K1, K2, K3, K4, K5 and K6.

[0322] Example I-35. A method for inducing an immune protective antibody response against an antigen in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of Examples I-6 to I-13 or Examples I-21 to I-34 or an immunogenic conjugate according to any one of Examples I-19 to I-33 in an excipient suitable for parenteral administration.

[0323] Example

[0324] The invention is illustrated in the following examples. The materials, methods, and examples are merely illustrative and not intended to be limiting. Various variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. Unless otherwise described in detail, the examples are performed using techniques and conventional methods well known to those skilled in the art.

[0325] Example from WO2018 / 126229

[0326] The examples in WO2018 / 126229 describe in detail the synthesis of unit site eCRM moieties (e.g., K11TAG). These moieties are expressed in cell-free protein synthesis (CFPS) extracts and are incorporated with pAMF instead of native Lys.

[0327] Variants of CRM containing multiple nnAAs per polypeptide were also expressed, with each protein having various numbers of Lys→pAMF substitutions. Generally, the more substitutions found, the higher the resulting vector-induced MW conjugate, but the lower the vector solubility. Vectors with six pAMF residues typically provide both good solubility (>>50 mg / mL) and immunogenicity. The high solubility is surprising because replacing charged Lys residues in the native sequence with hydrophobic pAMF residues increases the hydrophobicity of CRM197, a protein whose hydrophobicity has been reported to affect its solubility. This demonstrates that the same linker sites (i.e., Lys residues) already used in known CRM197 conjugates can be maintained without insoluble matter when charged residues are lost.

[0328] A particularly useful set of six Lys→pAMF substitutions was observed using K34, K213, K245, K265, K386, and K527 (numbered according to SEQ ID NO:3). This combination of pAMF substitution sites was surprisingly effective, especially since individual substitutions at positions K245 and K527 resulted in relatively poor expression levels.

[0329] This set of six substitutions can be combined with the disruption (RR→RN) of the Arg-Arg dipeptide at residues 192-193 of SEQ ID NO:1 to provide SEQ ID NO:4, where each X is pAMF.

[0330] Examples in WO2018 / 126229 further describe general protocols for sugar activation with sodium metaperiodate, for periodate oxidation of polysaccharides with DBCO, for sugar activation with CDAP, and for sugar-DBCO conjugation with eCRM. See also U.S. Serial No. 62 / 693,978, previously incorporated by reference.

[0331] Multivalent immunogenic compositions

[0332] Combinations of conjugates for each of the 24 pneumococcal serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) were prepared using a CRM197 derivative SEQ ID NO:4 (where X = pAMF) as the carrier polypeptide in each conjugate. The immunogenicity of this multivalent composition was confirmed by intramuscular injection of 0.25 mL of vaccine in a group of seven rabbits using a three-dose regimen. Each dose contained 24 μg of sugar (1 μg per serotype), yielding a concentration of 96 μg / mL.

[0333] Then, combinations of conjugates for each of the 32 pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B were prepared, and immunogenicity was confirmed in a similar manner.

[0334] For comparative purposes, the 13-valent Prevnar vaccine was also tested. TM Conjugated vaccines and 23-valent Pneumovax supplemented with unconjugated serotype 6A polysaccharide TMThe vaccine is a 24-valent unconjugated vaccine. These three compositions contain a comparable polysaccharide dose for each serotype (except for 6B, in which Prevnar...). TM (Including double dose), which involves diluting Prevnar TM and Pneumovax TM All three compositions contain aluminum phosphate adjuvant (60 μg Al per dose). +++ This involves adding an adjuvant to Pneumovax. TM The composition does not contain preservatives.

[0335] The 24-valent conjugate composition contains a lower amount of carrier peptide than the approved Prevnar-13. TM The vaccine, although the composition also contains capsular sugars from 11 additional serotypes. The total weight ratio of capsular sugars to carrier polypeptides in the 24-valent conjugate composition is approximately [value missing]. TM Twice as observed in [the study].

[0336] IgG and OPA responses in rabbits were measured. After the third dose, these responses were significantly greater in rabbits receiving both conjugated vaccines than in rabbits receiving the unconjugated vaccine. Furthermore, IgG and OPA responses using the 24-valent combination were compared with those using Prevnar in the 13 serotypes covered by the approved vaccine. TM The achieved response was comparable, but also superior to Prevnar. TM Eleven serotypes not included in the study. Surprisingly, there is no evidence that the vector used in the 24-valent composition induced epitope inhibition.

[0337] Figure 1 The 32 serotypes in the 32-valent conjugate composition are provided relative to the polysaccharide / alum formulation and Prevnar-13. TM The geometric mean titer.

[0338] Multivalent conjugate compositions can be usefully packaged into pre-filled sterile syringes, allowing them to be easily dispensed in unit dose form and then administered at the time of use without the need to transfer the contents of the vial into a syringe for injection, etc.

[0339] Replaceable positions in CRM197

[0340] Based on the work disclosed in WO2018 / 126229, various Asp, Asn, Glu, Gln, Lys, Arg, Ser, and Thr residues in the natural CRM197 sequence (SEQ ID NO:1) were replaced with pAMF by mutating their codons to TAG and expressing the protein in a cell-free system at 25°C, where this codon is recognized by tRNA incorporating nnAA. The mutant polypeptide was expressed using an N-terminal methionine and a downstream six-histidine tag linked via a Gly-Ser-Gly tripeptide linker. Residues in Asn270-Ile289, Ala320-Glu349, and Phe410-His-449 were avoided due to the T-cell epitopes recognized in these regions (see above).

[0341] By inspection 14 C-Leu incorporation into mutant proteins and examination of both total and soluble protein levels are used to assess expression efficiency. Generally, mutations in the catalytic domain of CRM197 result in lower expression levels relative to the unmodified CRM197 sequence, and mutants with optimal expression levels typically involve substitutions downstream of Arg-193, which can be used to delineate the ends of the catalytic domain.

[0342] The optimal 72 mutants showed increased expression levels of both total and soluble protein and had substitutions at the following residues numbered according to SEQ ID NO:1: Ser-198, Ser-200, Asp-211, Lys-212, Lys-218, Lys-221, Lys-229, Ser-231, Ser-233, Lys-236, Ser-239, Glu-240, Glu-248, Glu-249, Gln-252, Thr-253, G lu-256, Glu-259, Ser-261, Lys-264, Thr-265, Thr-267, Thr-269, Gln-287, Glu-292, Thr -293, Asp-295, Asn-296, Ser-297, Lys-299, Asp-352, Asn-359, Glu-362, Ser-374, Arg-37 7. Ser-381, Lys-385, Thr-386, Asp-392, Ser-397, Asn-399, Thr-400, Arg-407, Thr-408, Ser-451, Arg-455, Lys-456, Arg-460, Arg-462, Asp-465, Asp-467, Thr-469, Arg-472, Lys -474, Ser-475, Asn-481, Asn-486, Arg-493, Ser-494, Ser-495, Ser-496, Lys-498, Ser-5 01. Asn-502, Ser-505, Asp-507, Lys-516, Thr-517, Asp-519, Lys-522, Asn-524 and Lys-534.

[0343] The embodiments described herein are provided by way of example only, and various alternatives to the embodiments are not excluded when practicing them.

[0344] sequence list

[0345] SEQ ID NO:1 (Natural CRM197)

[0346] GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0347] SEQ ID NO:2 (CRM197 with Arg-Asn substitution)

[0348] GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0349] SEQ ID NO:3 (CRM197 with 6 preferred nnAA sites and N-terminal Met)

[0350] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS

[0351] SEQ ID NO:4 (with Arg-Asn substitution n 、6 preferred nnAA sites and CRM197 with N-terminal Met)

[0352] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSX LSLFFEIKS

[0353] SEQ ID NO:5 (Haemophilus influenzae protein D)

[0354] CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK sequence list <110> VAXCYTE <120> Improvement of immunogenic conjugates <130> PD01324A <150> US 62 / 693,981 <151> 2018-07-04 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 535 <212> PRT <213> Corynebacterium diphtheriae <400> 1 Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 180 185 190 Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 305 310 315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr 435 440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525 Leu Phe Phe Glu Ile Lys Ser 530 535 <210> 2 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> CRM197 with Arg-Asn substitution <400> 2 Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 180 185 190 Asn Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 305 310 315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr 435 440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525 Leu Phe Phe Glu Ile Lys Ser 530 535 <210> 3 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> CRM197 has 6 preferred nnAA sites and N-terminal Met. <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (213)..(213) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (245)..(245) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (265)..(265) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (386)..(386) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (527)..(527) <223> Xaa can be any non-naturally occurring amino acid. <400> 3 Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln Xaa Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40 45 Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala 50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln 165 170 175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val 180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 195 200 205 Val Ile Arg Asp Xaa Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Xaa Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu Ser Glu Leu Xaa Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295 300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Xaa Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 385 390 395 400 Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly 405 410 415 His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly 420 425 430 Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys 435 440 445 Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala 450 455 460 Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val 465 470 475 480 Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser 485 490 495 Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val 500 505 510 Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Xaa Leu 515 520 525 Ser Leu Phe Phe Glu Ile Lys Ser 530 535 <210> 4 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> CRM197 features Arg-Asn substitution, six preferred nnAA sites, and N-terminal Met. <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (213)..(213) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (245)..(245) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (265)..(265) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (386)..(386) <223> Xaa can be any non-naturally occurring amino acid. <220> <221> misc_feature <222> (527)..(527) <223> Xaa can be any non-naturally occurring amino acid. <400> 4 Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln Xaa Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40 45 Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala 50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln 165 170 175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val 180 185 190 Arg Asn Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 195 200 205 Val Ile Arg Asp Xaa Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Xaa Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu Ser Glu Leu Xaa Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295 300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Xaa Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 385 390 395 400 Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly 405 410 415 His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly 420 425 430 Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys 435 440 445 Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala 450 455 460 Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val 465 470 475 480 Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser 485 490 495 Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val 500 505 510 Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Xaa Leu 515 520 525 Ser Leu Phe Phe Glu Ile Lys Ser 530 535 <210> 5 <211> 346 <212> PRT <213> Haemophilus influenzae <400> 5 Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp 1 5 10 15 Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His 20 25 30 Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu 35 40 45 Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His 50 55 60 Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His 6 Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys 85 90 95 Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Lys Asp Gly 100 105 110 Lys Gln Ala Gln Val Tyr Pro Asn Arg Phe Pro Leu Trp Lys Ser His 115 120 125 Phe Arg Ile His Thr Phe Glu Asp Glu Ile Glu Phe Ile Gln Gly Leu 130 135 140 Glu Lys Ser Thr Gly Lys Lys Val Gly Ile Tyr Pro Glu Ile Lys Ala 145 150 155 160 Pro Trp Phe His His Gln Asn Gly Lys Asp Ile Ala Ala Glu Thr Leu 165 170 175 Lys Val Leu Lys Lys Tyr Gly Tyr Asp Lys Lys Thr Asp Met Val Tyr 180 185 190 Leu Gln Thr Phe Asp Phe Asn Glu Leu Lys Arg Ile Lys Thr Glu Leu 195 200 205 Leu Pro Gln Met Gly Met Asp Leu Lys Leu Val Gln Leu Ile Ala Tyr 210 215 220 Thr Asp Trp Lys Glu Thr Gln Glu Lys Asp Pro Lys Gly Tyr Trp Val 225 230 235 240 Asn Tyr Asn Tyr Asp Trp Met Phe Lys Pro Gly Ala Met Ala Glu Val 245 250 255 Val Lys Tyr Ala Asp Gly Val Gly Pro Gly Trp Tyr Met Leu Val Asn 260 265 270 Lys Glu Glu Ser Lys Pro Asp Asn Ile Val Tyr Thr Pro Leu Val Lys 275 280 285 Glu Leu Ala Gln Tyr Asn Val Glu Val His Pro Tyr Thr Val Arg Lys 290 295 300 Asp Ala Leu Pro Glu Phe Phe Thr Asp Val Asn Gln Met Tyr Asp Ala 305 310 315 320 Leu Leu Asn Lys Ser Gly Ala Thr Gly Val Phe Thr Asp Phe Pro Asp 325 330 335 Thr Gly Val Glu Phe Leu Lys Gly Ile Lys 340 345

Claims

1. A carrier polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; wherein the carrier polypeptide does not contain an Arg-Arg dipeptide sequence; and comprises six 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid residues substituted for K34, K213, K245, K265, K386, and K527 associated with SEQ ID NO:

4.

2. The carrier polypeptide of claim 1, wherein Arg-194 of SEQ ID NO: 4 is substituted with Asn.

3. An immunogenic conjugate comprising a carrier polypeptide according to any one of claims 1-2, wherein the carrier polypeptide is conjugated to an antigen via an nnAA residue in the carrier polypeptide, wherein the antigen is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae () capsular sugar.

4. The immunogenic conjugate of claim 3, wherein the antigen has an alkyne group conjugated to the nnAA via an azido group.

5. The immunogenic conjugate of claim 3, comprising a carrier polypeptide and a saccharide antigen, wherein the antigen is a saccharide covalently bound to the carrier polypeptide via at least one nnAA residue within SEQ ID NO:

4.

6. The immunogenic conjugate of claim 5, wherein the antigen has an alkyne group conjugated to the nnAA via an azido group.

7. The immunogenic conjugate of claim 3, wherein the antigen is a capsular saccharide of a S. pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

8. The immunogenic conjugate of claim 4, wherein the molecular weight of the conjugate is at least 500 kDa.

9. The immunogenic conjugate of claim 8, wherein the molecular weight of the conjugate is between 900 kDa and 5 MDa.

10. A pharmaceutical composition comprising two or more different immunogenic conjugates of claim 3.

11. The pharmaceutical composition of claim 10, wherein the ratio of saccharide to carrier polypeptide of the composition is greater than 1 by mass.

12. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition comprises: conjugates of capsular saccharides from 2-28 different S. pneumoniae serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

13. A pharmaceutical composition comprising two or more different immunogenic conjugates of any one of claims 4-9.

14. The pharmaceutical composition of claim 13, wherein the ratio of saccharide to carrier polypeptide of the composition is greater than 1 by mass.

15. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises: conjugates from 2-28 different pneumococcal serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

16. Use of the polypeptide carrier of any one of claims 1-2, the immunogenic conjugate of any one of claims 3-9, the pharmaceutical composition of any one of claims 10-12, and the pharmaceutical composition of any one of claims 13-15 in the manufacture of a medicament for eliciting an immunoprotective antibody response to capsular saccharide antigens from 2-28 serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

17. A pharmaceutical composition comprising immunogenic conjugates of capsular saccharide from each of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 16, 17F, 18C, 19A, 19F, 20C, 22F, 23A, 23B, 23F, 31, 33F, wherein the immunogenic conjugates comprise a carrier polypeptide having an amino acid sequence of SEQ ID NO: 4; wherein the carrier polypeptide is free of Arg-Arg dipeptide sequences and comprises 6 2-amino-3-(4-(azidomethyl)phenyl)propionic acid (pAMF) residues substituted for K34, K213, K245, K265, K386, and K527 associated with SEQ ID NO: 4, wherein Arg-194 of SEQ ID NO: 4 is substituted with Asn, binds to pneumococcal serotypes through the nnAA residue therein.

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