Improving immunogenic conjugates

Abstract

To provide various improvements concerning immunogenic conjugates which comprise a carrier polypeptide and a saccharide antigen.SOLUTION: In one embodiment, the present invention provides a sterile container (e.g. a vial) containing a pharmaceutical composition which comprises an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide. The container can contain a unit dose of the pharmaceutical composition. Sterile glass containers are preferred.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 693,981, filed July 4, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation of electronic text files submitted together The contents of the following text file, submitted electronically together, are incorporated herein by reference in their entirety: A computer-readable copy of the Sequence Listing (Filename: STRO_005_01WO_SeqList_ST25.txt, Date of Record: July 1, 2019, File Size: Approximately 23 KB) [Background technology]

[0003] Immune responses to "weak" saccharide antigens can be amplified by conjugation to known "strong" carrier polypeptide antigens, such as diphtheria toxoid, tetanus toxoid, H. influenzae protein D, or CRM197. International Publication No. WO 2018 / 126229 (SutroVax, Inc., Foster City, California) discloses methods, compositions, and techniques for producing conjugated vaccine antigens using carrier polypeptides containing unnatural amino acids (nnAA). Orthogonal conjugation chemistry via nnAA allows for the conjugation of antigens to carrier polypeptides, producing immunogenic conjugates useful for immunization.

[0004] It is an object of the present invention to provide variations and improvements to these methods, compositions, and techniques. The variations and improvements described below may be applied to or combined with any of the methods, compositions, or techniques disclosed in WO 2018 / 126229 or U.S. Provisional Patent Applications Serial Nos. 62 / 693,978 and 62 / 693,981, both filed July 4, 2018. The aforementioned patent applications are incorporated herein by reference in their entireties. Summary of the Invention

[0005] In one embodiment, a sterile container (e.g., a vial) is provided containing a pharmaceutical composition comprising an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. The container may contain a unit dose of the pharmaceutical composition. Preferably, the container is a sterile glass container.

[0006] In another embodiment, a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.) containing a pharmaceutical composition comprising an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen is provided, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. The delivery device can contain a unit dose of the pharmaceutical composition. The delivery device can be used to administer the pharmaceutical composition to a mammalian subject.

[0007] In another embodiment, there is provided a hermetically sealed container containing a pharmaceutical composition comprising an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bound to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. Containers suitable for hermetically sealing include, for example, vials. The contents are preferably sterilized at the time of hermetically sealing.

[0008] In another embodiment, a syringe is provided containing 0.25 to 0.75 mL (e.g., 0.3 to 0.75 mL, preferably 0.5 mL) of a pharmaceutical composition comprising two or more different immunogenic conjugates, each comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide.

[0009] In another embodiment, there is provided 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; (ii) the aluminum salt adjuvant is an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant; and (iii) the volume of the pharmaceutical composition is 0.25 to 0.75 mL (e.g., 0.3 to 0.75 mL, preferably 0.5 mL).

[0010] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural 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 in the composition can also be adsorbed to the aluminum phosphate adjuvant.

[0011] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, (ii) the carrier polypeptide does not comprise 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 in the composition can be adsorbed to the aluminum phosphate adjuvant.

[0012] In another embodiment, there is provided a pharmaceutical composition comprising two or more different immunogenic conjugates, (i) each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the volume of the pharmaceutical composition is 0.25 to 1.25 mL (e.g., 0.3 to 0.7 mL, preferably 0.5 mL). The composition may also comprise an aluminum phosphate adjuvant, and the conjugates in the composition may be adsorbed to the aluminum phosphate adjuvant.

[0013] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide.

[0014] In another embodiment, a preservative-free pharmaceutical composition is provided comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide.

[0015] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the composition has an osmolality of 200 to 400 mOsm / kg.

[0016] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural 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 include an aluminum salt adjuvant. The composition can include both sodium chloride and polysorbate 80 as excipients.

[0017] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total amount of carrier polypeptide in the n immunogenic conjugates is 3 nμg or less per dose of the pharmaceutical composition.

[0018] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total concentration of the carrier polypeptide in the n immunogenic conjugates is 6 n μg / mL or less in the pharmaceutical composition.

[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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total amount of saccharide antigen in the n immunogenic conjugates is 3 nμg or less 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total concentration of the saccharide antigens in the n immunogenic conjugates is 6 n μg / mL or less in the pharmaceutical composition.

[0021] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the average amount of carrier polypeptide per conjugate is 1 to 4 μg per dose of the pharmaceutical composition.

[0022] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the average concentration of the carrier polypeptide per conjugate is 2 to 8 μg / mL in the pharmaceutical composition.

[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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the average amount of saccharide antigen per conjugate is 1 to 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the average concentration of the saccharide antigen per conjugate is 2 to 8 μg / mL in the pharmaceutical composition.

[0025] 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 saccharide antigen, wherein the saccharide antigen is covalently attached 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 comprise a carrier polypeptide in unconjugated form, or (iv) the composition contains a carrier polypeptide in unconjugated form, wherein the mass of the carrier polypeptide in the composition in unconjugated form is <10% of the mass of the carrier polypeptide in the n immunogenic conjugates.

[0026] 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 saccharide antigen, wherein the saccharide antigen is covalently attached 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 comprise any saccharide antigen in unconjugated form, or (iv) the composition contains at least one saccharide antigen in unconjugated form, wherein the total mass of the saccharide antigen in unconjugated form in the composition is <40% (e.g., ≦30%, ≦20%, or ≦10%) of the total mass of the saccharide antigen in the n immunogenic conjugates.

[0027] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the total amount of carrier polypeptide per dose is <40 μg.

[0028] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the concentration of the carrier polypeptide per conjugate is ≦80 μg / mL.

[0029] In another embodiment, there is provided a method for preparing a plurality of unit dose pharmaceutical compositions, the method comprising: (i) the pharmaceutical composition comprising an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the method comprising the steps of preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into a plurality of separate containers. Ideally, the method is performed aseptically. The individual containers can be sealed after each unit dose is packaged therein. Ideally, the individual containers are syringes.

[0030] 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the composition is lyophilized.

[0031] In another embodiment, a method for preparing a pharmaceutical composition is provided, the pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, and (ii) the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, the method comprising one of the following steps: (A) separately adsorbing each of the immunogenic conjugates to an aluminum salt adjuvant and then mixing the individual adsorbed conjugates together; (B) sequentially adsorbing each of the immunogenic conjugates to an aluminum salt adjuvant; or (C) preparing a mixture of two or more (e.g., all) of the immunogenic conjugates and combining the mixture with an aluminum salt adjuvant. The adjuvant may be an aluminum phosphate adjuvant.

[0032] In another embodiment, a modified CRM197 carrier polypeptide is provided, comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue. That is, for example, Arg-192 and / or Arg-193 of SEQ ID NO: 1 can be deleted or substituted with a different amino acid. The nnAA residue can be introduced by substitution and / or insertion of an amino acid residue in SEQ ID NO: 1. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., of saccharide antigens) via the nnAA residues therein.

[0033] In another embodiment, a modified CRM197 carrier polypeptide is provided, the carrier polypeptide (i) having at least 80% sequence identity to SEQ ID NO: 1; and (ii) comprising an amino acid sequence including 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 ;Glu-240;Glu-248;Glu-249;Glu-256;Glu-259;Glu-292;Glu-362;Gln-252;Gln-287;Lys-212;Lys-218;Lys- 221;Lys-229;Lys-236;Lys-264;Lys-299;Lys-385;Lys-456;Lys-474;Lys-498;Lys-516;Lys-522;Lys-534; Arg-377;Arg-407;Arg-455;Arg-460;Arg-462;Arg-472;Arg-493;Ser-198;Ser-200;Ser-231;Ser-233;Ser- 239; Ser-261; Ser-374; Ser-381; Ser-297; Ser-397; Ser-451; Ser-475; Ser-494; Ser-495; Ser-496; Ser-501; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and / or Thr-517. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., of saccharide antigens) via the nnAA residues therein.

[0034] In another embodiment, a modified CRM197 carrier polypeptide is provided, the carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) includes an nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-211; A sp-295;Asp-352;Asp-392;Asp-465;Asp-467;Asp-507;Asp-519;Asn-296;Asn-359;Asn-399;Asn-481;Asn-486; Asn-502;Asn-524;Glu-240;Glu-248;Glu-249;Glu-256;Glu-259;Glu-292;Glu-362;Gln-252;Gln-287;Lys-212; Lys-218;Lys-221;Lys-229;Lys-236;Lys-264;Lys-299;Lys-385;Lys-456;Lys-474;Lys-498;Lys-516;Lys-522 ;Lys-534;Arg-377;Arg-407;Arg-455;Arg-460;Arg-462;Arg-472;Arg-493;Ser-198;Ser-200;Ser-231;Ser-23 3; 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., of saccharide antigens) via the nnAA residues therein.

[0035] In another embodiment, there is provided an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide comprises the amino acid sequence of SEQ ID NO: 4; and (ii) the saccharide antigen is covalently linked to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. There is also provided a pharmaceutical composition comprising two or more different immunogenic conjugates, each comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide in each conjugate comprises the amino acid sequence of SEQ ID NO: 4, and (ii) the saccharide antigen in each conjugate is covalently linked to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4.

[0036] In another embodiment, there is provided a syringe containing a pharmaceutical composition comprising two or more different immunogenic conjugates, each comprising a carrier polypeptide and a pneumococcal saccharide antigen, wherein the syringe is a non-siliconized syringe. Ideally, the pharmaceutical composition in the non-siliconized syringe comprises 13 or more different pneumococcal conjugates, and the carrier polypeptide may comprise an nnAA, but may alternatively be, for example, CRM197. Further details of the non-siliconized syringe are provided below. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 shows the geometric mean antibody titers for each of the 32 depicted serotypes in a 32-valent vaccine of the invention against a polysaccharide / alum formulation and Prevnar-13™, as described in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0038] Various details of methods, compositions and techniques for producing conjugated antigens are disclosed in International Patent Application WO 2018 / 126229, the entire contents of which are incorporated herein by reference.

[0039] immunogenic complex This invention relates generally to immunogenic conjugates. These conjugates comprise a carrier polypeptide covalently linked to an antigen. This linkage can convert a T-cell-independent immunogen (e.g., a saccharide) into a T-cell-dependent immunogen, thereby eliciting an enhanced immune response, particularly in children. As used herein, a conjugate comprises a covalent bond formed between the antigen and a non-natural amino acid ("nnAA") residue within the carrier polypeptide. These nnAA residues can provide a functional group that facilitates reaction with the antigen of interest.

[0040] Typically, one carrier polypeptide will be conjugated to multiple antigen molecules. The antigen can have one linking group (e.g., the reducing end of a sugar) per molecule that is conjugated to a carrier polypeptide, or multiple linking groups (e.g., multiple aldehyde or cyanate ester groups). When an antigen molecule has multiple linking groups, this generally leads to the formation of a high molecular weight crosslinked or lattice complex involving binding between multiple carrier polypeptides via the antigen. Crosslinked complexes are preferred herein (particularly for Streptococcus pneumoniae), and therefore antigens with multiple linking groups are also preferred.

[0041] The covalent bond is formed between the antigen and an nnAA residue in the carrier polypeptide. Preferably, the antigen is not bound to a lysine residue in the carrier polypeptide, and more preferably, the antigen is not bound to a natural amino acid residue in the carrier polypeptide.

[0042] Useful carrier polypeptides contain T cell epitopes. A variety of such carrier polypeptides are known in the art, and approved vaccines include diphtheria toxoid (a chemically engineered toxin from Corynebacterium diphtheriae; "Dt"), tetanus toxoid (a chemically engineered tetanospasmin toxin from Clostridium tetani; "Tt"), protein D ("PD" or "HiD") from Haemophilus influenzae, the outer membrane protein complex ("OMPC") of serogroup B strains of Neisseria meningitidis, and CRM197 mutant C. diphtheriae toxin.

[0043] A preferred carrier polypeptide on which the carrier of the present invention is based is CRM197. CRM197 is well known in the art (see, for example, Broker et al. 2011 Biologicals 39:195-204) and has the following amino acid sequence (SEQ ID NO: 1), where the underlined residue (Glu-52) differs from the native diphtheria toxin, whereby the substitution of Gly→Glu leads to the loss of toxic enzymatic activity in the protein: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK EFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKR GQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETA DNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESG HDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS.

[0044] The present invention does not use native CRM197. Instead of using CRM197 comprising SEQ ID NO: 1, it uses modified amino acid sequences that contain at least one nnAA. These modified CRM197 carrier polypeptides are described in more detail below.

[0045] In addition to CRM197, other detoxified mutant forms of diphtheria toxin can be used. For example, the non-toxic K51E / E148K double mutant can also be used as the carrier polypeptide in the conjugate (Pecetta et al. 2016 Vaccine 34:1405-11), and nnAA residues can be incorporated into the sequence of this double mutant in a similar manner as in CRM197.

[0046] Another carrier polypeptide of interest is PD from H. influenzae, which naturally has the following amino acid sequence (SEQ ID NO:5): CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK.

[0047] Instead of using natural PD, a modified amino acid sequence is used, which contains at least one nnAA. For example, one or more Lys residues in SEQ ID NO: 5 can be substituted with nnAA. Since there are 36 Lys residues in SEQ ID NO: 5, some can be substituted with nnAA and then used for binding. Prediction and recognition of T cell epitopes for PD has been reported by Hua et al. (2016) Clin Vaccine Immunol 23:155-61.

[0048] More generally, any polypeptide containing a T cell epitope can be used as a carrier polypeptide. 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 to the antigen or hapten bound to it (see, e.g., Costantino et al. 2011, Expert Opin Drug Discov 6:1045-66). Micoli et al. (2018) Molecules 23, 1451 provides an overview of various carrier polypeptides and their selection criteria. Tontini et al. (2016) Vaccine 34:4235-42 discuss the preclinical testing of 28 carrier polypeptides, including testing for their ability to induce antibodies against saccharide antigens. Polyepitope carrier polypeptides containing diverse, broadly reactive (i.e., immunogenic in the context of most human MHC class II molecules) human CD4+ T cell epitopes from various pathogen-derived antigens have been designed, e.g., N19 and other polypeptides disclosed in Falugi et al. (2001) Eur J Immunol 31:3816-24, Baraldo et al. (2004) Infect Immun 72:4884-7, and U.S. Patent Nos. 6,855,321 and 7,867,498. The ability to design these polypepitope carriers demonstrates the ability of those skilled in the art to identify suitable T cell epitopes from diverse sources and use them to design effective carrier polypeptides. See also U.S. Patent Application No. 2016-0101187. T cell epitopes present in known carriers (e.g., Tt, PD, CRM197) can be used. For example, various detoxified bacterial toxins, such as Tt, Dt, P. aeruginosa exotoxin, and C. difficile A and B toxins, have been successfully used as carriers.Many different carrier polypeptides have been used in conjunction with pneumococcal saccharides, such as CRM197 in Prevnar™, PD, Tt, and Dt in Synflorix™, and various peptides in Velasco et al. (1995) Infect Immun 63:961-8. The present invention allows for the use of any of these numerous carrier polypeptides, modified to contain at least one nnAA, to enhance the immunogenicity of an antigen of interest.

[0049] Carrier polypeptides containing nnAAs for use in the present invention can generally be prepared using the techniques disclosed in Section 6 of WO 2018 / 126229 ("Carrier Protein Production Methods"). Preferred carriers contain an nnAA in addition to at least one T-cell epitope of the carrier. If the T cell epitope region for a particular carrier is unknown, those skilled in the art can identify the epitope using standard techniques, including empirical and / or predictive approaches, see, for example, Reece et al. (1993) IJ Immunol 151:6175-84; Beissbarth et al. (2005) Bioinformatics 21 Suppl 1:i29-37; Maciel Jr et al. (2008) Virol 378:105-17; Fridman et al. (2012) Oncoimmunol 1:1258-70. It can also be confirmed that any particular modification of the sequence of the carrier polypeptide does not eliminate the desired T cell response to the bound antigen, such as the saccharide herein. A preferred group of carriers does not contain any modifications, including nnAA insertions or substitutions, within the T cell epitope. Particularly preferred carriers contain at least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs. Particularly preferred carriers can also have up to 10, 9, 8, 7, or 6 nnAAs. Particularly preferred ranges of nnAAs in the carrier polypeptide are 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 4-10, 4-9, 4-8, 4-7, and 4-6 nnAAs.

[0050] Various antigens can be included in the immunogenic conjugates used herein. Typically, the antigen is a saccharide. The term "saccharide" includes polysaccharides having 50 or more repeating units and oligosaccharides having fewer than 50 repeating units. Typically, the polysaccharide is from about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about 2,000 (or more) repeating units, and optionally from about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 repeating units to about 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 repeating units. Oligosaccharides are typically from about 6, 7, 8, 9 or 10 repeating units to about 15, 20, 25, 30 or 35 to about 40 or 45 repeating units.

[0051] Useful saccharides to incorporate into immunogenic conjugates include those present in bacteria. These may be non-capsular saccharides (such as exopolysaccharides, e.g., S. aureus exopolysaccharide), but are preferably bacterial capsular saccharides.

[0052] Bacterial capsular saccharides are high molecular weight saccharides present in the capsule of Gram-positive or Gram-negative bacteria and can be used as vaccine antigens. Such capsular saccharides are typically prepared from whole cell lysates or culture supernatants of the corresponding bacteria through processes involving diafiltration, protein removal, ethanol precipitation, nucleic acid removal and freeze-drying. The bacterial saccharides used in the invention may be intact, as present in the bacteria, or may be fragments obtained from the intact saccharide, for example obtained by hydrolysis of saccharides purified from the bacteria.

[0053] Saccharide antigens of particular interest include, but are not limited to: - Capsular saccharides of Streptococcus pneumoniae (S. pneumoniae) Further details of pneumococcal capsular saccharides useful as antigens for practicing the present invention are given below. - Sugars of group A hemolytic streptococcus (Streptococcus pyogenes) The antigen may be a saccharide from Group A Streptococcus (S. pyogenes). In one embodiment, the antigen is the capsular saccharide of Group A Streptococcus (S. pyogenes), which is made up of the high molecular weight polymer hyaluronic acid, the repeating unit of which has the structure: [→4)-β-D-GlcUAp-(1→3)-β-D-GlcpNAc-(→] This appears to be invariant between serotypes of S. pyogenes. In another embodiment, the antigen is a non-capsular saccharide from group A streptococcus (S. pyogenes), such as group A-strep cell wall saccharide, which comprises a backbone of poly-L-rhamnopyranosyl units linked by alternating α-L-(1→3) and α-L-(1→2) linkages, with N-acetyl-β-D-glucosamine residues attached to the 3-position of the rhamnose backbone. - Capsular saccharides of group B hemolytic streptococcus (Streptococcus agalactiae) The antigen may be a capsular saccharide from S. agalactiae (Group B Streptococcus or GBS). There are at least 10 GBS serotypes with different capsular saccharide repeating units (Ia, Ib, II-IX), but only a few serotypes usually cause disease. These include serotypes Ia, Ib, II, III and V, and conjugates of capsular saccharides from these serotypes can be prepared. - Capsular saccharides of Haemophilus influenzae The antigen may be a capsular saccharide from H. influenzae. There are at least six serotypes of H. influenzae (a to f) with different capsular saccharide chemical structures. However, only types a and b are considered to be "highly virulent" strains and therefore the preferred type of capsular saccharide of H. influenzae for use in the present invention is type b (Hib). - Capsular saccharides of Neisseria meningitidisThe antigen may be a capsular saccharide from N. meningitidis. There are at least 13 serogroups of N. meningitidis (serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z and Z') with different capsular saccharide chemical structures, but only six (A, B, C, W-135, X, Y) are considered to be lethal. Advantageously, the saccharide antigen is from any of serogroups A, C, W135, X or Y. - Capsular saccharides of Porphyromonas gingivalis The antigen may be a capsular saccharide from one of the six serotypes K1, K2, K3, K4, K5 and K6 of P. gingivalis. - Capsular saccharides of Salmonella typhi The antigen can be the Vi saccharide. Vi is the capsular saccharide of Salmonella typhi (the typhi serovar of S. enterica). The Vi saccharide is a linear homopolymer of hexosaminuronic acid, α1,4-N-acetylgalactosaminouronic acid, that is 60-90% acetylated at the C-3 position. - Staphylococcus aureus sugars The antigen may be a saccharide from S. aureus. The saccharide may be an exopolysaccharide of S. aureus, which is poly-N-acetylglucosamine (PNAG), or it may be a capsular saccharide of S. aureus, which may be, for example, serotype 5, serotype 8, or serotype 336. - Surface saccharides of Clostridium difficile The antigen can be a surface glycan from C. difficile, such as PS-I or PS-II. - Glucan The antigen can be a glucan containing β-1,3- and / or β-1,6-linkages, which can be useful in generating an antifungal immune response, for example, against Candida albicans.

[0054] Further details of these saccharide antigens can be found in WO 2018 / 126229.

[0055] Antigens often do not naturally contain functional groups that are suitable or ideal for conjugation. Therefore, the antigen may need to be functionalized prior to its conjugation to an nnAA. Further details of such functionalization are provided below.

[0056] Pneumococcal capsular saccharides A preferred antigen for use in the present invention is a capsular saccharide from Streptococcus pneumoniae. S. pneumoniae is a gram-positive, encapsulated bacterium that can cause pneumonia, bacteremia, and meningitis. There are at least 90 distinct documented serotypes of S. pneumoniae that carry capsular saccharides with serotype-specific repeating unit structures (see, e.g., Kalin, M. Thorax 1998;53:159-162). As will be appreciated by those skilled in the art, it has been proposed that serotype 20 of S. pneumoniae is actually made up of two closely related serotypes whose capsular polysaccharides are largely cross-protective (Calix et al. 2012 J Biol Chem 287:27885-94). That is, as will be further appreciated by those skilled in the art, serotype 20 refers to a saccharide that would previously have been classified in the art as serotype 20, and thus, as disclosed by Calix et al., may be structurally either 20A or 20B (from strains that would previously have been classified in the art as serotype 20, but which may genotypically be either 20A or 20B). For example, the strain used to produce the serotype 20 polysaccharide in Pneumovax™ (Merck & Co.) is considered herein to be serotype 20A. In some instances, 20A may be preferred. In other instances, 20B may be preferred. Prevalence in the target population may be the basis for selecting between these serotypes. Nevertheless, because strains classified as 20, 20A, and 20B are serologically similar, the choice between strains may not be critical, as they are largely cross-protective in vaccines.

[0057] The antigens used in the present invention include S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11 A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 2 The capsular saccharide may be from any of 0, 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 J Clin Microbiol 1995;33:2759-2762). However, because only a subset of these serotypes usually cause clinically significant bacterial infections, the antigen could be a capsular saccharide from any of S. 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. Because serotypes 6C, 7C, 15A, 15C, 16F, 20A, 20B, 23A, 23B, 24B, 31, 34, 35B, 35F, 37, and 38 have also become of clinical concern, the antigen could be a capsular saccharide from one of these S. pneumoniae serotypes.

[0058] Where the invention uses conjugates from different pneumococcal serotypes, it preferably comprises saccharides from at least 14 different S. pneumoniae serotypes (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). Where the composition comprises more than 14 serotypes, it preferably comprises the 13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. In addition to these 13 S. pneumoniae serotypes, it is preferred that the composition also comprises one or more of serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20 (alternatively 20A or 20B), 22F and / or 33F. Alternatively, in addition to the 13 serotypes listed above, the composition preferably includes one or more of S. 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 each of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and may also include serotype 8. Useful combinations of 20 or more (e.g., 21 or more) S. pneumoniae serotypes include 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 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.

[0059] The structures of the repeating units of the capsular saccharides of common pneumococcal serotypes are described by Jones et al. (Jones C et al. An Acad Bras Cienc. 2005 Jun;77(2):293-324): Type 1 [→3)-D-AAT-α-Galp-(1→4)-α-D-GalpA(2 / 3OAc)-(1→3)-α-D-GalpA-(1→] Type 2 [→4)-β-D-Glcp-(1→3)-[α-D-GlcpA-(1→6)-α-D-Glcp-(1→2)]-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)β-L-Rhap-(1→] Type 3 [→3)-β-D-GlcA-(1→4)-β-D-Glcp-(1→] Type 4 [→3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp2,3(S)Py-(1→] Type 5 [→4)-β-D-Glcp-(1→4)-[α-L-PnepNAc-(​​​​​​​​​​​​​​​​​​​[→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→] 18C type [→4)-β-D-Glcp-(1→4)-[α-D-Glcp(6OAc)(1→2)][Gro-(1→P→3)]-β-D-Galp-(1→4)-α-D-Glcp-(1→3)-β-L-Rhap-(1→] 19F type [→4)-β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap-(1→P→] 23F type [→4)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→]

[0060] A more extensive discussion of these sugars can be found in Geno et al. (2015) Clin. Microbiol. Rev. 28:871-99, which lists the structures of the 97 known serotypes in Table 1. The table also discloses the percentage of sugar residues that are acetylated when acetylation is less than complete.

[0061] Capsular saccharides may be O-acetylated. In some embodiments, capsular saccharides 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 comprise saccharides that are O-acetylated to a degree of 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 75 to 100%, 80 to 100%, 90 to 100%, 50 to 90%, 60 to 90%, 70 to 90% or 80 to 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 saccharides can be determined by proton NMR (see, e.g., Lemercinier & Jones (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247). Typically, saccharides used to prepare conjugates will retain at least 50% (e.g., 75% or 100%) of the O-acetylation level found in the starting capsular saccharide purified from the bacterium.

[0062] Capsular saccharides of S. pneumoniae may be obtained directly from the bacteria using isolation methods known to those skilled in the art (see, for example, the methods disclosed in US Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498, and WO 2008 / 118752). Alternatively, they may be obtained from commercial sources (e.g., ATCC).

[0063] Advantageously, pneumococcal capsular saccharide antigens for use in the present invention may have a molecular weight of 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 between 100 kDa and 2,000 kDa; 100 kDa and 1,750 kDa; 100 kDa and 1,500 kDa; 100 kDa and 1,250 kDa; 100 kDa and 1,000 kDa; 100 kDa and 750 kDa; 100 kDa and 500 kDa; 200 kDa and 4,000 kDa; 200 kDa and 3,500 kDa; 200 kDa and 200 kDa. The molecular weight may be from 200 kDa to 3,000 kDa; from 200 kDa to 2,500 kDa; from 200 kDa to 2,000 kDa; from 200 kDa to 2,000 kDa; from 200 kDa to 1,750 kDa; from 200 kDa to 1,500 kDa; from 200 kDa to 1,250 kDa; from 200 kDa to 1,000 kDa; from 200 kDa to 750 kDa; or from 200 kDa to 500 kDa. Further details and guidance regarding molecular weights are available in U.S. Serial No. 62 / 693,978, previously incorporated by reference herein.

[0064] Capsular saccharides may be chemically modified relative to naturally occurring capsular saccharides. For example, saccharides may be de-O-acetylated (partially or fully), de-N-acetylated (partially or fully), N-propionated (partially or fully), etc. De-acetylation may occur before, during or after activation, before, during or after derivatization, or before, during or after conjugation, but typically occurs before conjugation.

[0065] Some embodiments of the invention involve the use of two or more different conjugates. In the context of pneumococcal capsular saccharide conjugates, this means that each "different" conjugate has a saccharide from a different pneumococcal serotype (when using a single type of carrier polypeptide per conjugate).

[0066] Multivalent complexes Preferred compositions of the present invention involve the use of two or more different conjugates, e.g., within a single pharmaceutical composition. These embodiments are also referred to as multivalent. When any two conjugates are described as "different," i.e., resulting in different valencies in a "multivalent" composition, this refers to the differences in the carrier polypeptide and antigen combinations in the two conjugates. For example, when a single type of modified CRM197 (e.g., SEQ ID NO: 4) is conjugated to capsular saccharides from a single serotype of Streptococcus pneumoniae, the reaction product will contain many different types of molecules (different molecular weights, different linkage patterns within each molecule, etc.), but will be considered herein as a single conjugate. Those skilled in the art are familiar with this heterogeneity at the molecular level, and similarly, the antigen-carrier combination of a particular conjugate, along with other average properties (e.g., molecular weight) within the conjugate composition, defines the individual conjugates of a vaccine. Two "different" conjugates will have different carrier polypeptides (i.e., different amino acid sequences) and / or different antigens (i.e., different antigenic structures).

[0067] For example, capsular saccharide antigens may be purified from two different serotypes of Streptococcus pneumoniae. These two different capsular saccharides may be separately conjugated to a carrier polypeptide (which may be the same or different) to give two different conjugates. That is, in the case of bacterial capsular saccharide conjugates, the difference between two "different" conjugates is typically that one contains a capsular saccharide from a first serotype or serogroup of a bacterial species, while the other contains a capsular saccharide from a second serotype or serogroup of that bacterial species, for example capsular saccharides from different serotypes of Streptococcus pneumoniae, or capsular saccharides from different serogroups of Neisseria meningitidis. Two conjugates will also be "different" if they contain antigenically distinct capsular saccharides from more than one bacterial species, for example a Hib saccharide conjugate and a meningococcal saccharide conjugate.

[0068] Preferred multivalent compositions of the invention comprise n different immunogenic saccharide conjugates, where the saccharide antigen in each of the n immunogenic conjugates is different from the saccharide antigen in the other n-1 immunogenic conjugates. For example, if the composition comprises antigens from a single bacterial species, capsular saccharides from n different serotypes or serogroups of that species may be present.

[0069] This nomenclature, referring to "different" conjugates, is used in the field of conjugate vaccines. For example, Glesby et al. (2015) J Infect Dis 212:18-27 refer to the Prevnar™ PCV13 vaccine as containing "13 different conjugates" because it contains saccharide antigens from 13 different pneumococcal serotypes separately conjugated to CRM197. Similarly, European Patent Application Publication No. 2932979 (EP-A-2932979) refers to "immunogenic compositions comprising 13 different polysaccharide-protein conjugates."

[0070] That is, the PCV7 Prevnar™ vaccine has 7 different conjugates, the PCV13 Prevnar™ vaccine has 13 different conjugates, the Menveo™ vaccine has 4 different conjugates, the Menactra™ vaccine has 4 different conjugates, the Nimenrix™ vaccine has 4 different conjugates, the Menitorix™ vaccine has 2 different conjugates, the Menhibrix™ vaccine has 3 different conjugates, the Synflorix™ vaccine has 10 different conjugates, etc.

[0071] Preferably, multivalent compositions of pneumococcal conjugates comprise 13 or more different conjugates, for example 14, 15, 20, 21, 24, 25 or more. Suitable choices of serotypes for these greater than 13-valent compositions are discussed above.

[0072] For vaccines with high numbers of vaccines (e.g., containing more than 13 different conjugates), it may be preferable to use multiple carrier polypeptides to reduce the possibility of carrier suppression (e.g., WO 98 / 51339 and WO 2011 / 110241). For example, in a multivalent vaccine containing n different conjugates, a first carrier polypeptide would be coupled to ny different antigens (e.g., capsular saccharides from different bacterial serotypes or serogroups), and a second carrier polypeptide would be coupled to the remaining y antigens. In a similar manner, three, four, or more carriers could be used with n antigens divided among them. When multiple carriers are used, at least a first carrier is an nnAA-containing carrier polypeptide according to the invention. In a preferred embodiment, at least a first carrier and a second carrier are nnAA-containing carrier polypeptides according to the invention.

[0073] Unnatural amino acids As described above, a conjugate, as used herein, comprises a covalent bond between an antigen and a functional group within an nnAA residue in a carrier polypeptide. The side chain of the nnAA residue can provide a reactive functional group that is useful for attaching the antigen to a discrete site on the carrier polypeptide.

[0074] Generally speaking, an nnAA can be any amino acid that can be incorporated into a polypeptide during translation but that is not one of the 20 common amino acids. Advantageously, an nnAA can be incorporated into a polypeptide by converting a tRNA molecule such that its codon incorporates the nnAA rather than the natural cognate amino acid. One technique for achieving this involves the use of a "suppression codon," i.e., a nucleotide triplet that is introduced into a coding sequence at a desired position to be recognized by a specific tRNA that is capable of recognizing a natural stop codon (e.g., an amber, ochre, or opal stop codon), resulting in the incorporation of an nnAA (thereby suppressing the natural stop codon), while still allowing translation to continue.

[0075] The nnAA residue can be any of the nnAA residues described herein or others identified as compatible with cell-based or cell-free protein synthesis (see, e.g., Schultz et al. Annu Rev Biochem. 2010; 79:413-44, especially pp. 418-420; and Chin et al. Annu Rev Biochem. 2014; 83:5.1-5.30, which are incorporated herein by reference). Ideally, the nnAA does not naturally occur in cells by modification of one of the 20 common amino acids (e.g., pyrrolysine, selenocysteine, phosphotyrosine, formylmethionine, etc.).

[0076] In particular, preferred nnAAs used herein are those that can be incorporated during translation (in a cellular or cell-free system) and have side chains that provide functional groups not present in the side chains of any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into polypeptides are known, see, for example, Young & Schultz (2010) J Biol Chem 285:11039-44, Maza et al. (2015) Bioconjugate Chem. 26:1884-9, and Zimmerman et al. (2014) Bioconjugate Chem. 25:351-61. International Publication No. 2018 / 126229 provides detailed information on how nnAA residues can be incorporated into carrier polypeptides, for example, using cell-free expression mixtures, nnAA-specific orthogonal tRNA / aminoacyl-tRNA synthetase pairs, suppression codons, etc. See also U.S. Patent Application No. 2017 / 0267637.

[0077] The nnAA may contain chemical groups suitable for "click" chemistry reactions with corresponding groups on the antigen of interest. Chemical groups suitable for "click" chemistry include azide groups (-N3), alkyne groups (-C≡C-), alkene groups (-C=C-), and 1,2,4,5-tetrazine groups. [ka] and phosphine groups (e.g., -P(Ph)2).

[0078] The nnAA can be any of 2-amino-3-(4-azidophenyl)propanoic acid (para-azido-L-phenylalanine, or pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (para-azidomethyl-L-phenylalanine, or pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, or 2-amino-5-azidopentanoic acid.

[0079] The most preferred nnAA for use herein is pAMF: [ka] pAMF provides highly favorable reaction rates for generating conjugates (e.g., much faster than pAF when reacting with alkyne-containing saccharide antigens in the SPAAC method).

[0080] nnAA can be a 2,3-disubstituted propanoic acid bearing: an amino substituent at the 2-position; and an azide-containing, 1,2,4,5-tetrazinyl-, or ethynyl-containing substituent at the 3-position. Preferably, the substituent at the 3-position is an azide-containing substituent, particularly an azide-containing substituent containing a terminal azide group attached to the 3-position carbon atom via a linking group. For example, the linking group can include an optionally substituted, optionally heteroatom-containing arylene moiety. 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.

[0081] The nnAA can have the structure of the following general formula XII: [ka] (XII) wherein Ar comprises a 5- or 6-membered aromatic ring which may contain at least one heteroatom; W 5 is C1-C 10 selected from alkylene, -NH-, -O-, and -S-; Q1 is zero or 1; and W 6is selected from azide, 1,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl. In some embodiments, Ar does not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene group (i.e., Ar is -CH-). 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 particularly preferred embodiments, Q is 1 and W is 0. 5 is a lower alkylene; and W 6 is an azide.

[0082] The nnAA can be an azide-containing nnAA, for example an nnAA of the following general formula I: [ka] wherein D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-; each of W1, W2, and W3 is independently a single bond or lower alkylene; each X1 is independently -NH-, -O-, or -S-; each Y1 is independently a single bond, -NH-, or -O-; each Y2 is independently a single bond, -NH-, -O-, or N- or C-bonded pyrrolidinylene; Ar is [ka] and; and one of Z1, Z2 and Z3 is -N-, and the others of Z1, Z2 and Z3 are independently -CH-.

[0083] In other embodiments, the nnAA has the following general formula II: [ka] Where W4 is C1-C 10 It is alkylene.

[0084] The preparation of azide-containing amino acids according to general formulas I and II can be found, for example, in U.S. Patent Application Publication No. 2014-0066598A1 to Stafford et al., particularly paragraphs

[0331] to

[0333] , which are incorporated by reference. The method involves the substitution of a hydroxyl group for a chloride in a corresponding aryl amino acid derivative with thionyl chloride, followed by nucleophilic substitution of the chloride with an azide. Suitable aryl side chain containing amino acids are also commercially available.

[0085] The nnAA can be a 1,2,4,5-tetrazine-containing nnAA, for example, of the following general formula III: [ka] In the formula, Ar is [ka] V is a single bond, lower alkylene, or -W1-W2-; one of W1 and W2 is absent or lower alkylene, and the other is -NH-, -O-, or -S-; each one of Z1, Z2, and Z3 is independently -CH- or -N-; and X1 is independently -NH-, -O-, or -S-; and R is lower alkyl.

[0086] The preparation of 1,2,4,5-tetrazine-containing amino acids according to general formula III can be found, for example, in U.S. Patent Application Publication No. 2016 / 0251336 to Yang et al., particularly paragraphs

[0341] to

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

[0087] The nnAA can be an alkyne-containing nnAA. In one embodiment, it is a propargyl group. Various propargyl-containing amino acids, including their syntheses, can be found in Beatty et al. Angew. Chem. Int. Ed. 2006, 45, 7364-7; Beatty et al. J. Am. Chem. Soc. 2005(127):14150-1; Nguyen et al. JACS 2009(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.

[0088] As used herein, nnAAs are generally α-amino acids with an asymmetric center at the α-carbon, and are preferably L-stereoisomers.

[0089] Polypeptide carriers used in the present invention contain at least one nnAA residue. Preferably, the carrier polypeptide should contain multiple nnAA residues, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues (or even more). Carrier polypeptides with fewer than 10 nnAA residues are preferred. That is, the polypeptide may contain 2 to 9 nnAA residues, preferably 4 to 6 nnAA residues.

[0090] When a 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 for each nnAA simultaneously. If it is desired to conjugate two different antigens to one carrier molecule, this can be achieved by using different nnAA species within a single carrier and conjugating each antigen with a different nnAA, but conjugating with a single species of nnAA within a carrier is preferred. Furthermore, when multiple different conjugates are used (e.g., different pneumococcal serotypes), it may be preferable for each conjugate to contain the same single species of nnAA. Also, when a composition contains multiple different conjugates (e.g., different pneumococcal serotypes), it may be preferable for each conjugate to contain the same carrier polypeptide.

[0091] An nnAA can be incorporated into a carrier polypeptide by substitution or insertion (or by C- or N-terminal extension). In one embodiment, an nnAA residue is incorporated by substitution. Advantageously, a lysine residue in a native polypeptide can be substituted with an nnAA. For example, in CRM197, substitutions can be made at one or more of positions K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522, and K526 in SEQ ID NO: 1 or 2. Substitutions of an nnAA (e.g., pAMF) at each of K33, K212, K244, K264, K385, and K526 (and in one embodiment, no other positions) are preferred.

[0092] However, substitutions incorporating nnAAs are not limited to lysine positions, and other amino acids can also be substituted with nnAAs, for example, Phe, Asp, Asn, Glu, Gln, Arg, Ser, and / or Thr.

[0093] The nnAA in the carrier polypeptide is ideally a surface-accessible residue, which can be assessed using the 3D structure of the polypeptide or by making extensive substitutions of natural amino acids for the nnAA followed by binding studies to assess the availability of each site.

[0094] To preserve the function of the carrier polypeptide, it is preferable not to incorporate nnAAs within T cell activation epitopes of the carrier polypeptide. The use of nnAAs allows for selective placement of the binding site, thereby preventing T cell activation epitopes of the carrier polypeptide from becoming sites for antigen binding. As described above, these epitopes are easily identifiable. For example, in studies of CRM197 by Raju et al., Bixler et al., Leonard et al., and Pillai et al. (e.g., Eur J Immunol. 1995 Dec;25(12):3207-14, WO 89 / 06974), various T-cell epitopes have been identified, for example, within residues P271-D290, V321-G383, and Q411-I457. Thus, it is preferable to avoid introducing nnAAs within these regions of SEQ ID NO: 1.

[0095] join Conjugation involves the formation of a covalent bond between the nnAA residue and the antigen. This requires reactive functional groups on both the nnAA and the antigen. Generally, an nnAA will be selected for the carrier polypeptide because the nnAA already has a suitable functional group (e.g., the azide group of pAMF), whereas antigens often do not naturally contain a functional group that is suitable or ideal for conjugation. That is, the antigen may need to be functionalized prior to its conjugation to the nnAA.

[0096] Detailed technical information on conjugation can be found in Bioconjugate Techniques (Greg T Hermanson, 3rd edition, 2013). WO 2018 / 126229 discloses in detail how antigens can be functionalized and then conjugated to nnAAs. As described above, useful nnAAs contain functional groups (e.g., azide groups) that are suitable for "click" chemistry reactions with functional groups on antigens. That is, functionalized antigens ideally contain groups suitable for such "click" reactions.

[0097] Thus, generally, conjugation is accomplished by a method comprising the following three steps: (a) activating the antigen; (b) optionally derivatizing the activated antigen (e.g., with a linker or nucleophilic group) to introduce reactive functional groups not normally present on the antigen; and (c) conjugating the antigen to a carrier polypeptide via step (a) or, if present, via the groups introduced in step (b). In some embodiments, step (a) comprises an initial step of removing blocking groups on the antigen to make certain functional groups (e.g., hydroxyl, amine, thiol) more accessible to activation. Steps (a)-(c) can occur essentially simultaneously (e.g., when a reactive moiety such as N-hydroxysuccinimide is added to the antigen), while in other embodiments, two or more of steps (a)-(c) are interspersed, including optional purification steps between steps.

[0098] As noted above, since cross-linked conjugates are preferred, it is also preferable to introduce multiple reactive functional groups per antigen molecule. For example, multiple aldehyde or cyanate groups can be introduced during activation of the saccharide molecule. These groups can then be derivatized to introduce, for example, reactive cyclooctynes ​​that can then react with the azide groups in the nnAA.

[0099] Antigens can be activated using a variety of chemistries, including, but not limited to, periodate oxidation (e.g., oxidation of a hydroxyl group at an adjacent carbon atom to give a reactive aldehyde group), as disclosed in WO 2011 / 110531; cyanylation, for example, with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP); hydroxyl activation with 1,1'-carbonyldiimidazole (CDI) followed by nucleophilic addition; or unmasking of endogenous aldehydes (e.g., the reducing end of a saccharide).

[0100] Periodate oxidation and cyanylation with CDAP are two preferred activation techniques. Periodate oxidation has been shown to be particularly useful for activating pneumococcal serotypes 1, 2, 3, 7F, 8, 9N, and 11A. CDAP cyanylation has been shown to be particularly useful for activating pneumococcal serotypes 3, 7F, and 10A.

[0101] While activated antigens can be directly coupled to nnAAs, the activated groups are usually derivatized to introduce functional groups that exhibit favorable reactivity with the functional groups of nnAAs. For example, alkynyl groups can be introduced. Bifunctional reagents bearing amino and alkyne groups can react with aldehyde groups introduced into antigens (e.g., via reductive amination), thereby leaving pendant alkynes that can react with nnAAs. For example, bifunctional reagents containing amino and DBCO functional groups can be used.

[0102] In one embodiment, the nnAA reacts with an alkynyl group (e.g., a propargyl group) in the antigen. The alkyne group in the antigen is ideal for reaction with the azide group in the nnAA using reactions known in the art, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC), ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), or Huisgen's azide-alkyne 1,3-dipolar cycloaddition. The alkynyl group may have a molecular environment that enhances its reactivity; for example, it may be endocyclic. For example, an alkylene may be in a cyclooctyne ring (which may contain heteroatoms), such as a diaryl-strained cyclooctyne ring (e.g., DBCO). This reaction may be a [3 + 2] cycloaddition, referred to in the art as strain-promoted azide-alkyne cycloaddition (SPAAC). DIFO- and DBCO-based reagents are readily available for these reactions.

[0103] Alkyne-containing rings useful in SPAAC reactions include difluorinated cyclooctynes ​​(DIFOs) and dibenzocyclooctynes, which are available with pendant functional groups for conjugation to activated antigens (e.g., with pendant amino groups for conjugation to aldehydes or cyanide esters), using, for example, any of the following reagents: [ka]

[0104] The value of "n" in "PEGn" represents the number of oxyethylene repeating units. The value of n is in the range of 1 to 20, for example, 2 to 18, 3 to 16, or 4 to 14. That is, n can be, for example, 4, 5, 11, 12, or 13.

[0105] Other click chemistry reactions that can be used to conjugate antigens and nnAAs include, but are not limited to, tetrazine-alkene ligation and Staudinger ligation between phosphines and azides.

[0106] The complexes of the present invention can have a molecular weight of at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa or more. In some embodiments, the complexes have a molecular weight of about 750 kDa to about 5,000 kDa. In some embodiments, the complexes have a molecular weight of about 800 kDa to about 2,800 kDa. In some embodiments, the complexes have a molecular weight of about 850 kDa to about 2,800 kDa. In some embodiments, the complexes have a molecular weight of about 900 kDa to about 2,800 kDa. In some embodiments, the complexes have a molecular weight of about 950 kDa to about 2,800 kDa. In some embodiments, the complexes have a molecular weight of about 1,000 kDa to about 2,800 kDa. The molecular weight of the complex is calculated by size exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALS).

[0107] The conjugates of the present invention comprise an antigen (e.g., a saccharide) and a carrier polypeptide, and the weight ratio of these two components can be used as a parameter to define the conjugate. A higher antigen:carrier weight ratio for a saccharide-carrier conjugate allows for more saccharide antigen to be delivered with a smaller amount of carrier polypeptide. For pneumococcal conjugate vaccines, this ratio typically ranges from 0.3 to 3.0, but this can vary depending on the serotype and the conjugation chemistry (Annex 2: Recommendations for the production and control of pneumococcal conjugate vaccines; WHO Technical Report Series, No. 927, 2005). The commercially available vaccine, Prevnar-13™, has a ratio of 0.9. For compositions containing conjugates of multiple pneumococcal serotypes (e.g., 13 or more serotypes), the ratio for the complete composition will ideally be greater than 1.0 (i.e., excess weight of pneumococcal saccharide antigen), and preferably 1.5 or more (e.g., in the range of 1.5 to 3.0, or preferably 1.5 to 2.0).

[0108] Modified CRM197 carrier polypeptide As noted above, the carrier polypeptide of primary interest herein is a modified form of CRM197. As such, preferred carrier polypeptides for use in the present invention comprise an amino acid sequence that is at least 80% sequence identity (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%) to SEQ ID NO: 1. For example, a carrier polypeptide can comprise the amino acid sequence of SEQ ID NO: 1, except that up to 10 nnAAs are present, as noted above.

[0109] SEQ ID NO: 1 contains an Arg-Arg dipeptide sequence at positions 192-193. This sequence may be subject to proteolytic cleavage in some circumstances. If desired, this site can be modified to prevent cleavage and improve yield. That is, in some embodiments, the modified CRM197 carrier polypeptide used herein does not contain an Arg-Arg dipeptide sequence. For example, Arg-192 and / or Arg-193 of SEQ ID NO: 1 can be deleted or substituted with a different amino acid. Thus, a preferred carrier polypeptide comprises an amino acid sequence that (i) has at least 80% (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%) sequence identity to SEQ ID NO: 1, (ii) does not contain an Arg-Arg dipeptide sequence, and (iii) contains at least one nnAA residue (e.g., at least two, and preferably more, as described above).

[0110] One such amino acid sequence is SEQ ID NO:2 below, which differs from SEQ ID NO:1 by having an Arg→Asn substitution at position 193: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVR N SVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS.

[0111] Any embodiment described herein or in WO 2018 / 126229 that references SEQ ID NO: 1 can be implemented using SEQ ID NO: 2 instead.

[0112] That is, a carrier polypeptide is provided that comprises the amino acid sequence of SEQ ID NO:2, which has been modified to contain 1 to 10 (e.g., 3 to 9, or 2 to 8, or 2 to 6, or 3 to 6, or 4 to 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 six Lys-to-nnAA substitutions). Preferably, residue Asn-193 of SEQ ID NO:2 is not substituted with nnAA. This carrier polypeptide can be used to prepare immunogenic conjugates (e.g., of saccharide antigens) via the nnAA residues therein.

[0113] In some embodiments, these carrier polypeptides comprise amino acid sequences upstream and / or downstream of SEQ ID NO: 1 or 2. That is, for example, they can comprise a methionine residue upstream of the N-terminal amino acid residue of SEQ ID NO: 1 or 2. This methionine residue can be formylated. While no methionine residue is present at this position in wild-type CRM197, it can be included herein to initiate translation without requiring the entire native leader sequence (e.g., in a cell-free polypeptide synthesis system). In some embodiments, the carrier polypeptide (i) does not comprise any amino acid sequence upstream of the N-terminus of SEQ ID NO: 1 or 2, except for the optional methionine, and (ii) does not comprise any amino acids downstream of the C-terminus of SEQ ID NO: 1 or 2.

[0114] Preferably, at least one Lys residue in SEQ ID NO: 1 or 2 is substituted with an nnAA residue. Preferably, one or more residues in SEQ ID NO: 1 or 2 are substituted with an nnAA, and ideally, only one residue in SEQ ID NO: 1 is substituted with an nnAA, e.g., only the Lys residue is substituted. When more than one residue in SEQ ID NO: 1 is substituted with an nnAA, it is preferred that the same nnAA is used at each position, e.g., each substitution position is pAMF. As noted above, in some embodiments, residues other than Lys are substituted.

[0115] Carrier polypeptides comprising the amino acid sequence of SEQ ID NO: 1 or 2 with 2 to 9 substitutions with nnAA residues (e.g., Lys→nnAA substitution, preferably Lys→pAMF) are preferred, and ideally with 2 to 8, 2 to 6, 3 to 8, 3 to 6, 4 to 9, 4 to 8, or 4 to 6 nnAA substitutions, e.g., 4, 5, or 6 nnAA residues. This allows for a wider range of conjugation of antigen to the carrier than using a single nnAA, thereby increasing the antigen:carrier ratio while avoiding excessive disruption of the native sequence and structure, which may result in insolubility.

[0116] Structural studies of CRM197 reveal two general three-dimensional regions within SEQ ID NO: 1 or 2: the first region extends from the N-terminus to Asn-373, and the second region extends from Ser-374 to the C-terminus. The first of these regions roughly corresponds to the domains known as "C" and "T" (catalytic and transmembrane), and the second region roughly corresponds to the domain "R" (receptor-binding). Ideally, the carrier polypeptide contains at least one nnAA in the first region and at least one nnAA in the second region, e.g., at least two nnAAs in each region, or at least three nnAAs in each region. This allows the bound antigen to be spatially separated when bound to the carrier. A carrier containing three nnAAs in the first region and three nnAAs in the second region is useful.

[0117] The first region contains 27 Lys residues, and the second region contains 12 Lys residues, i.e., one or more (e.g., three) Lys residues within the N-terminal 374 amino acids and one or more (e.g., three) Lys residues within the C-terminal 162 amino acids of SEQ ID NO: 1 or 2 can be substituted with nnAA, for example, in pAMF.

[0118] A preferred embodiment of a CRM197-based nnAA-containing carrier has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, in which one or more of residues K24, K33, K37, K39, K212, K214, K227, K264, K385, K522, and K526 have been replaced with an nnAA (such as pAMF). One such sequence is SEQ ID NO: 3 below, in which each X represents an nnAA (preferably the same nnAA, such as pAMF): MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ XGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO: 3).

[0119] Another such sequence is SEQ ID NO: 4 below, in which each X represents an nnAA (preferably the same nnAA, such as in pAMF): MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO: 4).

[0120] SEQ ID NOs: 3 and 4 are very expressible in cell-free protein synthesis systems while retaining good solubility when combined with pneumococcal capsular saccharides to provide a good immunogenic response. SEQ ID NO: 4 lacks the natural Arg-Arg dipeptide.

[0121] A polypeptide consisting of SEQ ID NO: 4, wherein each X is pAMF, is another preferred carrier polypeptide for use in the present invention.

[0122] WO 2018 / 126229 describes several amino acid residues that are 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, 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;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;Ar g-493;Ser-198;Ser-200;Ser-231;Ser-233;Ser-239;Ser-261;Ser-374;Ser-381;Ser-29 7;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.

[0123] Also provided is a polypeptide comprising an amino acid sequence that (i) has at least 80% (e.g., ≧85%, ≧90%, ≧95%, ≧96%, ≧97%, or preferably ≧98%) sequence identity to SEQ ID NO:1, (ii) does not contain an Arg-Arg dipeptide sequence, and (iii) includes at least one nnAA residue, wherein the polypeptide has an N-terminal methionine and / or is in a monomeric form.

[0124] These CRM197-derived carrier polypeptides can be used in the same conjugation methods as CRM197 has been used in the prior art (see, e.g., Broker et al. 2011, supra; WO 2015 / 117093, etc.), but with the improvement of allowing site-specific conjugation via nnAA residues. They will generally be used in monomeric form rather than forming polypeptide multimers with other CRM197 or CRM197-derived subunits. Similarly, they 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.

[0125] Also provided are immunogenic conjugates comprising any of these various carrier polypeptides conjugated to a saccharide antigen via at least one of its nnAA residues. The carrier polypeptides are particularly useful for conjugating pneumococcal capsular saccharides via the nnAA residues therein. Immunogenic conjugates prepared in this manner can be combined to form multivalent compositions, as discussed elsewhere herein.

[0126] Namely, an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen is provided, wherein (i) the carrier polypeptide has the amino acid sequence of SEQ ID NO: 4, e.g., where each X is pAMF, and (ii) the saccharide antigen is covalently linked to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. Also provided is a multivalent pharmaceutical composition comprising two or more such immunogenic conjugates.

[0127] That is, a pharmaceutical composition is provided that includes a plurality of different conjugates (e.g., different pneumococcal serotypes), each of which includes a carrier polypeptide having the amino acid sequence of SEQ ID NO:4.

[0128] Also provided is an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide has the amino acid sequence of SEQ ID NO: 4, (ii) the saccharide antigen is covalently linked to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4, and (iii) the saccharide antigen is a capsular saccharide from any of 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 create multivalent pharmaceutical compositions of the invention.

[0129] Also provided are polynucleotides encoding the carrier polypeptides described herein. In another embodiment, the disclosure provides expression vectors comprising the polynucleotides. In another embodiment, the disclosure provides host cells comprising the expression vectors.

[0130] Adjuvants The pharmaceutical compositions of the present invention can include an aluminum salt adjuvant, which can enhance the immunogenicity of the conjugate within the pharmaceutical composition, and the conjugate within the composition can be adsorbed to the aluminum salt adjuvant.

[0131] Useful aluminum salt adjuvants include, but are not limited to, aluminum hydroxide adjuvants and aluminum phosphate adjuvants, which are described, for example, in Chapters 8 and 9 of Vaccine Design···(1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.

[0132] Adjuvants commonly known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide, which can be represented by the general formula AlO(OH), exhibits a high spectral response in infrared (IR) spectroscopy, particularly at 1070 cm -1 and the absorption band at 3090-3100 cm -1 It can be distinguished from other aluminum compounds, such as Al(OH)3, by the presence of a strong shoulder at half maximum (WHH) and a strong peak at 0.05°C (Powell & Newman, Chapter 9). The crystallinity of aluminum hydroxide adjuvants is reflected by the width of the diffraction band at half maximum (WHH), with particles with lower crystallinity exhibiting greater line broadening due to their smaller crystal size. As WHH increases, the surface area increases, and adjuvants with higher WHH values ​​have been shown to have greater antigen adsorption capacity. The morphology of the fibers (e.g., as seen in transmission electron micrographs) is typically that of needle-like particles, e.g., about 2 nm in diameter, for aluminum hydroxide adjuvants. The pI of aluminum hydroxide adjuvants is typically about 11, i.e., at physiological pH, the adjuvant itself has a positive surface charge. For aluminum hydroxide adjuvants, at pH 7.4, the Al +++ Adsorption capacities of 1.8 to 2.6 mg of protein per mg have been reported.

[0133] Adjuvants commonly known as "aluminum phosphate" are typically aluminum hydroxyphosphates, which often also contain small amounts of sulfate (i.e., aluminum hydroxyphosphate sulfate). They can be obtained by precipitation, and the reaction conditions and concentrations during precipitation affect the degree of substitution of phosphate for hydroxyl groups in the salt. Hydroxyphosphates generally have a PO4 / Al molar ratio of 0.3 to 1.2. Hydroxyphosphates can be distinguished from strict AlPO4 by the presence of hydroxyl groups. For example, -1 The presence of certain IR spectral bands (e.g., when heated to 200°C) suggests the presence of structural hydroxyl groups (Powell & Newman, Chapter 9).

[0134] Aluminum phosphate adjuvant PO4 / Al 3+ The molar ratio of aluminum phosphate to hydroxyphosphate will generally be 0.3 to 1.2, preferably 0.8 to 1.2, and more preferably 0.95±0.1. The aluminum phosphate will generally be amorphous, particularly the hydroxyphosphate. A typical adjuvant is 0.6 mg Al 3+ The aluminum phosphate adjuvant is amorphous aluminum hydroxyphosphate with a PO4 / Al molar ratio of 0.84 to 0.92, containing 0.01% / ml. The aluminum phosphate will generally be granular (e.g., plate-like morphology when viewed in transmission electron micrographs, with primary particles in the 50 nm range). Typical diameters of the particles range from 0.5 to 20 μm (e.g., about 5 to 10 μm) after any antigen adsorption. The aluminum phosphate adjuvant contains 0.01% Al at pH 7.4. +++ Adsorption capacities of 0.7 to 1.5 mg of protein per mg have been reported.

[0135] The point of zero charge (PZC) of aluminum phosphate is inversely related to the degree of substitution of phosphate for hydroxyl groups, and this degree of substitution can be varied depending on the reaction conditions and concentrations of reactants used to prepare the salt by precipitation. The PZC can also be varied by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer, such as a histidine buffer (making the PZC more basic). The aluminum phosphates used in accordance with the present invention will generally have a PZC of 4.0 to 7.0, more preferably 5.0 to 6.5, e.g., about 5.7.

[0136] The concentration of aluminum ions in the composition administered to a patient is preferably less than 2.5 mg / ml, e.g., ≦2 mg / ml, ≦1 mg / ml, etc. A preferred maximum concentration is ≦1.7 mg / ml. +++ The range may be 0.3 to 1 mg / ml or 0.3 to 0.5 mg / ml. A maximum of 0.85 mg / dose is preferred.

[0137] In solution, both aluminum phosphate and aluminum hydroxide adjuvants tend to form stable porous aggregates 1 to 10 μm in diameter. Compositions can include mixtures of both aluminum hydroxide and aluminum phosphate adjuvants.

[0138] When a composition contains multiple conjugates, each of which is adsorbed to an aluminum salt adjuvant, each conjugate can be adsorbed individually to the aluminum salt and then mixed, or they can be added sequentially to the aluminum salt, thereby forming a mixed conjugate composition. Mixtures of either approach can be used.

[0139] Pharmaceutical composition excipients Pharmaceutical compositions of the invention will generally include one or more pharmaceutically acceptable excipients. A comprehensive discussion of such excipients can be found in Handbook of Pharmaceutical Excipients (ed. Rowe et al.), 6th edition 2009.

[0140] Pharmaceutical compositions are preferably in aqueous form, particularly at the time of administration, although they can also be in dry form (e.g., as a lyophilisate) which can be converted into aqueous form for administration.

[0141] The pharmaceutical composition may contain a buffer or pH adjuster. The buffer may be selected from the group consisting of phosphate buffer, acetate buffer, histidine buffer, citrate buffer, succinate buffer, Tris buffer, HEPES buffer, etc. Buffer salts will typically be included in the range of 5 to 20 mM.

[0142] The pharmaceutical composition can include a saline solution, such as a sodium salt, for example, to control tonicity. Sodium chloride (NaCl) is typical, which may be present at 1 to 20 mg / ml, e.g., 10±2 mg / ml or 9 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, 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.

[0143] The pharmaceutical composition can include an organic acid, such as acetic acid or succinic acid, which can be part of a buffer system.

[0144] The pharmaceutical composition may include a sugar alcohol, such as mannitol or sorbitol. The pharmaceutical composition may include a sugar, such as sucrose or glucose.

[0145] The pharmaceutical composition may include 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), e.g., 0.01 to 0.03%. Polysorbate 80 is a preferred surfactant.

[0146] The pharmaceutical composition may include a preservative, such as thiomersal or 2-phenoxyethanol. Preferably, the composition should be substantially free of mercury materials (e.g., <10 μg / ml), e.g., free of thiomersal. Mercury-free compositions are more preferred. The inclusion of a preservative can be particularly useful when the composition includes aluminum salt adjuvants, as their insolubility means that the composition is generally a cloudy-appearing suspension that can prevent adverse effects on bacterial growth. Preservatives are also particularly useful when the composition is to be used multiple times, such as in a multi-use vial. However, pharmaceutical compositions can often be preservative-free.

[0147] The pharmaceutical composition may have an osmolality of 200 mOsm / kg to 400 mOsm / kg, for example, 240 to 360 mOsm / kg, or 290 to 310 mOsm / kg.

[0148] Pharmaceutical compositions typically have a pH of 5.0 to 9.5, for example 5.0 to 8.0 or 6.0 to 8.0.

[0149] Pharmaceutical compositions are preferably non-pyrogenic, eg containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose.

[0150] The pharmaceutical composition may have an osmolality of 200 to 400 mOsm / kg, for example 240 to 360 mOsm / kg, or 280 to 320 mOsm / kg.

[0151] Preferably, the pharmaceutical composition is gluten-free.

[0152] The pharmaceutical compositions are suitable for administration to animal (and, in particular, human) patients, ie, both human and veterinary use is included.

[0153] The pharmaceutical composition can be prepared in the form of a unit dose. In some embodiments, the unit dose can be 0.1 to 1.0 ml, for example, about 0.25 ml, or preferably about 0.5 ml. Such a volume is ideal for injection in humans.

[0154] complex concentration Pharmaceutical compositions may comprise multiple immunogenic conjugates. Currently licensed meningococcal conjugate vaccines contain capsular saccharides from four different serogroups, and licensed pneumococcal conjugate vaccines contain capsular saccharides from seven, ten or thirteen different serotypes. Thus, a composition of the invention could contain, for example, from 3 to 50 different conjugates (e.g. 14, 15, 20, 21, 24, 25 or more). For example, each of these conjugates may contain capsular saccharides from different serotypes or serogroups of the same species (e.g. multiple meningococcal serogroups or multiple pneumococcal serotypes).

[0155] When a pharmaceutical composition contains n different immunogenic complexes, the total amount of carrier polypeptides in the n complexes can be 3nμg or less per dose.In other words, the average amount of carrier polypeptides per complex is less than 3μg.The total amount can be, for example, n to 2.5nμg per dose.

[0156] When a pharmaceutical composition contains n different immunogenic conjugates, the total amount of saccharide antigens in the n conjugates can be 4.4 nμg or less per dose. In other words, the average amount of saccharides per conjugate is less than 4.4 μg. The total amount can be, for example, 0.4 n to 4.4 nμg per dose, e.g., 1.1 n to 2.2 nμg.

[0157] When a pharmaceutical composition contains n different immunogenic conjugates, the total concentration of the carrier polypeptides for the n conjugates can be 6n μg / mL or less. In other words, the average concentration of the carrier polypeptides per conjugate is less than 6 μg / mL. The total concentration can be, for example, n to 4n μg / mL.

[0158] When a pharmaceutical composition contains n different immunogenic conjugates, the total concentration of saccharide antigens for the n conjugates can be 8.8 nμg / mL or less. In other words, the average concentration of saccharides per conjugate is less than 8.8 μg / mL. The total concentration can be, for example, 0.8 n to 8.8 nμg / mL, e.g., 2.2 n to 4.4 nμg / mL.

[0159] In some embodiments, the total amount of conjugated carrier polypeptide in a unit dose of a multivalent pharmaceutical composition of the invention can be 4 to 128 μg, e.g., 8 to 64 μg or 16 to 48 μg. The concentration of conjugated carrier polypeptide in a multivalent pharmaceutical composition of the invention can be 8 to 256 μg / mL, e.g., 16 to 128 μg / mL or 32 to 96 μg / mL.

[0160] In some embodiments, the total amount of conjugated saccharide antigens in a unit dose of a multivalent pharmaceutical composition of the invention may be 10 to 120 μg, for example 20 to 90 μg or 30 to 60 μg. The concentration of conjugated saccharide antigens in a multivalent pharmaceutical composition of the invention may be 20 to 240 μg / mL, for example 40 to 180 μg / mL or 60 to 120 μg / mL.

[0161] unbound component As noted above, a pharmaceutical composition may comprise a plurality of immunogenic conjugates, for example between 3 and 50 different conjugates (e.g. 14, 15, 20, 21, 24, 25 or more), each of which may comprise capsular saccharides from different serotypes or serogroups of the same bacterial species.

[0162] In some embodiments, the composition does not contain the complexed carrier polypeptide in unconjugated form, while in other embodiments, unconjugated carrier polypeptide is present at low levels, provided that the mass of unconjugated carrier polypeptide in the composition is <10% (e.g., <5% or <2%) of the mass of carrier polypeptide in the n immunogenic complexes of the composition as a whole.

[0163] In some embodiments, the composition does not contain conjugated saccharides in unconjugated form, while in other embodiments, unconjugated saccharides are present at low levels, provided that the mass of unconjugated saccharides in the composition is <10% (e.g., <5% or <2%) of the total mass of saccharides in the n immunogenic conjugates of the composition.

[0164] Containers, delivery devices, etc. The pharmaceutical composition comprising the immunogenic complex may be packaged in a sterile container, delivery device, etc. Sterility may be maintained by hermetically sealing the container so that it is airtight. Suitable containers include, but are not limited to, vials, syringes, nebulizers, sprays, inhalers, skin patches, etc. Vials and syringes are preferred.

[0165] The immunogenic composition is often contained in a vial. The vial is preferably made of a plastic material or preferably glass. The vial is sealed after filling, but the seal can be broken just before use. The vial is preferably sterilized before the composition is added thereto and then sealed. To avoid problems with latex-sensitive patients, the vial may be sealed with a latex-free stopper, and it is preferred that all packaging materials be latex-free. The vial ideally contains a single unit dose of the composition, but it may also contain more than one dose (multi-use vial), e.g., 10 doses. Preferred vials are made of colorless glass.

[0166] The vial may have a closure (e.g., a luer lock) adapted to allow a syringe to be inserted into the closure, facilitating transfer of material (in both directions) between the vial and the syringe. After removing the syringe from the vial, a needle can then be attached, and the composition can be administered to a subject. The closure is preferably located inside the seal or cover, so that the seal or cover must be removed before the closure is accessible. The vial may have a closure that allows for aseptic removal of its contents, particularly for multi-dose vials.

[0167] The composition can be contained within a delivery device ready to be administered to a subject. The composition can be transferred to the delivery device (e.g., from a vial) just before use, or the composition can be placed in the delivery device during manufacturing (e.g., in the form of a pre-filled syringe).

[0168] Syringes used in the present invention can be made of glass or plastic (e.g., cycloolefin polymer or cycloolefin copolymer). Syringes (especially glass syringes) can be siliconized. Non-siliconized syringes can also be used, for example, using Terumo's i-Coating™ system, which is available for Terumo's PLAJEX™ syringes, or Daikyo Seiko's CZ™ syringes with ethylene tetrafluoroethylene (ETFE) copolymer, or TriboGlide™ syringes with perfluoropolyether (PFPE). Instead of siliconization, a carbon membrane can be used (see, for example, JP 2001190665 A). Silicon-free syringes are also disclosed in JP 2011212183 A. Non-siliconized syringes containing plunger stoppers such as those disclosed in EP 0 375 778 A1 may also be used, i.e., in this case the stopper comprises a thermoplastic elastomer at least partially covered with a thermoplastic resin layer having a low coefficient of dynamic friction.

[0169] If the composition is contained in a syringe, the syringe may have a needle attached thereto for injecting the contents of the syringe into a subject or a container. The syringe may be supplied with the needle already attached. If the needle is not attached, a separate needle may be supplied with the syringe for assembly or use, or the needle may be of a separate origin. Such needles must be sterilized at the time of use and may be wrapped. Safety needles may be used. 1 inch, 23 gauge needles, 1 inch, 25 gauge needles, and 5 / 8 inch, 25 gauge needles are typical. 1 / 2 inch to 1 1 / 2 inch, 22 to 25 gauge needles may be used. If the syringe and needle are packaged separately, the needle is preferably fitted with a butyl rubber shield.

[0170] Syringes can be provided with peel-off labels that can be printed with lot numbers and expiration dates to facilitate record-keeping. The plungers in the syringes can be stoppered to prevent accidental removal of the plunger during aspiration. The syringes can have latex rubber caps and / or plungers, although latex-free rubbers, such as latex-free cyclobutyl rubber or latex-free isoprene bromobutyl rubber, can be used. Syringes generally have tip caps to seal the tip prior to needle attachment, and the tip cap is preferably made of butyl rubber, such as latex-free isoprene bromobutyl rubber. Useful syringes are, for example, those commercially available under the trade name "Tip-Lok" (trademark).

[0171] The container may be marked to indicate half-dose amounts, for example, to facilitate delivery to children. For example, a syringe containing a 0.5 ml dose may be marked to indicate a 0.25 ml volume. The syringe itself may have a volume greater than the dose, for example, a 1 ml syringe can be used to contain a 0.5 ml dose of the pharmaceutical composition. Disposable or pre-filled syringes typically contain a single dose of vaccine.

[0172] If glass containers (eg, syringes or vials) are used, they are preferably made from borosilicate glass rather than soda-lime glass.

[0173] The container may be packaged (e.g., in the same box) with a leaflet containing details of the vaccine, e.g., administration instructions, details of the antigens in the vaccine, etc. The instructions may also contain warnings, e.g., to have adrenaline solution readily available in case of an anaphylactic reaction after vaccination. Multiple containers may be packaged together, e.g., in the same box.

[0174] The pharmaceutical composition can be in unit dose form, with a single dose per container (e.g., per syringe or vial). Rather than individually manufacturing each unit dose, a bulk composition is prepared and the unit doses are removed and packaged individually within their containers. That is, for example, multiple unit doses are removed from the bulk and each unit dose is transferred to a separate container, such as a syringe or vial.

[0175] Increased immune response The immunogenic complexes can be administered to a mammalian subject to elicit a protective immune response against the antigen in the complex. They are administered in the form of a pharmaceutical composition. The composition can contain multiple immunogenic complexes, as described elsewhere herein, so that protective immune responses against multiple antigens can be elicited simultaneously.

[0176] That is, a method is provided for eliciting a protective antibody response in a mammalian subject against one or more antigens by administering to the subject a complex of antigens.

[0177] Also provided is a conjugate as disclosed herein for use in eliciting a protective antibody response.

[0178] Also provided is the use of a conjugate as disclosed herein in the manufacture of a medicament for eliciting a protective antibody response.

[0179] Also provided are (i) methods of eliciting a protective antibody response in a mammalian subject against multiple antigens by administering to the subject a multivalent composition of the invention; (ii) multivalent compositions of the invention for use in eliciting a protective antibody response; and (iii) the use of multiple conjugates as disclosed herein in the manufacture of a multivalent pharmaceutical composition for eliciting protective antibody responses against multiple antigens.

[0180] The ability to elicit a protective immune response means that the conjugates can be used, for example, to prevent invasive disease caused by S. pneumoniae, to prevent otitis media caused by S. pneumoniae, to provide active immunization to prevent pneumonia caused by S. pneumoniae, to provide active immunization in subjects at risk of exposure to N. meningitidis to prevent invasive disease, etc.

[0181] The pharmaceutical composition can be prepared in various forms. For example, the composition can be prepared as an injection, such as a solution or suspension. Injections for intramuscular administration are typical. For humans, an injection volume of about 0.5 ml is preferred. That is, a preferred unit dose volume is about 0.5 ml. Intramuscular administration is typically given, for example, into the anterolateral aspect of the thigh in infants or into the deltoid muscle in infants, children, and adults.

[0182] The conjugates will typically be administered according to a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. Administration of multiple doses (typically two doses) is particularly useful in immunonaive patients. Multiple doses will typically be administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, etc.).

[0183] General The term "comprising" encompasses "consisting" as well as "including", e.g., a composition "comprising" X may consist exclusively of X, or may additionally include something, e.g., X+Y.

[0184] The word "about" in connection with a numerical value x is optional and means, for example, x±10%.

[0185] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the invention.

[0186] The term "sequence identity" in the context of two amino acid sequences refers to two sequences that are identical or have a specified percentage of identical amino acid residues when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm (e.g., BLASTP). The percentage of identity is determined relative to a full-length reference sequence disclosed herein, such as the reference sequence set forth in SEQ ID NO: 1 or 2. A method for calculating sequence identity as provided herein is the BLASTP program with its default settings of a word length (W) of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff & Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). See, e.g., the BLAST alignment tool available on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi or elsewhere.

[0187] As used herein, and unless otherwise specified, the term "lower alkyl" refers to a saturated, straight-chain or branched-chain hydrocarbon having from 1 to 6 carbon atoms, i.e., a C1 to C6 alkyl. In some embodiments, a lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both saturated and unsaturated moieties. See also U.S. Patent Publication No. 2014 / 0066598. The term "lower alkylene" refers to an alkylene radical of a lower alkyl.

[0188] Unless otherwise specified, all technical and scientific terms used herein have the meanings that are commonly understood. In particular, practitioners are guided by Green & Sambrook (eds.) Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), and Ausubel, FM, et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012), and Plotkin, SA, Orenstein, WA, & Offit, PA, Vaccines, 6th ed., Elsevier, London (2013).

[0189] Methods for cell-free synthesis are described in Spirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. Methods for incorporating unnatural amino acids into proteins using cell-free synthesis are also described in Shimizu et al. (2006) FEBS Journal, 273, 4133-4140 and Chong (2014) Curr Protoc Mol Biol. 108:16.30.1-11.

[0190] In some embodiments, the invention does not include compositions in which SEQ ID NO:3 is used as a carrier polypeptide for conjugates from 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 (exemplified in WO 2018 / 126229). More generally, in some embodiments, the invention does not include compositions in which SEQ ID NO:3 is used as a carrier polypeptide for each conjugate in a multivalent composition.

[0191] Enumeration of Embodiments Embodiment I-1 A sterile container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide.

[0192] Embodiment I-2: A hermetically sealed container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bound to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. Containers suitable for hermetically sealing include, for example, vials. The contents are preferably sterilized at the time of hermetically sealing.

[0193] Embodiment I-3 The container of embodiment I-1 or I-2, which is a sterile glass container, such as a vial.

[0194] Embodiment I-4 A delivery device containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide.

[0195] Embodiment I-5 The container of embodiment I-1 or I-2 or the delivery device of embodiment I-4, which is a syringe.

[0196] Embodiment I-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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the aluminum salt adjuvant is an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant.

[0197] Embodiment I-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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the concentration of aluminum ions in the composition is ≦2.5 mg / mL.

[0198] Embodiment I-8 A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein (i) each immunogenic conjugate comprises a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the volume of the pharmaceutical composition is between 0.25 and 1.25 mL.

[0199] Embodiment 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide.

[0200] Embodiment I-10. A preservative-free pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide.

[0201] Embodiment I-11 A pharmaceutical composition comprising two or more different immunogenic conjugates, (i) each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the composition has an osmolality of 200 to 400 mOsm / kg.

[0202] Embodiment I-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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural 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.

[0203] Embodiment I-13: A pharmaceutical composition comprising n different immunogenic conjugates (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached 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 carrier polypeptide in the n immunogenic conjugates is 3 nμg or less per dose; (iv) the total concentration of the carrier polypeptide in the n immunogenic complexes is 6 nμg / ml or less; (v) the total amount of saccharide antigens in the n immunogenic conjugates is 3 nμg or less per dose; (vi) the total concentration of the saccharide antigens in the n immunogenic conjugates is 6 nμg / mL or less; (vii) the average amount of carrier polypeptide per conjugate is 1 to 4 μg per dose; (viii) the average concentration of carrier polypeptide per conjugate is 2 to 8 μg / mL; (ix) the average amount of saccharide antigen per conjugate is 1 to 4 μg per dose; (x) the average concentration of saccharide antigen per conjugate is 2 to 8 μg / mL; (xi) the composition does not contain a carrier polypeptide in unconjugated form; (xii) the composition contains a carrier polypeptide in unconjugated form, and the mass of the carrier polypeptide in unconjugated form in the composition is <10% of the mass of the carrier polypeptide in the n immunogenic complexes; (xiii) the composition does not contain saccharide antigens in unconjugated form; and / or (xiv) The pharmaceutical composition, wherein the composition contains at least one saccharide antigen in unconjugated form, and the total mass of the saccharide antigen in unconjugated form in the composition is <10% of the total mass of the saccharide antigen in the n immunogenic complexes.

[0204] Embodiment I-14 A method of preparing a plurality of unit dose pharmaceutical compositions, wherein (i) the pharmaceutical compositions comprise an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the method comprises preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into a plurality of separate containers.

[0205] Embodiment I-15 A method of preparing a pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, and (ii) the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, the method comprising the steps of: (A) separately adsorbing each of the immunogenic conjugates to an aluminum salt adjuvant and then mixing the individual adsorbed conjugates together; or (B) sequentially adsorbing each of the immunogenic conjugates to an aluminum salt adjuvant.

[0206] Embodiment I-16 A carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue.

[0207] Embodiment I-17 A carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO:1; and (ii) includes an nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO:1): Asp-211; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys 212; Lys 218; Lys 221; Lys 229;Lys 236;Lys 264;Lys 299;Lys 385;Lys 456;Lys 474;Lys 498;Lys 516;Lys 522;Lys 534;Arg 377;Arg 407;Arg 455;Arg 460;Arg 462;Arg 472;Arg 493;Ser 198;Ser 200;Ser 231;Ser 233;Ser 239;Ser 261;Ser 374;Ser 381;Ser 297;Ser 397;Ser 451;Ser 475;Ser 494;Ser 495;Ser 496;Ser 501;Ser 505;Thr 253;Thr 265;Thr 267;Thr 269;Thr 293;Thr 386;Thr 400;Thr 408;Thr-469;and / or Thr 517.

[0208] Embodiment I-18 The carrier polypeptide of embodiment I-16 or I-17, wherein Arg-193 in SEQ ID NO: 1 has been substituted with a different amino acid, such as Asn.

[0209] Embodiment I-19 An immunogenic complex comprising the carrier polypeptide of embodiment I-16 or I-17 or I-18 conjugated to an antigen via an nnAA residue in the carrier polypeptide.

[0210] Embodiment I-20. An immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide comprises the amino acid sequence of SEQ ID NO: 4, and (ii) the saccharide antigen is covalently attached to the carrier polypeptide via at least one nnAA residue in the SEQ ID NO: 4.

[0211] Embodiment I-21 A pharmaceutical composition comprising two or more different immunogenic conjugates according to embodiment I-20.

[0212] Embodiment I-22 The container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein the carrier polypeptide comprises 4 to 9 nnAA residues.

[0213] Embodiment I-23. The container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein a lysine in the native sequence of the carrier polypeptide is substituted with at least one nnAA.

[0214] Embodiment I-24. The container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein the carrier polypeptide has at least 90% sequence identity to SEQ ID NO:1.

[0215] Embodiment I-25 The container, device, composition, method, polypeptide or complex of embodiment I-24, wherein K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and / or K526 in SEQ ID NO: 1 or 2 are substituted with at least one nnAA.

[0216] Embodiment I-26. The container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein the carrier polypeptide comprises the amino acid sequence of SEQ ID NO:14.

[0217] Embodiment I-27 A container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein the nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0218] Embodiment I-28 The container, device, composition, method, polypeptide, or complex of any of the preceding embodiments, wherein the antigen has an alkyne group that is attached to the nnAA via an azide group.

[0219] Embodiment I-29 A container, device, composition, method, polypeptide, or conjugate according to any of the preceding embodiments, wherein the antigen is a bacterial capsular saccharide, for example a capsular saccharide from a bacterium selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis.

[0220] Embodiments I-30. The container, device, composition, method, polypeptide, or conjugate of any of the preceding embodiments, wherein the antigen is a capsular saccharide of a serotype of S. pneumoniae 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.

[0221] Embodiment I-31 A container, device, composition, method, polypeptide or conjugate according to any of the preceding embodiments, wherein the ratio of saccharide to carrier polypeptide (w / w) in the conjugate is greater than 1.

[0222] Embodiment I-32 The container, device, composition, method, polypeptide, or conjugate of any of the preceding embodiments, wherein the carrier polypeptide comprises three or more nnAA residues and the conjugate has a molecular weight of at least 500 kDa.

[0223] Embodiment I-33 A container, device, composition, method, polypeptide or complex according to any of the preceding embodiments, wherein the complex has a molecular weight of between 900 kDa and 5 MDa.

[0224] Embodiment I-34 The pharmaceutical composition comprises: a complex of capsular saccharides from two or more 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; a complex of capsular saccharides from 14 or more 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; a complex of capsular saccharides from 15 or more 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; a complex of capsular saccharides from 20 or more 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; a complex of capsular saccharides from 21 or more 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; a complex of capsular saccharides from 24 or more 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; a complex of capsular saccharides from 25 or more 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; a complex of capsular saccharides from four or more different meningococcal serogroups selected from the group consisting of serogroups A, C, W135, X, and Y; or A container, device, composition, or method of any one of embodiments I-1 to I-15 or I-21 to I-33, comprising a conjugate of capsular saccharides from two or more different P. gingivalis serotypes selected from the group consisting of serotypes K1, K2, K3, K4, K5, and K6.

[0225] Embodiment I-35 A method of eliciting an immunoprotective antibody response against an antigen in a subject, comprising administering to the subject a pharmaceutical composition according to any one of embodiments I-6 to I-13 or I-21 to I-34, or an immunogenic conjugate according to any one of embodiments I-19 to I-33, in an excipient suitable for parenteral administration. [Example]

[0226] The following examples illustrate the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. The examples are carried out using techniques well known and routine to those skilled in the art, unless otherwise described in detail.

[0227] Examples from WO 2018 / 126229 The Examples in WO 2018 / 126229 fully detail the synthesis of eCRM moieties (e.g., K11TAG), which were expressed in cell-free protein synthesis (CFPS) extracts and incorporated pAMF in place of the native Lys.

[0228] CRM variants containing multiple nnAA residues per polypeptide were also expressed with varying numbers of Lys→pAMF substitutions per protein. In general, a larger number of substitutions resulted in carriers that led to higher molecular weight conjugates but also to lower carrier solubility. Carriers with six pAMF residues generally provided both good solubility (>50 mg / mL) and immunogenicity. The high solubility was surprising because substituting hydrophobic pAMF residues for charged Lys residues in the native sequence increased the hydrophobicity of CRM197, a protein whose hydrophobicity has previously been reported to affect its solubility. This demonstrated that it was possible to maintain the same attachment site (i.e., Lys residue) used in known CRM197 conjugates without causing insolubility by losing the charged residues.

[0229] A particularly useful set of six Lys→pAMF substitutions was found, using K34, K213, K245, K265, K386, and K527 (numbered according to SEQ ID NO: 3). Surprisingly, this combination of pAMF substitution sites was effective, especially since the individual substitutions at positions K245 and K527 led to relatively low levels of expression.

[0230] This set of six substitutions can be combined with disruption of the Arg-Arg dipeptide at residues 192 to 193 (RR→RN) of SEQ ID NO:1 to yield SEQ ID NO:4, where each X is pAMF.

[0231] The Examples of WO 2018 / 126229 further describe general protocols for saccharide activation with sodium meta-periodate, derivatization of periodate-oxidized polysaccharides with DBCO, saccharide activation with CDAP, and conjugation of saccharide-DBCO to eCRM. See also U.S. Serial No. 62 / 693,978, previously incorporated by reference.

[0232] Multivalent immunogenic compositions Conjugate combinations 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 the CRM197 derivative SEQ ID NO: 4 (where X = pAMF) as the carrier polypeptide in each conjugate. The immunogenicity of this multivalent composition was confirmed using a three-dose regimen of 0.25 mL intramuscular injections in groups of seven rabbits. Each dose contained 24 μg of saccharide (1 μg per serotype) at a concentration of 96 μg / mL.

[0233] Next, conjugate combinations 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 their immunogenicity was confirmed using the same method.

[0234] For comparison purposes, a 13-valent Prevnar™ conjugate vaccine was also tested, along with a 24-valent unconjugated vaccine made of a 23-valent Pneumovax™ vaccine supplemented with unconjugated serotype 6A polysaccharide. These three compositions had equivalent polysaccharide doses per serotype (except for 6B, which contained a double dose of Prevnar™), but this involved dilution of Prevnar™ and Pneumovax™. All three compositions contained aluminum phosphate adjuvant (60 μg Al per dose). +++ ), which involved adding this adjuvant to Pneumovax™. The composition did not contain a preservative.

[0235] The 24-valent conjugate composition contained less carrier polypeptide than in the licensed Prevnar-13™ vaccine, even though it also contained capsular saccharides from 11 additional serotypes. The total weight ratio of capsular saccharide to carrier polypeptide in the 24-valent conjugate composition was approximately twice that seen in Prevnar™.

[0236] IgG and OPA responses were measured in rabbits. After the third dose, both responses were much greater in rabbits receiving the two conjugate vaccines than in rabbits receiving the non-conjugate vaccine. Furthermore, IgG and OPA responses using the 24-valent composition were comparable to those achieved with Prevnar™ for the 13 serotypes covered by licensed vaccines, but were also superior for the 11 serotypes not included in Prevnar™. Surprisingly, there was no evidence of carrier-induced epitope suppression using the 24-valent composition.

[0237] FIG. 1 shows the geometric mean titers for each of the 32 serotypes in the 32-valent conjugate composition for polysaccharide / alum formulations and Prevnar-13™.

[0238] Multivalent conjugate compositions can be advantageously packaged in pre-filled sterile syringes so that they can be easily delivered in unit dose form and then administered at the point of use without the need to transfer the contents of a vial into an injection syringe or the like.

[0239] Substitutable positions in CRM197 Based on the work disclosed in WO 2018 / 126229, various Asp, Asn, Glu, Gln, Lys, Arg, Ser, and Thr residues in the native CRM197 sequence (SEQ ID NO: 1) were individually substituted in pAMF by mutating their codons to TAG and expressing the protein at 25°C in a cell-free system in which this codon is recognized by tRNAs incorporating nnAA. The mutant polypeptides were expressed with an N-terminal methionine and a downstream hexahistidine tag attached via a Gly-Ser-Gly tripeptide linker. Residues within Asn270-Ile289, Ala320-Glu349, and Phe410-His-449 were avoided due to T cell epitopes recognized in these regions (see above).

[0240] to mutant proteins 14 Expression efficiency was assessed by confirming C-Leu incorporation and examining both total and soluble protein. Generally, mutations in the catalytic domain of CRM197 led to decreased expression levels compared to the unmodified CRM197 sequence, and mutations with the best expression levels generally included substitutions downstream of Arg-193, which can be used to delineate the end of the catalytic domain.

[0241] The best 72 mutations 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-23 9, Glu-240, Glu-248, Glu-249, Gln-252, Thr-253, Glu-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, As n-359, Glu-362, Ser-374, Arg-377, 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-4 69, Arg-472, Lys-474, Ser-475, Asn-481, Asn-486, Arg-493, Ser-494, Ser-495, Ser-496, Lys-498 , Ser-501, Asn-502, Ser-505, Asp-507, Lys-516, Thr-517, Asp-519, Lys-522, Asn-524 and Lys-534.

[0242] The embodiments described herein are provided as examples only and do not exclude various alternatives to the embodiments when implementing the embodiments described herein.

[0243] Sequence Listing SEQ ID NO: 1 (native CRM197) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0244] SEQ ID NO: 2 (CRM197 containing Arg-Asn substitution) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0245] SEQ ID NO: 3 (CRM197 containing six preferred nnAA sites and an N-terminal Met (methionine)) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS

[0246] SEQ ID NO: 4 (Arg-Asn subset n CRM197, which contains six preferred nnAA sites and an N-terminal Met (methionine). MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH XTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS

[0247] SEQ ID NO: 5 (H. influenzae protein D) CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK Furthermore, the present invention encompasses the following aspects. 1. A sterile container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bound to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. 2. A hermetically sealed container containing a pharmaceutical composition comprising an immunogenic complex 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; suitable containers for hermetically sealing include, for example, vials, and the contents are preferably sterilized at the time of hermetically sealing. 3. The container according to item 1 or 2, which is a sterile glass container such as a vial. 4. A delivery device containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. 5. The container according to item 1 or 2, or the delivery device according to item 4, which is a syringe. 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the aluminum salt adjuvant is an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant. 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the concentration of aluminum ions in the composition is ≦2.5 mg / mL. 8. A pharmaceutical composition comprising two or more different immunogenic conjugates, (i) each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the volume of the pharmaceutical composition is between 0.25 and 1.25 mL. 9. A pharmaceutical composition comprising two or more different immunogenic conjugates and a preservative, wherein each immunogenic conjugate comprises a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide. 10. A preservative-free pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide. 11. A pharmaceutical composition comprising two or more different immunogenic conjugates, (i) each immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide, and (ii) the composition has an osmolality of 200 to 400 mOsm / kg. 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 saccharide antigen, wherein the saccharide antigen is covalently attached to the carrier polypeptide via an unnatural 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. 13. A pharmaceutical composition comprising n different immunogenic complexes, (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently attached 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 carrier polypeptide in the n immunogenic conjugates is 3 nμg or less per dose; (iv) the total concentration of the carrier polypeptide in the n immunogenic complexes is 6 nμg / ml or less; (v) the total amount of saccharide antigens in the n immunogenic conjugates is 3 nμg or less per dose; (vi) the total concentration of saccharide antigens in the n immunogenic conjugates is 6 nμg / mL or less; (vii) the average amount of carrier polypeptide per conjugate is 1 to 4 μg per dose; (viii) the average concentration of carrier polypeptide per conjugate is 2 to 8 μg / mL; (ix) the average amount of saccharide antigen per conjugate is 1 to 4 μg per dose; (x) the average concentration of saccharide antigen per conjugate is 2 to 8 μg / mL; (xi) the composition does not contain a carrier polypeptide in unconjugated form; (xii) the composition contains a carrier polypeptide in unconjugated form, wherein the mass of the carrier polypeptide in unconjugated form in the composition is <10% of the mass of the carrier polypeptide in the n immunogenic complexes; (xiii) the composition does not contain saccharide antigens in unconjugated form; and / or (xiv) The pharmaceutical composition, wherein the composition contains at least one of the saccharide antigens in unconjugated form, and the total mass of the saccharide antigen in unconjugated form in the composition is <10% of the total mass of the saccharide antigen in the n immunogenic complexes. 14. A method for preparing a plurality of unit dose pharmaceutical compositions, wherein (i) the pharmaceutical compositions comprise an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently bound to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; and (ii) the method comprises preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into a plurality of separate containers. 15. A method for preparing a pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, and (ii) the saccharide antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, the method comprising the steps of: (A) separately adsorbing each of the immunogenic conjugates to an aluminum salt adjuvant and then mixing the individual adsorbed conjugates together; or (B) sequentially adsorbing each of the immunogenic conjugates to the aluminum salt adjuvant. 16. A carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue. 17. A carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity to SEQ ID NO: 1; and (ii) includes an nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-211; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys 212; Lys 218; Lys 221; Lys 229;Lys 236;Lys 264;Lys 299;Lys 385;Lys 456;Lys 474;Lys 498;Lys 516;Lys 522;Lys 534;Arg 377;Arg 407;Arg 455;Arg 460;Arg 462;Arg 472;Arg 493;Ser 198;Ser 200;Ser 231;Ser 233;Ser 239;Ser 261;Ser 374;Ser 381;Ser 297;Ser 397;Ser 451;Ser 475;Ser 494;Ser 495;Ser 496;Ser 501;Ser 505;Thr 253;Thr 265;Thr 267;Thr 269;Thr 293;Thr 386;Thr 400;Thr 408;Thr-469;and / or Thr 517. 18. The carrier polypeptide of item 16 or 17, wherein Arg-193 in SEQ ID NO: 1 has been substituted with a different amino acid, such as Asn. 19. An immunogenic complex comprising the carrier polypeptide of paragraph 16, paragraph 17, or paragraph 18, conjugated to an antigen via an nnAA residue in the carrier polypeptide. 20. An immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide comprises the amino acid sequence of SEQ ID NO: 4, and (ii) the saccharide antigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. 21. A pharmaceutical composition comprising two or more different immunogenic complexes according to item 20. 22. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 21, wherein the carrier polypeptide comprises 4 to 9 nnAA residues. 23. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 22, wherein a lysine in the native sequence of the carrier polypeptide is substituted with at least one nnAA. 24. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 23, wherein the carrier polypeptide has at least 90% sequence identity to SEQ ID NO:1. 25. The container, device, composition, method, polypeptide, or complex of item 24, wherein K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522, and / or K526 in SEQ ID NO: 1 or 2 are substituted with at least one nnAA. 26. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 25, wherein the carrier polypeptide comprises the amino acid sequence of SEQ ID NO:4. 27. The container, device, composition, method, polypeptide, or complex of any one of items 1 to 26, wherein the nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid. 28. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 27, wherein the antigen has an alkyne group that is attached to the nnAA via an azide group. 29. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 28, wherein the antigen is a bacterial capsular saccharide, for example, a capsular saccharide from a bacterium selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis. 30. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 29, wherein the antigen is a capsular saccharide of a serotype of S. pneumoniae 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. 31. The container, device, composition, method, polypeptide, or conjugate of any one of paragraphs 1 to 30, wherein the ratio (w / w) of saccharide to carrier polypeptide in the conjugate is greater than 1. 32. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 1 to 31, wherein the carrier polypeptide comprises three or more nnAA residues and the complex has a molecular weight of at least 500 kDa. 33. The container, device, composition, method, polypeptide, or complex described in any one of paragraphs 1 to 32, wherein the complex has a molecular weight of 900 kDa to 5 MDa. 34. A pharmaceutical composition comprising: a complex of capsular saccharides from two or more 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; a complex of capsular saccharides from 14 or more 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; a complex of capsular saccharides from 15 or more 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; a complex of capsular saccharides from 20 or more 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; a complex of capsular saccharides from 21 or more 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; a complex of capsular saccharides from 24 or more 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; a complex of capsular saccharides from 25 or more 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; a complex of capsular saccharides from four or more different meningococcal serogroups selected from the group consisting of serogroups A, C, W135, X, and Y; or 34. The container, device, composition, or method of any one of paragraphs 1 to 15 or 21 to 33, comprising a complex of capsular saccharides from two or more different P. gingivalis serotypes selected from the group consisting of serotypes K1, K2, K3, K4, K5, and K6. 35. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 30 to 34, wherein serotype 20 is serotype 20B. 36. The container, device, composition, method, polypeptide, or complex of any one of paragraphs 30 to 34, wherein serotype 20 is serotype 20A. 37. A method for eliciting an immunoprotective antibody response to an antigen in a subject, the method comprising administering to the subject a pharmaceutical composition described in any one of paragraphs 6 to 13 or paragraphs 21 to 34, or an immunogenic complex described in any one of paragraphs 19 to 33, in an excipient suitable for parenteral administration.

Claims

1. 1. A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a saccharide antigen, wherein the saccharide antigen is covalently linked to the carrier polypeptide via an unnatural amino acid residue in the carrier polypeptide; each carrier polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:1, does not contain an Arg-Arg dipeptide sequence, and contains 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid residues at K33, K212, K244, K264, K385, and K526 relative to SEQ ID NO:1; and (ii) is at least one of the following: a) the pharmaceutical composition further comprises an aluminum salt adjuvant which is an aluminum hydroxide or aluminum phosphate adjuvant; b) the pharmaceutical composition further comprises an aluminum salt adjuvant, and the aluminum ion concentration in the composition is <2.5 mg / mL; c) the volume of the pharmaceutical composition is 0.25 mL to 1.25 mL; d) the pharmaceutical composition has an osmolality of 200 to 400 mOsm / kg; or e) the pharmaceutical composition comprises at least one excipient selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein each carrier polypeptide has the same amino acid sequence.

3. 2. The pharmaceutical composition of claim 1, wherein the saccharide antigens of the two or more different immunogenic conjugates are capsular saccharides of a bacterium selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Group A hemolytic streptococcus, Streptococcus pyogenes, Group B hemolytic streptococcus, and Porphyromonas gingivalis.

4. 4. The pharmaceutical composition of claim 3, wherein the saccharide antigens of the two or more different immunogenic conjugates are capsular saccharides of a serotype of S. pneumoniae 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.

5. 10. The pharmaceutical composition of claim 1, further comprising a preservative.

6. A pharmaceutical composition according to any one of claims 1 to 5 for use in eliciting an immunoprotective antibody response against an antigen.

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