Improvement of immunogenic complexes

Abstract

The present invention provides a carrier polypeptide and an immunogenic complex comprising a carrier polypeptide and a sugar antigen. [Solution] The carrier polypeptide is a modified CRM197 carrier polypeptide having an amino acid sequence that has 80% sequence identity with a specific amino acid sequence and does not contain an Arg-Arg dipeptide sequence. The sugar antigen is covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide, forming an immunogenic complex.

Description

[Technical Field]

[0001] Cross-references to related applications The priority of this application is the benefit of U.S. Provisional Patent Application No. 62 / 693,981, filed on 4 July 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Supporting the electronic text files submitted together The contents of the following text file, submitted electronically, 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 submission: July 1, 2019, file size approximately 23 kilobytes). [Background technology]

[0003] The immune response to "weak" sugar antigens can be amplified by binding to known "strong" carrier polypeptide antigens, such as diphtheria toxoid, tetanus toxoid, Haemophilus influenzae protein D, or CRM197. International Publication No. 2018 / 126229 (SutroVax, Inc., Foster City, California) discloses methods, compositions, and techniques for the production of conjugated vaccine antigens using carrier polypeptides containing non-natural amino acids (nnAAs). The chemistry of orthogonal binding via nnAAs allows for the binding of the antigen to the carrier polypeptide, producing an immunogenic complex useful for immunization.

[0004] It is the object of the present invention to provide variations and improvements on these methods, compositions, and techniques. The variations and improvements described below can be applied to or combined with any of the methods, compositions, or techniques disclosed in International Publication No. 2018 / 126229, or in U.S. Provisional Patent Applications Nos. 62 / 693,978 and 62 / 693,981, both filed on July 4, 2018. The aforementioned patent applications are incorporated herein by reference in their entirety. [Overview of the Initiative]

[0005] One embodiment provides a sterile container (e.g., a vial) containing a pharmaceutical composition comprising an immunogenic complex 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 within the carrier polypeptide. This container may contain a unit dose of the pharmaceutical composition. Preferably, it is a sterile glass container.

[0006] In another embodiment, a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.) is provided that contains a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide. This delivery device may contain a unit dose of the pharmaceutical composition. The pharmaceutical composition can be administered to a mammalian subject using this delivery device.

[0007] In another embodiment, an airtight container is provided containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide. Suitable containers for airtight sealing include, for example, vials. The contents are preferably sterilized during airtight 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 complexes, each containing a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bound to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide.

[0009] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic complexes and an aluminum salt adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural 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 amount of the pharmaceutical composition is 0.25 to 0.75 mL (for example, 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 complexes and an aluminum phosphate adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within 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 should be ≤1.7 mg / mL. The complexes in the composition can also be adsorbed onto the aluminum phosphate adjuvant.

[0011] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic complexes and an aluminum phosphate adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide, (ii) the carrier polypeptide does not contain 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 should be ≤1.7 mg / mL. The complexes in the composition can be adsorbed onto the aluminum phosphate adjuvant.

[0012] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic complexes, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, and (ii) the amount 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 contain an aluminum phosphate adjuvant, and the complexes in the composition can be adsorbed to the aluminum phosphate adjuvant.

[0013] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic complexes and a preservative, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

[0014] In another embodiment, a preservative-free pharmaceutical composition is provided, comprising two or more different immunogenic complexes, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

[0015] In another embodiment, a pharmaceutical composition comprising two or more different immunogenic complexes is provided, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide, and (ii) the composition having a weight osmolality of 200 to 400 mOsm / kg.

[0016] Another embodiment provides a pharmaceutical composition comprising two or more different immunogenic complexes and at least one excipient, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide, and (ii) at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80. The pharmaceutical composition may also contain an aluminum salt adjuvant. The composition may contain both sodium chloride and polysorbate 80 as excipients.

[0017] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic complexes, wherein (i) each of the n immunogenic complexes comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total amount of carrier polypeptide in the n immunogenic complexes is 3 n μg or less per dose of the pharmaceutical composition.

[0018] In another embodiment, there is provided a pharmaceutical composition comprising n different immunogenic conjugates, wherein (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, and the saccharide antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the total concentration of the carrier polypeptides in the n immunogenic conjugates is 6n μg / mL or less in the pharmaceutical composition.

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

[0020] In another embodiment, there is provided a pharmaceutical composition comprising n different immunogenic conjugates, wherein (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a saccharide antigen, and the saccharide antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue within 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 6n μg / mL or less in the pharmaceutical composition.

[0021] In another embodiment, there is provided a pharmaceutical composition comprising two or more different immunogenic conjugates, wherein (i) each immunogenic conjugate comprises a carrier polypeptide and a saccharide antigen, and the saccharide antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide; and (ii) the average amount of the carrier polypeptide per conjugate is 1 to 4 μg per dose of the pharmaceutical composition.

[0022] In another embodiment, there is provided a pharmaceutical composition comprising two or more different immunogenic conjugates, wherein (i) each immunogenic conjugate comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide; and (ii) the average concentration of the carrier polypeptide per conjugate is from 2 to 8 μg / mL in the pharmaceutical composition.

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

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

[0025] In another embodiment, there is provided a pharmaceutical composition comprising n different immunogenic conjugates, wherein (i) each of the n immunogenic conjugates comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the composition either does not contain the carrier polypeptide in unbound form or (iv) the composition contains the carrier polypeptide in unbound form, and the mass of the carrier polypeptide in unbound form in the composition is < 10% of the mass of its carrier polypeptides in the n immunogenic conjugates, whichever is applicable.

[0026] In another embodiment, a pharmaceutical composition is provided comprising n different immunogenic complexes, wherein (i) each of the n immunogenic complexes comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) the composition does not contain sugar antigens in an unbound form, or (iv) the composition contains at least one sugar antigen in an unbound form, the total mass of sugar antigens in an unbound form in the composition is <40% (e.g., ≤30%, ≤20%, or ≤10%) of the total mass of sugar antigens in the n immunogenic complexes.

[0027] In another embodiment, a pharmaceutical composition comprising 14 or more different immunogenic complexes is provided, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bound to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; and (ii) the total amount of carrier polypeptide per dose is <40 μg.

[0028] In another embodiment, a pharmaceutical composition comprising 14 or more different immunogenic complexes is provided, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bound to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; and (ii) the concentration of the carrier polypeptide per unit is ≤80 μg / mL.

[0029] In another embodiment, a method is provided for preparing multiple unit dose pharmaceutical compositions, wherein (i) the pharmaceutical composition comprises an immunogenic complex comprising a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the method comprises the steps of preparing a bulk composition comprising the immunogenic complex and packaging individual unit doses from the bulk composition into a plurality of separate containers. This method is ideally carried out aseptically. The individual containers can be sealed after each unit dose has been packaged in the individual container. The individual containers are ideally syringes.

[0030] In another embodiment, a pharmaceutical composition comprising two or more different immunogenic complexes is provided, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; and (ii) the composition is freeze-dried.

[0031] Another embodiment provides a method for preparing a pharmaceutical composition, the pharmaceutical composition comprising two or more different immunogenic complexes and an aluminum salt adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and (ii) the sugar antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide, the method comprising one of the following steps: (A) separately adsorbing each immunogenic complex onto an aluminum salt adjuvant and then mixing the individual adsorbed complexes together; (B) sequentially adsorbing each immunogenic complex onto an aluminum salt adjuvant; or (C) preparing a mixture of two or more (e.g., all of them) immunogenic complexes and combining this 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, which (i) has at least 80% sequence identity with SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue. That is, for example, Arg-192 and / or Arg-193 in SEQ ID NO: 1 may be deleted or substituted with a different amino acid. The nnAA residue may be introduced by substitution and / or insertion of an amino acid residue in SEQ ID NO: 1. An immunogenic complex (e.g., of a sugar antigen) can be prepared using the modified CRM197 carrier polypeptide via the nnAA residue therein.

[0033] Another embodiment provides a modified CRM197 carrier polypeptide, the carrier polypeptide having (i) at least 80% sequence identity with SEQ ID NO: 1; and (ii) an amino acid sequence containing an nnAA substitution in 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. Using a modified CRM197 carrier peptide, immunogenic complexes (e.g., of sugar antigens) can be prepared via nnAA residues within it.

[0034] In another embodiment, a modified CRM197 carrier polypeptide is provided, which (i) has at least 80% sequence identity with SEQ ID NO: 1; (ii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains an amino acid sequence in which one or more of the following amino acid residues (numbered according to SEQ ID NO: 1) have an nnAA substitution: 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. Using a modified CRM197 carrier peptide, immunogenic complexes (e.g., of sugar antigens) can be prepared via nnAA residues within it.

[0035] Another embodiment provides an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein (i) the carrier polypeptide comprises the amino acid sequence of SEQ ID NO: 4; and (ii) the sugar antigen is covalently bonded to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. Also provided is a pharmaceutical composition comprising two or more different immunogenic complexes, each comprising a carrier polypeptide and a sugar antigen, wherein (i) the carrier polypeptide in each complex comprises the amino acid sequence of SEQ ID NO: 4; and (ii) the sugar antigen in each complex is covalently bonded to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4.

[0036] In another embodiment, a syringe is provided which contains a pharmaceutical composition comprising two or more different immunogenic complexes, each comprising a carrier polypeptide and a pneumococcal sugar antigen, and which is a non-silicone-treated syringe. Ideally, the pharmaceutical composition in the non-silicone-treated syringe has 13 or more different pneumococcal complexes, and the carrier polypeptide may contain nnAA, but may instead be, for example, CRM197. Further details of the non-silicone-treated syringe are provided below. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1 shows the geometric mean antibody titers for each of the 32 serotypes shown in the 32-valent vaccine of the present invention against polysaccharide / alum preparations and Prevnar-13 (trademark), as described in the examples. [Modes for carrying out the invention]

[0038] Various details of methods, compositions, and techniques relating to the production of binding antigens are disclosed in International Publication No. 2018 / 126229 of the International Patent Application, which is incorporated herein by reference in its entirety.

[0039] immunogenic complex This invention relates, in general, to immunogenic complexes. These complexes include a carrier polypeptide that covalently binds to an antigen. This binding can convert a T cell-independent immunogen (e.g., sugars) into a T cell-dependent immunogen, thereby enhancing the elicited immune response (particularly in children). The complexes used herein include a covalent bond formed between the antigen and non-natural amino acid ("nnAA") residues within the carrier polypeptide. These nnAA residues can provide functional groups that facilitate the reaction with the antigen of interest.

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

[0041] The covalent bond is formed between the antigen and the nnAA residue in the carrier polypeptide. Preferably, the antigen does not bind to lysine residues in the carrier polypeptide, and more preferably, the antigen does not bind to native amino acid residues in the carrier polypeptide.

[0042] Useful carrier polypeptides contain T cell epitopes. Various such carrier polypeptides are known in the art, and approved vaccines include diphtheria toxoid (chemically treated toxin from Corynebacterium diphtheriae; "Dt"), tetanus toxoid (chemically treated tetanospasmin toxin from Clostridium tetani; "Tt"), protein D from Haemophilus influenzae ("PD" or "HiD"), outer membrane protein complex of serotype B meningococcal strain ("OMPC"), and CRM197 variant diphtheriae toxin.

[0043] A preferred carrier polypeptide that forms the basis of the carrier of the present invention 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 that of the natural diphtheria toxin, and thereafter the Gly→Glu substitution leads to the loss of toxic enzymatic activity in the protein: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK EFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKR GQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETA DNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESG HDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS.

[0044] This invention does not use natural (native) CRM197. Instead of using CRM197 containing SEQ ID NO: 1, it uses a modified amino acid sequence which contains at least one nnAA. These modified CRM197 carrier polypeptides are described in more detail below.

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

[0046] Another carrier polypeptide of interest is PD from Haemophilus 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 SEQ ID NO: 5 contains 36 Lys residues, some can be substituted with nnAA and then used for binding. Prediction and recognition of T cell epitopes related to PD have been reported by Hua et al. (2016) Clin Vaccine Immunol 23:155-61.

[0048] More broadly, any polypeptide containing a T cell epitope can be used as a carrier polypeptide. T cell epitopes can bind to MHC class II cells and interact with T cell receptors on the surface of CD4+ T cells, thereby enhancing the antibody response to the antigen or hapten bound to them (see, for example, Costantino et al. 2011, Expert Opin Drug Discov 6:1045-66). Micoli et al. (2018) Molecules 23,1451 outlines various carrier polypeptides and their selection criteria. Tontini et al. (2016) Vaccine 34:4235-42 discusses preclinical studies of 28 carrier polypeptides, including tests on their ability to induce antibodies against sugar antigens. Polyepitope carrier polypeptides containing diverse, broadly reactive (i.e., immunogenicity in the context of most human MHC class II molecules) human CD4+ T cell epitopes from various pathogen-derived antigens have been designed, such as 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. Patents 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 to design effective carrier polypeptides using them. See also U.S. Patent Application No. 2016-0101187. T cell epitopes present in known carriers (e.g., Tt, PD, CRM197) can be used. Various detoxified bacterial toxins have been successfully used as carriers, such as Tt, Dt, Pseudomonas aeruginosa exotoxin, and C. difficile A and B toxins.Many different carrier polypeptides have been used with respect to 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. This invention makes it possible to enhance the immunogenicity of an antigen of interest using any of these numerous carrier polypeptides, modified to contain at least one nnAA.

[0049] The nnAA-containing carrier polypeptides intended for use in the present invention can generally be prepared using the techniques disclosed in Section 6 of International Publication No. 2018 / 126229 ("Carrier Protein Production Methods"). Preferred carriers contain nnAA in addition to at least one T cell epitope of the carrier. If the T cell epitope region is unknown for a particular carrier, those skilled in the art can identify the epitope using standard techniques; 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 (including empirical and / or predictive approaches). It can also be confirmed that any particular modification of the carrier polypeptide sequence does not eliminate the desired T cell response to the binding antigen, such as sugars as described herein. The preferred group of carriers does not contain any modifications within the T cell epitope, including nnAA insertions or substitutions. Particularly preferred carriers contain at least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs. Particularly preferred carriers may 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 may be contained within the immunogenic complexes used herein. Typically, the antigens are saccharides. The term "saccharide" includes polysaccharides having 50 or more repeating units and oligosaccharides having fewer than 50 repeating units. Typically, polysaccharides have approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to approximately 2,000 (and sometimes more), and also, arbitrarily, approximately 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1,000 repeating units to approximately 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, or 1,900 repeating units. Oligosaccharides typically consist of approximately 6, 7, 8, 9, or 10 repeating units, or approximately 15, 20, 25, 30, or 35 repeating units, or approximately 40 or 45 repeating units.

[0051] Useful sugars incorporated into immunogenic complexes include those present in bacteria. These may be non-capsular sugars (such as exopolysaccharides, for example, Staphylococcus aureus exopolysaccharide), but bacterial capsular sugars are preferred.

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

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

[0054] Further details on these sugar antigens can be found in International Publication No. 2018 / 126229.

[0055] Antigens often do not inherently contain functional groups that are suitable or ideal for binding. Therefore, antigens may need to be functionalized before their binding to nnAA. Further details of such functionalization are given below.

[0056] Capsular sugars of Streptococcus pneumoniae The preferred antigen used in this invention is a capsular polysaccharide from Streptococcus pneumoniae. Streptococcus pneumoniae is a capsular, Gram-positive bacterium that can cause pneumonia, bacteremia, and meningitis. There are at least 90 different recorded serotypes of Streptococcus pneumoniae that carry capsular polysaccharides with serotype-specific repeating unit structures (see, for example, Kalin, M. Thorax 1998;53:159-162). As will be understood by those skilled in the art, it has been proposed that serotype 20 of Streptococcus pneumoniae is actually composed of two closely related serotypes, and their capsular polysaccharides exhibit significant cross-protection (Calix et al. 2012 J Biol Chem 287:27885-94). That is, as will be further recognized by those skilled in the art, serotype 20 refers to the sugars that have been previously classified in the art as serotype 20, and therefore, as disclosed by Calix et al., it may be either structurally 20A or 20B (a strain that has been previously classified in the art as serotype 20, but which may be genotypely either 20A or 20B). For example, the strain used to produce the serotype 20 polysaccharide in Pneumovax (Merck) is considered to be serotype 20A. In some cases, 20A may be preferred. In other cases, 20B may be preferred. Prevalence in the target population can be the basis for selection among these serotypes. Nevertheless, because the strains classified as 20, 20A, and 20B are serologically similar, selection among strains will not be of significant importance because they provide significant cross-protection 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 Capsular sugars can be derived from any of the following: 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, since only a subset of these serotypes typically causes clinically significant bacterial infections, the antigen can be a capsular sugar from any of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. Serotypes 6C, 7C, 15A, 15C, 16F, 20A, 20B, 23A, 23B, 24B, 31, 34, 35B, 35F, 37, and 38 have also become clinical concerns, so the antigen can be a capsular sugar from any of these Streptococcus pneumoniae serotypes.

[0058] When the present invention uses a complex of different serotypes of Streptococcus pneumoniae, it is preferable that the composition contains sugars from at least 14 different serotypes of Streptococcus pneumoniae (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). When the composition contains 14 or more serotypes, it is preferable that these include 13 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In addition to these 13 serotypes of Streptococcus pneumoniae, it is preferable that the composition contains one or more of the serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20 (or 20A or 20B), 22F, and / or 33F. Alternatively, in addition to the 13 serotypes mentioned above, the composition preferably contains one or more serotypes of Streptococcus pneumoniae (S. pneumoniae) 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. A useful combination of 15 or more (e.g., 16 or more) serotypes may include each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and may also include serotype 8. A useful combination of 20 or more (e.g., 21 or more) serotypes of Streptococcus pneumoniae includes each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. A useful combination of 24 or more serotypes includes each of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0059] The structure of the repeating units of capsule sugars in common serotypes of Streptococcus pneumoniae is described by Jones et al. (Jones C et al. An Acad Bras Cienc. 2005 Jun;77(2):293-324): Type 1 [→3)-D-AAT-α-Galp-(1→4)-α-D-GalpA(2 / 3OAc)-(1→3)-α-D-GalpA-(1→] Type 2 [→4)-β-D-Glcp-(1→3)-[α-D-GlcpA-(1→6)-α-D-Glcp-(1→2)]-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)β-L-Rhap-(1→] Type 3 [→3)-β-D-GlcA-(1→4)-β-D-Glcp-(1→] Type 4 [→3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp2,3(S)Py-(1→] Type 5 [→4)-β-D-Glcp-(1→4)-[α-L-PnepNAc-(1→2)-β-D-GlcpA-(1→3)]-α-L-FucpNAc-(1→3)-β-D-Sugp-(1→] Type 6B [→2)-α-D-Galp-(1→3)-α-D-Glcp-(1→3)-α-L-Rhap-(1→4)-D-Rib-ol-(5→P→] Type 9N [→4)-α-D-GlcpA-(1→3)-α-D-Glcp-(1→3)-β-D-ManpNAc-(1→4)-β-D-Glcp-(1→4)-α-D-GlcpNAc-(1→] Type 9V [→4)-α-D-GlcpA(2 / 3OAc)-(1→3)-α-D-Galp-(1→3)-β-D-ManpNAc(4 / 6OAc)-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→] Type 12F [→4)-[α-D-Galp-(1→3)]α-L-FucpNAc-(1→3)-β-D-GlcNAc-(1→4)-[α-D-Glc-(1→2)-α-D-Glc-(1→3)]-β-D-ManNAcA-(→] Type 14 [→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, where Table 1 shows the structures of 97 known serotypes. This table also discloses the percentage of acetylated sugar residues where acetylation is incomplete.

[0061] Capsular sugars can be O-acetylated. In some embodiments, capsular sugars from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F include sugars with a degree of O-acetylation 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 sugars can be determined by proton NMR (see, for example, Lemercinier & Jones (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247). Typically, the sugars used to prepare the complex retain at least 50% (e.g., 75%, or 100%) of the O-acetylation level found in the starting capsule sugars purified from bacteria.

[0062] Capsular sugars of Streptococcus pneumoniae can be obtained directly from the bacteria using isolation methods known to those skilled in the art (see, for example, the methods disclosed in U.S. Patent Publications 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498, and International Publication 2008 / 118752). Alternatively, they may be obtained from commercially available suppliers (e.g., ATCC).

[0063] The pneumococcus capsule sugar antigen used in the present invention is beneficial if it has a molecular weight of 10 kDa to 4,000 kDa, for example, 50 kDa to 3,000 kDa, or 100 kDa to 2,000 kDa. For example, such molecular weights are: 100 kDa to 2,000 kDa; 100 kDa to 1,750 kDa; 100 kDa to 1,500 kDa; 100 kDa to 1,250 kDa; 100 kDa to 1,000 kDa; 100 kDa to 750 kDa; 100 kDa to 500 kDa; 200 kDa to 4,000 kDa; 200 kDa to 3,500 kDa; 200 kDa The molecular weights may range from 3,000 kDa; 200 kDa to 2,500 kDa; 200 kDa to 2,000 kDa; 200 kDa to 2,000 kDa; 200 kDa to 1,750 kDa; 200 kDa to 1,500 kDa; 200 kDa to 1,250 kDa; 200 kDa to 1,000 kDa; 200 kDa to 750 kDa; or 200 kDa to 500 kDa. Further details and guidance regarding molecular weights are available in U.S. serial number 62 / 693,978, which is previously referenced herein.

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

[0065] Some embodiments of the present invention involve the use of two or more different complexes. With respect to pneumococcal capsular sugar complexes, this means that each "different" complex has sugars from different pneumococcal serotypes (when a single type of carrier polypeptide is used for each complex).

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

[0067] For example, the capsular sugar antigen may be purified from two different serotypes of Streptococcus pneumoniae. These two different capsular sugars can separately bind to a carrier polypeptide (which may be the same or different) to produce two different complexes. That is, with respect to bacterial capsular sugar complexes, the difference between two "different" complexes is typically that one contains capsular sugars from a first serotype or serogroup of a bacterial species, while the other contains capsular sugars from a second serotype or serogroup of that species, for example, capsular sugars from different serotypes of Streptococcus pneumoniae or capsular sugars from different serogroups of Neisseria meningitidis. The two complexes are also "different" if they contain antigenically different capsular sugars from multiple bacterial species, for example, the Hib sugar complex and the Neisseria meningitidis sugar complex.

[0068] A preferred polyvalent composition of the present invention contains n different immunogenic sugar complexes, wherein the sugar antigen in each of the n immunogenic complexes is different from the sugar antigen in the other n-1 immunogenic complexes. For example, if the composition contains antigens from a single bacterial species, then capsular sugars from n different serotypes or n serogroups of that species may be present.

[0069] This naming convention, related to "different" complexes, is used in the field of conjugate vaccines. For example, Glesby et al. (2015) J Infect Dis 212:18-27 refers to the Prevnar PCV13 vaccine as containing "13 different complexes" because it contains sugar antigens from 13 different pneumococcal serotypes, each individually conjugated to CRM197. Similarly, European Patent Application Publication No. 2932979 (EP-A-2932979) refers to an "immunogenic composition containing 13 different polysaccharide-protein complexes."

[0070] Specifically, the PCV7 Prevnar vaccine has 7 different complexes, the PCV13 Prevnar vaccine has 13 different complexes, the Menveo vaccine has 4 different complexes, the Menactra vaccine has 4 different complexes, the Nimenrix vaccine has 4 different complexes, the Menitorix vaccine has 2 different complexes, the Menhibrix vaccine has 3 different complexes, and the Synflorix vaccine has 10 different complexes, and so on.

[0071] The polyvalent composition of pneumococcal complexes preferably contains 13 or more different complexes, for example, 14, 15, 20, 21, 24, 25 or more. The preferred selection of serotypes for these compositions with more than 13 valents is described above.

[0072] For vaccines with a high number of complexes (e.g., containing more than 13 different complexes), it may be preferable to use multiple carrier polypeptides to reduce the possibility of carrier suppression (e.g., International Publication 98 / 51339 and International Publication 2011 / 110241). For example, in a polyvalent vaccine containing n different complexes, the first carrier polypeptide binds to ny different antigens (e.g., capsular sugars from different bacterial serotypes or serogroups), and the second carrier polypeptide binds to the remaining y antigens. In a similar manner, three, four or more carriers can be used with n antigens separated among them. When multiple carriers are used, at least the first carrier is an nnAA-containing carrier polypeptide according to the present invention. In preferred embodiments, at least the first and second carriers are nnAA-containing carrier polypeptides according to the present invention.

[0073] Non-natural amino acids As described above, the complex used herein includes a covalent bond between the antigen and a functional group within an nnAA residue in the carrier polypeptide. The side chain of the nnAA residue can provide a reactive functional group useful for binding the antigen to separate sites in the carrier polypeptide.

[0074] In general terms, nnAAs can be any amino acid that is not one of the usual 20 amino acids, but can be incorporated into a polypeptide during translation. Conveniently, nnAAs can be incorporated into polypeptides by modifying a tRNA molecule so that its codon incorporates an nnAA instead of a naturally occurring congener amino acid. One technique to achieve this involves using a "suppression codon," that is, a nucleotide triplet that is introduced into the coding sequence at a desired position and recognized by a specific tRNA capable of recognizing a natural stop codon (e.g., amber, ochre, or opal stop codon), but translation can continue, with the incorporation of an nnAA (thereby suppressing the natural stop codon).

[0075] The nnAA residue may be any of the nnAA residues described herein, or other residues identified as compatible with cell-based or cell-free protein synthesis (see, for example, Schultz et al. Annu Rev Biochem. 2010;79:413-44, particularly pp.418-420; and Chin et al. Annu Rev Biochem. 2014;83:5.1-5.30, which are incorporated herein by reference). Ideally, nnAAs do not occur naturally in cells through modifications of one of the usual 20 amino acids (e.g., pyrrolicin, selenocysteine, phosphotyrosine, formylmethionine, etc.).

[0076] In particular, preferred nnAAs used herein are those that have a side chain providing a functional group not present in any of the 20 natural amino acids and that can be incorporated during translation (in cell or cell-free systems). 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 discloses in detail a method for incorporating nnAA residues into carrier polypeptides using, for example, 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] nnAA may contain chemical groups suitable for "click" chemistry reactions at corresponding groups in the antigen of interest. Suitable chemical groups for "click" chemistry include azide groups (-N3), alkyne groups (-C≡C-), alkene groups (-C=C-), and 1,2,4,5-tetrazine groups. [ka] This includes, but is not limited to, phosphine groups (e.g., -P(Ph)2).

[0078] nnAA may be any of the following: 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)pyridine-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridine-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridine-3-yl)propanoic acid, or 2-amino-5-azidopentaic acid.

[0079] The most preferred nnAA used herein is the following pAMF: [ka] pAMF provides a very favorable reaction rate for complex formation (for example, much faster than using pAF when reacting with alkyne-containing sugar antigens in the SPAAC method).

[0080] nnAA may be a 2,3-disubstituted propanoic acid that retains: an amino substituent at the 2-position; and an azide-containing substituent, a 1,2,4,5-tetradinyl-containing substituent, or an ethynyl-containing substituent at the 3-position. Preferably, the substituent at the 3-position is an azide-containing substituent, in particular an azide-containing substituent that includes a terminal azide group bonded to the carbon atom at the 3-position via a bonding group. For example, the bonding group may include an arylene moiety that is optionally substituted and optionally contains heteroatoms. For example, the bonding group may include a 5 or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 nonhydrogen ring substituents.

[0081] nnAA can have the structure of the following general formula XII: [ka] (XII) In the formula, Ar comprises a five-membered or six-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 6Ar is selected from azide, 1,2,4,5-tetradinyl which may be C-substituted with a lower alkyl group, and ethynyl. In some embodiments, Ar does not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene group (i.e., Ar is -C6H4-). In other embodiments, Ar contains a nitrogen heteroatom and at least one further heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described below, and Ar may be, for example, pyridine or pyridazine. In a particularly preferred embodiment, Q1 is 1 and W 5 It is a lower alkylene, and also W 6 It is Azid.

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

[0083] In other embodiments, nnAA has the following general formula II: [ka] In the formula, 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 paragraphs

[0331] to

[0333] of U.S. Patent Application Publication No. 2014-0066598A1 by Stafford et al., which are incorporated by reference. This method involves the substitution of a hydroxyl group to the chloride in the corresponding aryl amino acid derivative using thionyl chloride, followed by the nucleophilic substitution of the chloride with the azide. Suitable aryl side chains containing amino acids are also available commercially.

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

[0086] Preparation of 1,2,4,5-tetrazine-containing amino acids relating to general formula III can be found, for example, in paragraphs

[0341] to

[0377] of U.S. Patent Application Publication No. 2016 / 0251336 by Yang et al., which are incorporated by reference. This method involves introducing Ar by Negishi coupling with aminopyridyl bromide, an amino / carboxyl-protected derivative of (R)-2-amino-3-iodopropanoic acid, followed by a reaction with a methylthio-1,2,4,5-tetrazine derivative that introduces a tetrazine moiety into the amino acid.

[0087] The nnAA may be an alkyne-containing nnAA. In one embodiment, this is a propargyl group. Various propargyl-containing amino acids, including their synthesis, are described 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. These 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] The nnAAs used herein are generally α-amino acids having a chiral center at the α-carbon, and it is preferable that they are L-stereoisomers.

[0089] The polypeptide carrier used in the present invention contains at least one nnAA residue. Preferably, the carrier polypeptide needs to contain multiple nnAAs, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues (or more). A carrier polypeptide having fewer than 10 nnAA residues is 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 that it contains only one species of nnAA (for example, the only nnAA in the carrier is pAMF). This allows the same conjugation chemistry to be used simultaneously for each nnAA. 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 to a different nnAA, but it is preferable to conjugate to a single species of nnAA within a carrier. Furthermore, when multiple different complexes are used (for example, different serotypes of Streptococcus pneumoniae), it may be preferable that each complex contains the same single species of nnAA. Also, when a composition contains multiple different complexes (for example, different serotypes of Streptococcus pneumoniae), it may be preferable that each complex contains the same carrier polypeptide.

[0091] nnAA can be incorporated into a carrier polypeptide by substitution or insertion (or by C-terminal or N-terminal elongation). In one embodiment, the nnAA residue is incorporated by substitution. Conveniently, lysine residues in natural polypeptides can be substituted with nnAA. For example, in CRM197, substitution can be made at one or more of the following positions in SEQ ID NO: 1 or 2: K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522, and K526. Substitution of nnAA (e.g., pAMF) at K33, K212, K244, K264, K385, and K526 (and, in one embodiment, not other positions) is preferred.

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

[0093] Ideally, nnAAs within a carrier polypeptide are surface-accessible residues. This allows for the evaluation of the usefulness of each site using the polypeptide's 3D structure, or by performing a wide range of substitutions of native amino acids for the nnAAs, followed by binding tests.

[0094] To preserve the function of the carrier polypeptide, it is preferable not to incorporate nnAA into the T cell activation epitope of the carrier polypeptide. The use of nnAA allows for the selective placement of binding sites, thereby preventing the T cell activation epitope of the carrier polypeptide from becoming an antigen-binding site. As described above, these epitopes are easily identifiable. For example, in the CRM197 study 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, International Publication No. 89 / 06974), various T-cell epitopes have been identified, for example, within residues P271-D290, V321-G383, and Q411-I457. That is, it is preferable to avoid introducing nnAA within the range of these regions of Sequence ID No. 1.

[0095] join Binding involves the formation of a covalent bond between the nnAA residue and the antigen. This requires reactive functional groups in both the nnAA and the antigen. Generally, a specific nnAA is selected for the carrier polypeptide because the nnAA already possesses a suitable functional group (e.g., the azide group of pAMF), while the antigen often does not inherently contain a functional group that is suitable or ideal for binding. In other words, the antigen may need to be functionalized before its binding to the nnAA.

[0096] Detailed technical information on conjugation can be found in Bioconjugate Techniques (Greg T Hermanson, 3rd edition, 2013). International Publication No. 2018 / 126229 discloses in detail how antigens can be functionalized and then conjugated to nnAAs. As mentioned above, useful nnAAs contain functional groups (e.g., azide groups) that are suitable for "click" chemistry reactions at functional groups in the antigen. That is, the functionalized antigen ideally contains groups suitable for such "click" reactions.

[0097] Therefore, broadly speaking, the binding is carried out by a method comprising the following three steps: (a) activating the antigen; (b) optionally derivatizing the activated antigen (e.g., by a linker or nucleophile) to introduce a reactive functional group not normally present on the antigen; and (c) binding the antigen to a carrier polypeptide via the group introduced in step (a) or, if present, in step (b). In some embodiments, step (a) includes an initial step of removing a blocking group on the antigen so that a specific functional group (e.g., hydroxyl, amine, thiol) is more readily activated. Steps (a) to (c) may essentially occur simultaneously (e.g., when adding a reactive moiety such as N-hydroxysuccinimide to the antigen), but in other embodiments, two or more of steps (a) to (c) are dispersed, with optional purification between steps.

[0098] As described above, since cross-linked complexes are preferred, it is also preferable to introduce multiple reactive functional groups per antigen molecule. For example, when activating a sugar molecule, it is possible to introduce multiple aldehyde groups or cyanate ester groups. These groups can then be derivatized, for example, to introduce reactive cyclooctyne that can subsequently react with the azide group in nnAA.

[0099] Antigens can be activated using a variety of chemical methods, including but not limited to: periodic acid oxidation (e.g., oxidation of a hydroxyl group on an adjacent carbon atom to give a reactive aldehyde group), as disclosed in International Publication No. 2011 / 110531; cyanylation, for example, using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP); hydroxyl activation with 1,1'-carbonyldiimidazole (CDI) followed by nucleophilic addition; or unmasking of endogenous aldehydes (e.g., reducing ends of sugars).

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

[0101] Activated antigens can directly bind to nnAA, but typically, the activated group is derivatized to introduce a functional group that exhibits good reactivity with the functional group of nnAA. For example, an alkynyl group can be introduced. A bifunctional reagent containing an amino group and an alkyne group can react with the aldehyde group introduced into the antigen (e.g., via reductive amination), thereby leaving behind a pendant alkyne that can react with nnAA. For example, a bifunctional reagent containing an amino group and a DBCO functional group can be used.

[0102] In one embodiment, nnAA reacts with an alkynyl group (e.g., a propargyl group) in the antigen. The alkyne group in the antigen is ideally suited to react with an azide group in nnAA using reactions known in the art, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC), ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), or azide-alkyne 1,3-dipolar cycloaddition of hysgen. The alkynyl group may have a molecular environment that increases its reactivity, for example, it may be intracyclic. For example, alkylenes may be intracyclic (may contain heteroatoms), such as a diaryl-strained cyclooctin ring (e.g., DBCO). This reaction may be a [3+2] cycloaddition, known in the art as strain-enhanced azide-alkyne cycloaddition (SPAAC). DIFO and DBCO reagents are readily available for these reactions.

[0103] Alkyne-containing rings useful in the SPAAC reaction include difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne. These can be used with pendant functional groups for binding to activated antigens (e.g., with pendant aminos for binding to aldehydes or cyanide esters) using, for example, one 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 any of the following, for example, 4, 5, 11, 12, or 13.

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

[0106] The composites 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 composite has a molecular weight of about 750 kDa to about 5,000 kDa. In some embodiments, the composite has a molecular weight of about 800 kDa to about 2,800 kDa. In some embodiments, the composite has a molecular weight of about 850 kDa to about 2,800 kDa. In some embodiments, the composite has a molecular weight of about 900 kDa to about 2,800 kDa. In some embodiments, the composite has a molecular weight of about 950 kDa to about 2,800 kDa. In some embodiments, the composite has a molecular weight of about 1,000 kDa to about 2,800 kDa. The molecular weight of the complex is calculated using molecular sieve chromatography (SEC) combined with multi-angle laser scattering (MALS).

[0107] The complex of the present invention comprises an antigen (e.g., sugars) and a carrier polypeptide, and the weight ratio of these two components can be used as a parameter defining the complex. A higher antigen-to-carrier weight ratio for sugar-carrier complexes allows for the supply of more sugar antigen with a smaller amount of carrier polypeptide. For pneumococcal conjugate vaccines, this ratio is typically in the range of 0.3 to 3.0, but can vary depending on the serotype and the nature of the conjugation chemistry (Annex 2: Recommendations for the production and control of pneumococcal conjugate vaccines; WHO Technical Report Series, No. 927, 2005). The ratio for the commercially available vaccine Prevnar-13 (trademark Prevnar-13) is 0.9. With respect to compositions containing a complex of multiple serotypes of Streptococcus pneumoniae (e.g., 13 or more serotypes), the ratio with respect to the complete composition is ideally greater than 1.0 (i.e., excess weight of pneumococcal glycoantigen), preferably 1.5 or higher (e.g., in the range of 1.5 to 3.0, or preferably 1.5 to 2.0).

[0108] Modified CRM197 carrier polypeptide As described above, the carrier polypeptide of primary interest in this specification is a modified form of CRM197. Therefore, preferred carrier polypeptides used in the present invention contain an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 1 (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%). For example, the carrier polypeptide may contain the amino acid sequence of SEQ ID NO: 1, except that it contains up to 10 nnAAs, as described above.

[0109] Sequence ID 1 contains an Arg-Arg dipeptide sequence at positions 192-193. This sequence can be subjected to proteolytic cleavage under certain 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 the Arg-Arg dipeptide sequence. For example, Arg-192 and / or Arg-193 in Sequence ID 1 may be deleted or substituted with a different amino acid. Therefore, a preferred carrier polypeptide contains an amino acid sequence that (i) has at least 80% sequence identity with respect to Sequence ID 1 (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%), (ii) does not contain the Arg-Arg dipeptide sequence, and (iii) contains at least one (e.g., at least two, and preferably more, as described above) nnAA residues.

[0110] One such amino acid sequence is sequence number 2, which differs from sequence number 1 in that it has an Arg→Asn substitution at position 193: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVR N SVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS.

[0111] Any embodiment described herein or in International Publication No. 2018 / 126229 that references Sequence ID No. 1 can be implemented using Sequence ID No. 2 instead.

[0112] In other words, a carrier polypeptide containing the amino acid sequence of SEQ ID NO: 2 is provided, wherein SEQ ID NO: 2 is modified to include 1 to 10 (e.g., 3 to 9, 2 to 8, 2 to 6, 3 to 6, or 4 to 6) nnAA residues. These modifications of nnAA residues can be incorporated into SEQ ID NO: 2 as insertions and / or substitutions (e.g., SEQ ID NO: 4, which includes 6 Lys→nnAA substitutions). It is preferable that the residue Asn-193 of SEQ ID NO: 2 is not substituted with nnAA. Using this carrier polypeptide, immunogenic complexes (e.g., of sugar antigens) can be prepared via the nnAA residues therein.

[0113] In some embodiments, these carrier polypeptides contain an amino acid sequence upstream and / or downstream of SEQ ID NO: 1 or 2. That is, for example, they may contain a methionine residue upstream of the N-terminal amino acid residue of SEQ ID NO: 1 or 2. This methionine residue may be formylated. While a methionine residue is not present at this position in wild-type CRM197, it may 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 contain an amino acid sequence upstream of the N-terminus of SEQ ID NO: 1 or 2, except for an optional methionine, and (ii) does not contain an amino acid 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 nnAA, and ideally, only one residue in SEQ ID NO: 1 is substituted with nnAA, for example, only a Lys residue is substituted. When one or more residues in SEQ ID NO: 1 are substituted with nnAA, it is preferable to use the same nnAA at each position, for example, each substitution position is pAMF. As described above, in some embodiments, residues other than Lys are substituted.

[0115] A carrier polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, wherein 2 to 9 substitutions by nnAA residues (e.g., Lys → nnAA substitution, preferably Lys → pAMF), is preferred, and ideally, comprises 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 broader binding of the 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 could 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. Of these, the first region 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. That is, one or more (e.g., three) Lys residues in the 374 amino acids at the N-terminus of Sequence ID No. 1 or 2, and one or more (e.g., three) Lys residues in the 162 amino acids at the C-terminus, can be substituted with nnAA, for example, within pAMF.

[0118] A preferred embodiment of the 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 residues K24, K33, K37, K39, K212, K214, K227, K264, K385, K522, and K526 are substituted with nnAA (e.g., 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 (Sequence ID 3).

[0119] Another sequence of these sequences is sequence number 4 below, in which each X represents nnAA (preferably the same nnAA such as pAMF): MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (Sequence ID 4).

[0120] Sequence IDs 3 and 4 maintain good solubility and provide a good immunogenic response when bound to the capsular sugars of Streptococcus pneumoniae, while being very well expressible in cell-free protein synthesis systems. Sequence ID 4 lacks the native Arg-Arg dipeptide.

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

[0122] International Publication No. 2018 / 126229 describes several amino acid residues suitable for nnAA substitution (e.g., Lys-24, Lys-33, Lys-37, Lys-39, Lys-212, Lys-214, Lys-227, Lys-244, Lys-264, Lys-385, Lys-522, Lys-526, Phe-12, Phe-53, Phe-123, Phe-127, Phe-140, Phe-167, Phe-250, Phe-389, Phe-530, or Phe-531, numbered according to Sequence ID No. 1 herein). Other substitutable residues 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] The present invention also provides a polypeptide that (i) has at least 80% sequence identity (for example, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, or preferably ≥98%) with respect to Sequence ID No. 1, (ii) does not contain an Arg-Arg dipeptide sequence, and (iii) contains an amino acid sequence comprising at least one nnAA residue, and is having an N-terminal methionine and / or is in monomeric form.

[0124] These CRM197-derived carrier polypeptides can be used in the same binding methods as CRM197 has been used in prior art (see, for example, Broker et al. 2011, International Publication No. 2015 / 117093, etc.), but with the improvement of enabling site-specific binding via nnAA residues. They are generally used in monomeric form rather than forming polypeptide polymers with other CRM197 or CRM197-derived subunits. Similarly, they generally contain at least one disulfide crosslink, for example, between Cys-186 and Cys-201 (numbered according to Sequence ID No. 1), and optionally between Cys-461 and Cys-471.

[0125] Furthermore, the present invention provides immunogenic complexes comprising any of these various carrier polypeptides, each of which is bound to a sugar antigen via at least one of its nnAA residues. These carrier polypeptides are particularly useful for binding to the capsular sugars of Streptococcus pneumoniae via their nnAA residues. The immunogenic complexes prepared in this manner can be combined to form the polyvalent compositions discussed elsewhere in this specification.

[0126] In other words, the present invention provides an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein (i) the carrier polypeptide has the amino acid sequence of SEQ ID NO: 4, for example, the amino acid sequence of SEQ ID NO: 4 in which each X is pAMF, and (ii) the sugar antigen is covalently bonded to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. The present invention also provides a polyvalent pharmaceutical composition comprising two or more such immunogenic complexes.

[0127] In other words, the present invention provides a pharmaceutical composition comprising multiple different complexes (for example, different serotypes of Streptococcus pneumoniae), wherein each of the complexes comprises a carrier polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0128] The present invention also provides an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein (i) the carrier polypeptide has the amino acid sequence of SEQ ID NO: 4, (ii) the sugar antigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4, and (iii) the sugar antigen is a capsular sugar from any of the 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 of Streptococcus pneumoniae. These individual complexes can be combined to produce the polyvalent pharmaceutical compositions of the present invention.

[0129] Furthermore, this disclosure provides polynucleotides encoding carrier polypeptides described herein. In another embodiment, this disclosure provides an expression vector comprising such polynucleotide. In another embodiment, this disclosure provides a host cell comprising such expression vector.

[0130] Adjuvant The pharmaceutical composition of the present invention may contain an aluminum salt adjuvant. This adjuvant can enhance the immunogenicity of the complex within the pharmaceutical composition. The complex within the composition can be adsorbed onto the aluminum salt adjuvant.

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

[0132] The adjuvant commonly known as "aluminum hydroxide" is typically an aluminum oxyhydroxide salt, which is usually at least partially crystalline. Aluminum oxyhydroxide, which can be represented by the general formula AlO(OH), has, in infrared (IR) spectroscopy, particularly an absorption band at 1070 cm -1 and a strong intensity shoulder at 3090 - 3100 cm -1 which allows it to be distinguished from other aluminum compounds such as, for example, Al(OH)3 (Chapter 9 of Powell & Newman). The crystallinity of the aluminum hydroxide adjuvant is reflected by the width of the diffraction band at the half-height width (WHH), and particles with low crystallinity show a greater broadening of the spectral line width due to their smaller crystal size. As the WHH increases, the surface area increases, and it has been found that adjuvants with high WHH values have greater antigen adsorption performance. The fiber morphology (as seen, for example, in transmission electron micrographs) is typically, for aluminum hydroxide adjuvants, needle-like particles with a diameter of about 2 nm. The pI of the aluminum hydroxide adjuvant is typically about 11, that is, at physiological pH, the adjuvant itself has a positive surface charge. For the aluminum hydroxide adjuvant, an adsorption performance of 1.8 to 2.6 mg of protein per 1 mg of Al +++ has been reported at pH 7.4.

[0133] Adjuvants commonly known as "aluminum phosphate" are typically aluminum hydroxyphosphate, 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 the phosphate for the hydroxyl group 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 the hydroxyl group. For example, 3164 cm⁴ -1 The IR spectral band (for example, when heated to 200°C) suggests the presence of a structural hydroxyl group (Powell & Newman, Chapter 9).

[0134] PO4 / Al aluminum phosphate adjuvant 3+ The molar ratio is generally 0.3 to 1.2, preferably 0.8 to 1.2, and more preferably 0.95 ± 0.1. The aluminum phosphate is generally amorphous, and is particularly amorphous in the hydroxyphosphate form. A typical adjuvant is 0.6 mg Al 3+ This is amorphous aluminum hydroxyphosphate with a PO4 / Al molar ratio of 0.84 to 0.92, containing 1 ml / ml. Aluminum phosphate is generally granular (e.g., plate-like in form when viewed in transmission electron microscopy, with primary particles within a 50 nm range). Typical particle diameters are in the range of 0.5 to 20 μm (e.g., about 5 to 10 μm) after adsorption of any antigen. In aluminum phosphate adjuvants, at pH 7.4, Al +++ It has been reported that it can adsorb 0.7 to 1.5 mg of protein per 1 mg.

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

[0136] The concentration of aluminum ions in the composition administered to the patient is preferably less than 2.5 mg / ml, for example, ≤2 mg / ml, ≤1 mg / ml, etc. The preferred maximum concentration is ≤1.7 mg / mL. +++ The 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 with a diameter of 1 to 10 μm. Compositions can include mixtures of both aluminum hydroxide adjuvants and aluminum phosphate adjuvants.

[0138] If a composition contains multiple complexes, each of which adsorbs to an aluminum salt adjuvant, then each complex can be adsorbed individually to the aluminum salt and then mixed, or they can be added sequentially to the aluminum salt to form a mixed complex composition. Mixtures from either approach can be used.

[0139] Excipients for pharmaceutical compositions The pharmaceutical compositions of the present invention generally comprise one or more pharmacologically acceptable excipients. A comprehensive discussion of such excipients can be found in the Handbook of Pharmaceutical Excipients (ed. Rowe et al.), 6th edition 2009.

[0140] Pharmaceutical compositions are preferably in aqueous form, especially when administered, but they can also exist in a dry form (e.g., as a lyophilized product) that can be converted to an aqueous form for administration.

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

[0142] Pharmaceutical compositions may contain physiological saline, such as sodium salts, to control tonicity. Sodium chloride (NaCl) is typical, and can be present in concentrations of 1 to 20 mg / ml, for example, 10 ± 2 mg / ml or 9 mg / ml. Other possible salts include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, and calcium chloride. 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 may contain organic acids such as acetic acid or succinic acid. These may be part of a buffering system.

[0144] The pharmaceutical composition may contain sugar alcohols such as mannitol or sorbitol. The pharmaceutical composition may contain sugars such as sucrose or glucose.

[0145] The pharmaceutical composition may contain a surfactant. Preferred surfactants include, but are not limited to, polysorbate 20, polysorbate 80, and sodium dodecyl sulfate (SDS). In some embodiments, the surfactant is present in a concentration of 0.0003% to 0.3% (w / w), for example, 0.01% to 0.03%. Polysorbate 80 is a preferred surfactant.

[0146] Pharmaceutical compositions may contain preservatives such as thiomersal or 2-phenoxyethanol. Preferably, the composition should be substantially free of mercury materials (e.g., <10 μg / ml), for example, free of thiomersal. Compositions that do not contain mercury are more preferable. The inclusion of preservatives may be particularly useful when the composition contains aluminum salt adjuvants, as their insolubility means that the composition is a cloudy-looking suspension that can block the composition from generally adversely affecting bacterial growth. Preservatives are also particularly useful when the composition is to be used multiple times, for example, in a multi-use vial. However, pharmaceutical compositions are often free of preservatives.

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

[0148] The pharmaceutical composition typically has a pH of 5.0 to 9.5, for example, 5.0 to 8.0 or 6.0 to 8.0.

[0149] The pharmaceutical composition is preferably non-pyrogenic and contains, for example, <1 EU (endotoxin unit, standard scale) per dose, and more preferably <0.1 EU per dose.

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

[0151] The pharmaceutical composition is preferably gluten-free.

[0152] The pharmaceutical composition is suitable for administration to patients, which are animals (and especially humans), and includes both human and veterinary use.

[0153] Pharmaceutical compositions can be prepared in the form of unit doses. In some embodiments, the unit dose may be 0.1 to 1.0 ml, for example, about 0.25 ml or preferably about 0.5 ml. Such amounts are ideally for injection in humans.

[0154] complex concentration The pharmaceutical composition may contain multiple immunogenic complexes. Currently approved meningococcal conjugate vaccines contain capsular sugars from four different serogroups, and approved pneumococcal conjugate vaccines contain capsular sugars from seven, ten, or thirteen different serotypes. That is, the composition of the present invention may contain, for example, three to fifty different complexes (e.g., 14, 15, 20, 21, 24, 25, or more). For example, each of these complexes may contain capsular sugars from different serotypes or serogroups of the same bacterial 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 these n complexes may be 3 n μg or less per dose. In other words, the average amount of carrier polypeptide per complex is less than 3 μg. The total amount may be, for example, n to 2.5 n μg per dose.

[0156] When a pharmaceutical composition contains n different immunogenic complexes, the total amount of sugar antigens in these n complexes may be 4.4 nμg or less per dose. In other words, the average amount of sugars per complex is less than 4.4 μg. The total amount may be, for example, 0.4 n to 4.4 nμg per dose, and for example, 1.1 n to 2.2 n.

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

[0158] When a pharmaceutical composition contains n different immunogenic complexes, the total concentration of sugar antigens related to those n complexes may be 8.8 n μg / mL or less. In other words, the average concentration of sugars per complex is less than 8.8 μg / mL. The total concentration may be, for example, between 0.8 n and 8.8 n μg / mL, or between 2.2 n and 4.4 n μg / mL.

[0159] In some embodiments, the total amount of bound carrier polypeptide in a unit dose of the polyvalent pharmaceutical composition of the present invention may be 4 to 128 μg, for example, 8 to 64 μg or 16 to 48 μg. The concentration of bound carrier polypeptide in the polyvalent pharmaceutical composition of the present invention may be 8 to 256 μg / mL, for example, 16 to 128 μg / mL or 32 to 96 μg / mL.

[0160] In some embodiments, the total amount of bound sugar antigen in a unit dose of the polyvalent pharmaceutical composition of the present invention may be 10 to 120 μg, for example, 20 to 90 μg or 30 to 60 μg. The concentration of bound sugar antigen in the polyvalent pharmaceutical composition of the present 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 described above, the pharmaceutical composition may contain multiple immunogenic complexes, for example, 3 to 50 different complexes (e.g., 14, 15, 20, 21, 24, 25, or more). For example, each of these complexes may contain capsular sugars from different serotypes or serogroups of the same bacterial species.

[0162] In some embodiments, the composition does not contain the carrier polypeptide of the complex in an unbound form. In other embodiments, the unbound carrier polypeptide is present at a low level, however, the mass of the unbound carrier polypeptide in the composition is <10% (e.g., <5% or <2%) of the total mass of the carrier polypeptide in the n immunogenic complex of the composition.

[0163] In some embodiments, the composition does not contain complex sugars in an unbound form. In other embodiments, unbound sugars are present at low levels, however, the mass of unbound sugars in the composition is <10% (e.g., <5% or <2%) of the total mass of sugars in the n immunogenic complexes of the composition.

[0164] Containers, delivery devices, etc. Pharmaceutical compositions containing immunogenic complexes can be packaged in sterile containers, delivery devices, etc. Sterilization can be maintained by airtight sealing of the container. Suitable containers include, but are not limited to, vials, syringes, nebulizers, sprays, inhalers, skin patches, etc. Vials and syringes are preferred.

[0165] Immunogenic compositions are often contained in vials. Vials are preferably made of plastic or, more preferably, glass. Vials are sealed after filling, but the seal can be broken just before use. Vials are preferably sterilized before the composition is added and then sealed. To avoid problems in latex-sensitive patients, vials may be sealed with latex-free stoppers, and preferably all packaging materials are latex-free. Vials ideally contain a single dose of the composition, but may also contain more than one dose (multi-use vials), for example, 10 doses. Preferred vials are made of colorless glass.

[0166] The vial may have a lid (e.g., a Luer lock) fitted to allow insertion of a syringe into the lid, facilitating the movement of material between the vial and the syringe (in both directions). After removing the syringe from the vial, a needle can then be attached, and the composition can be administered to the subject. It is preferable that the lid be positioned inside the seal or cover such that the seal or cover must be removed before the lid can be accessed. The vial may have a lid that allows for sterile removal of its contents, particularly for multi-dose vials.

[0167] The composition can be contained within a delivery device in a state ready for administration to the target at any time. The composition can be transferred to the delivery device (e.g., from a vial) immediately before use, or the composition can be placed into the delivery device during the manufacturing stage (e.g., in the form of a pre-filled syringe).

[0168] The syringes used in this invention may be made of glass or plastic (for example, of a cycloolefin polymer or cycloolefin copolymer). The syringe (especially a glass syringe) may be silicon-treated. Non-silicon-treated syringes can also be used, for example, with Terumo's i-Coating™ system, which is available in Terumo's PLAJEX™ syringes, or with Daikyo's CZ™ syringes having an ethylene tetrafluoroethylene (ETFE) copolymer, or with TriboGlide™ syringes having a perfluoropolyether (PFPE). A carbon film may be used instead of silicon treatment (see, for example, JP 2001190665). Silicon-free syringes are also disclosed in JP 2011212183. A non-silicone treated syringe including a plunger stopper, such as the one disclosed in European Patent Application Publication No. 0375778, may also be used, namely, the stopper having a thermoplastic elastomer that is at least partially covered with a thermoplastic resin layer having a low coefficient of dynamic friction.

[0169] When a composition is contained in a syringe, the syringe may have a needle that attaches to the syringe for injection or injecting the contents of the syringe into a container. The syringe may be supplied with a needle that is already attached. If a 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 sterile at the time of use and may be sealed. Safety needles may be used. Typical needles are 1 inch, 23 gauge, 1 inch, 25 gauge, and 5 / 8 inch, 25 gauge. Needles of 1 / 2 inch to 1 and 1 / 2 inches, 22 to 25 gauge can be used. If the syringe and needle are packaged separately, it is preferable that the needle be fitted with a butyl rubber shield.

[0170] The syringes are provided with peel-off labels on which the lot number and the expiration date of the contents can be printed, facilitating record keeping. A stopper can be provided on the plunger inside the syringe to prevent accidental detachment of the plunger during aspiration. The syringes may have a latex rubber cap and / or plunger, but latex-free rubber such as latex-free cyclobutyl rubber or latex-free isoprene bromobutyl rubber can be used. Generally, the syringes have a tip cap to seal the tip before needle insertion, and the tip cap is preferably made of butyl rubber such as latex-free isoprene bromobutyl rubber. Useful syringes include, for example, those commercially available under the trade name "Tip-Lok" (trademark).

[0171] The container may have markings indicating half-dose amounts, for example, to facilitate delivery to children. For example, a syringe containing a 0.5 ml dose may have a marking indicating a 0.25 ml dose. The syringe itself may have a volume exceeding 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] When using glass containers (e.g., syringes or vials), it is preferable that they be made of borosilicate glass rather than soda-lime glass.

[0173] The container may be packaged with a leaflet containing vaccine details, such as administration instructions and details of the antigens in the vaccine (for example, in the same box). These instructions may also include warnings, such as the importance of having an adrenaline solution readily available in case of anaphylactic reactions after vaccination. Multiple containers may be packaged together, for example, in the same box.

[0174] Pharmaceutical compositions can exist in the form of single doses per container (e.g., per syringe or per vial). Rather than manufacturing each unit dose individually, a bulk composition is prepared, and the unit doses are extracted and individually packaged in those containers. That is, for example, multiple unit doses are extracted from the bulk and each unit dose is transferred to a separate container, for example, a syringe or vial.

[0175] Increased immune response It is possible to administer immunogenic complexes to mammalian subjects to elicit a protective immune response against the antigens in the complexes. These are administered in the form of pharmaceutical compositions. Since the compositions may contain multiple immunogenic complexes, as described elsewhere in this specification, it is possible to simultaneously elicit protective immune responses against multiple antigens.

[0176] In other words, the present invention provides a method for inducing a protective antibody response against one or more antigens in a mammalian subject by administering an antigen complex to that subject.

[0177] Furthermore, the present invention provides a complex, such as the one disclosed herein, for use in eliciting a protective antibody response.

[0178] Furthermore, this specification provides for the use of a complex as disclosed herein in the manufacture of pharmaceuticals for eliciting a protective antibody response.

[0179] Furthermore, the present invention provides (i) a method for inducing a protective antibody response against multiple antigens in a mammalian subject by administering the polyvalent composition of the present invention to said subject; (ii) the polyvalent composition of the present invention for use in inducing a protective antibody response; and (iii) the use of multiple complexes as disclosed herein in the manufacture of a polyvalent pharmaceutical composition for inducing a protective antibody response against multiple antigens.

[0180] The ability to elicit a protective immune response means that the complex can be used to prevent invasive diseases caused by Streptococcus pneumoniae, otitis media caused by Streptococcus pneumoniae, pneumonia caused by Streptococcus pneumoniae, and to provide active immunity to individuals at risk of exposure to Neisseria meningitidis, thereby preventing invasive diseases.

[0181] Pharmaceutical compositions can be prepared in various forms. For example, a composition can be prepared as an injectable, such as a solution or suspension. Injectable preparations for intramuscular administration are typical. For humans, an injection volume of about 0.5 ml is preferred. That is, the preferred unit dose is about 0.5 ml. Administration by intramuscular injection is typically done, for example, in the anterolateral aspect of the thigh of an infant, or in the deltoid muscle of the upper arm of a toddler, child, or adult.

[0182] The complex is typically administered according to a multi-dose schedule. Multi-dose administration may also be used in the primary immunization schedule and / or booster immunization schedule. Administration of multi-dose administration (typically two doses) is particularly useful in immunonatal patients. Multi-dose administration is typically spaced at least one week apart (e.g., approximately 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, etc.).

[0183] General The word "comprising" encompasses not only "including" but also "consisting." For example, a composition that "comprises" X may consist exclusively of X, or it may include something additional, such as X + Y.

[0184] The word "approximately" in relation to the numerical value x is optional; for example, it can mean x ± 10%.

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

[0186] In the context of two amino acid sequences, the term "sequence identity" refers to two sequences that, when aligned with the maximum match across a comparison window using a sequence comparison algorithm (e.g., BLASTP), are identical or have a specific proportion of identical amino acid residues. The proportion of identity is determined relative to the full-length reference sequences disclosed herein, such as the reference sequence shown in SEQ ID NO: 1 or 2. A method for calculating sequence identity as provided herein is the BLASTP program, which has default values ​​set to word length (W) = 3 and expected value (E) = 10, and a BLOSUM62 score matrix (see, for example, Henikoff & Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). See, for example, 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 saturated straight-chain or branched-chain hydrocarbons having 1 to 6 carbon atoms, i.e., C1 to C6 alkyls. In some embodiments, the lower alkyl is a primary, secondary, or tertiary hydrocarbon. The term includes both saturated and unsaturated parts. See also U.S. Patent Publication No. 2014 / 0066598. The term “lower alkylene” refers to the alkylene radical of a lower alkyl.

[0188] Unless otherwise specified, all technical and scientific terms used herein have their commonly understood meanings. In particular, practitioners are directed to 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] A cell-free synthesis method is described in Spirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. A method for incorporating unnatural amino acids into proteins using cell-free synthesis is described in Shimizu et al. (2006) FEBS Journal, 273, 4133-4140, and also in Chong (2014) Curr Protoc Mol Biol. 108:16.30.1-11.

[0190] In some embodiments, the present invention does not include compositions in which SEQ ID NO: 3 is used as a carrier polypeptide for each of the 24 serotypes of Streptococcus pneumoniae: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F (as illustrated in International Publication No. 2018 / 126229). More generally, in some embodiments, the present invention does not include compositions in which SEQ ID NO: 3 is used as a carrier polypeptide for each of the complexes in a polyvalent composition.

[0191] List of embodiments Embodiment I-1 A sterile container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

[0192] Embodiment I-2 An airtight container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide. Suitable containers for airtight sealing include, for example, vials. The contents are preferably sterilized during airtight sealing.

[0193] Embodiment I-3: The container of Embodiment I-1 or I-2, for example, a sterile glass container such as a vial.

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

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

[0196] Embodiment I-6 A pharmaceutical composition comprising two or more different immunogenic complexes and an aluminum salt adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bound to the carrier polypeptide via non-natural amino acid residues within 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 complexes and an aluminum phosphate adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural amino acid residue within 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 complexes, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide, and (ii) the amount of the pharmaceutical composition is 0.25 to 1.25 mL.

[0199] Embodiment I-9 A pharmaceutical composition comprising two or more different immunogenic complexes and a preservative, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

[0200] Embodiment I-10 A preservative-free pharmaceutical composition comprising two or more different immunogenic complexes, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

[0201] Embodiment I-11 A pharmaceutical composition comprising two or more different immunogenic complexes, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide, and (ii) the composition having a weight osmolality of 200 to 400 mOsm / kg.

[0202] Embodiment I-12 A pharmaceutical composition comprising two or more different immunogenic complexes and at least one excipient, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, and (ii) 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 complexes (i) Each of the n immunogenic complexes comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; (ii) n is an integer from 3 to 50, and (iii) The total amount of carrier polypeptides in the n-type immunogenic complex is 3 nμg or less per dose; (iv) The total concentration of carrier polypeptides in the n-type immunogenic complex is 6 n μg / ml or less; (v) The total amount of sugar antigens in the n-type immunogenic complex is 3 nμg or less per dose; (vi) The total concentration of sugar antigens in the n-type immunogenic complex is 6 n μg / mL or less; (vii) The average amount of carrier polypeptide per complex is 1 to 4 μg per dose; (viii) The average concentration of the carrier polypeptide per complex is 2 to 8 μg / mL; (ix) The average amount of sugar antigen per complex is 1 to 4 μg per dose; (x) The average concentration of sugar antigen per complex is 2 to 8 μg / mL; (xi) The composition does not contain carrier polypeptides in an unbound form; (xii) The composition contains a carrier polypeptide in an unbound form, and the mass of the carrier polypeptide in the composition is <10% of the mass of the carrier polypeptide in the n immunogenic complexes; (xiii) The composition does not contain sugar antigens in an unbound form; and / or (xiv) The pharmaceutical composition comprising at least one sugar antigen in an unbound form, wherein the total mass of the sugar antigen in the composition in an unbound form is <10% of the total mass of the sugar antigen in the n immunogenic complexes.

[0204] Embodiment I-14 A method for preparing multiple unit dose pharmaceutical compositions, wherein (i) the pharmaceutical composition comprises an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the method comprises the steps of preparing a bulk composition comprising the immunogenic complex and packaging individual unit doses from the bulk composition into a plurality of separate containers.

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

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

[0207] Embodiment I-17 (i) having at least 80% sequence identity with SEQ ID NO: 1; and (ii) a carrier polypeptide comprising an amino acid sequence in which one or more of the following amino acid residues (numbered according to SEQ ID NO: 1) contain an nnAA substitution: 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 according to Embodiment I-16 or I-17, wherein Arg-193 in SEQ ID NO: 1 is substituted with a different amino acid, such as Asn.

[0209] Embodiment I-19 An immunogenic complex comprising the carrier polypeptide described in Embodiment I-16, I-17, or I-18, which is bound to an antigen via an nnAA residue within the carrier polypeptide.

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

[0211] Embodiment I-21 A pharmaceutical composition comprising two or more different immunogenic complexes as described in Embodiment I-20.

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

[0213] Embodiment I-23 A container, device, composition, method, polypeptide, or complex according to any of the embodiments described above, wherein the lysine in the natural sequence of the carrier polypeptide is substituted with at least one nnAA.

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

[0215] Embodiment I-25 A container, device, composition, method, polypeptide, or complex according to 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 replaced with at least one nnAA.

[0216] Embodiment I-26 A container, device, composition, method, polypeptide, or complex according to any of the embodiments described above, 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 according to any of the embodiments described above, wherein nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

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

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

[0220] Embodiment I-30 A container, device, composition, method, polypeptide, or complex according to any of the embodiments described above, wherein the antigen is a capsular sugar of a serotype of Streptococcus 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 complex according to any of the embodiments described above, wherein the ratio (w / w) of sugars to the carrier polypeptide in the complex is greater than 1.

[0222] Embodiment I-32 A container, device, composition, method, polypeptide, or complex according to any of the embodiments described above, wherein the carrier polypeptide comprises three or more nnAA residues, and the complex 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 embodiments described above, wherein the complex has a molecular weight from 900 kDa to 5 MDa.

[0224] Embodiment I-34 The pharmaceutical composition is A complex of capsular sugars from two or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A complex of capsular sugars from 14 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A complex of capsular sugars from 15 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from more than 20 different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A complex of capsular sugars from 21 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 24 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 25 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A complex of capsular sugars 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 according to any one of Embodiments I-1 to I-15 or Embodiments I-21 to I-33, comprising a complex of capsular sugars from two or more different serotypes of P. gingivalis selected from the group consisting of serotypes K1, K2, K3, K4, K5, and K6.

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

[0226] The present invention will be illustrated by the following examples. The materials, methods, and examples are illustrative and not intended to limit the invention. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. Unless otherwise described in detail, the examples are carried out using art that is well known and common to those skilled in the art.

[0227] Examples from International Publication No. 2018 / 126229 The examples in International Publication No. 2018 / 126229 describe in full detail the synthesis of a single-site eCRM moiety (e.g., K11TAG). These were expressed in cell-free protein synthesis (CFPS) extracts, and pAMF was incorporated instead of the native Lys.

[0228] CRM variants containing multiple nnAAs per polypeptide were also expressed with various different numbers of Lys→pAMF substitutions per protein. Generally, a higher number of substitutions resulted in carriers leading to higher molecular weight complexes, but these carriers exhibited lower solubility. Carriers with six pAMF residues generally yielded both good solubility (>>50 mg / mL) and immunogenicity. The high solubility was surprising, given that replacing the charged Lys residues in the native sequence with hydrophobic pAMF residues increases the hydrophobicity of CRM197, a protein whose hydrophobicity has already been reported to affect its solubility. In other words, it demonstrated that it is possible to maintain the same addition site (i.e., Lys residue) used in known CRM197 complexes without causing insolubility, even after losing the charged residues.

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

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

[0231] The examples in International Publication No. 2018 / 126229 further describe general protocols for saccharide activity using sodium metaperiodate, derivatization of periodate-oxidized polysaccharides using DBCO, saccharide activity using CDAP, and binding of sugars-DBCO to eCRM. See also U.S. serial number 62 / 693,978, which was previously referenced.

[0232] Polyvalent immunogenic composition Complex combinations for 24 serotypes of Streptococcus pneumoniae (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 (X=pAMF) as the carrier polypeptide in each complex. The immunogenicity of these polyvalent compositions was confirmed using a three-dose regimen of 0.25 mL of vaccine administered intramuscularly to a group of seven rabbits. Each dose contained 24 μg of sugars (1 μg per serotype) at a concentration of 96 μg / mL.

[0233] Next, complex combinations were prepared for each of the serotypes of 32 types of Streptococcus pneumoniae (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), and their immunogenicity was confirmed using the same method.

[0234] For comparative purposes, a 13-valent Prevnar® conjugated vaccine was also tested alongside a 24-valent unconjugated vaccine made from a 23-valent Pneumovax® vaccine supplemented with unconjugated serotype 6A polysaccharide. These three compositions had equivalent polysaccharide doses per serotype (except for 6B, where Prevnar® contained twice the dose), but this involved dilution of Prevnar® and Pneumovax®. All three compositions contained aluminum phosphate adjuvant (60 μg of Al per dose). +++ This included [a specific adjuvant], but this involved adding this adjuvant to Pneumovax (trademark). The composition did not contain any preservatives.

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

[0236] The IgG and OPA responses were measured in rabbits. After the third dose, both responses were much greater in rabbits that received the two conjugate vaccines than in rabbits that received the unconjugated vaccine. Furthermore, the IgG and OPA responses using the 24-valent composition were comparable to those achieved using Prevnar (trademark), a 13-valent vaccine covering the 13 serotypes covered by the approved vaccine, but were additionally higher against the 11 serotypes not included in Prevnar (trademark). Surprisingly, there was no evidence of suppression of the carrier-induced epitopes using the 24-valent composition.

[0237] Figure 1 is the geometric mean titer for each of the 32 serotypes in the 32-valent conjugate composition against the polysaccharide / alum formulation and Prevnar 13 (trademark).

[0238] The multivalent conjugate composition can advantageously be packaged in pre-filled sterile syringes, making it easily deliverable in unit dose form and then administrable without the need to transfer the contents of the vial to an injection syringe or the like immediately prior to use.

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

[0240] Into the mutant protein 14 C-Leu incorporation was confirmed and expression efficiency was evaluated by looking at both the whole protein and the soluble protein. Generally, mutations in the catalytic domain of CRM197 led to a decrease in expression level compared to the unmodified CRM197 sequence, and the mutations with the best expression levels generally included substitutions downstream of Arg-193 that could be used to delineate the end of the catalytic domain.

[0241] The best 72 mutations increased the expression levels of both the whole protein and the soluble protein and had substitutions at the following residues numbered according to SEQ ID NO: 1: Ser-198, Ser-200, Asp-211, Lys-212, Lys-218, Lys-221, Lys-229, Ser-231, Ser-233, Lys-236, Ser-239, Glu-240, Glu-248, Glu-249, Gln-252, Thr-253, 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, Asn-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-469, 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 for illustrative purposes only and do not preclude various alternatives to the embodiments when carrying out the embodiments described herein.

[0243] Sequence List Sequence ID 1 (natural CRM197) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0244] Sequence ID 2 (CRM197 with Arg-Asn substitution) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0245] Sequence ID 3 (CRM197 containing 6 preferred nnAA sites and N-terminal Met (methionine)) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFF MICKS

[0246] Sequence ID 4 (Arg-Asn subset) n , six preferred nnAA sites, and CRM197 containing N-terminal Met (methionine) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH XTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFF MICKS

[0247] Sequence ID No. 5 (Haemophilus influenzae protein D) CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

Claims

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 bonded to the carrier polypeptide via a non-natural amino acid residue within the carrier polypeptide.

2. An airtight container containing a pharmaceutical composition comprising an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, and suitable containers for airtight sealing include, for example, vials, and the contents are preferably sterilized during airtight sealing.

3. The container according to claim 1 or 2, for example, a sterile glass container such as a vial.

4. A delivery device comprising a pharmaceutical composition containing an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

5. A syringe, the container according to claim 1 or claim 2, or the delivery device according to claim 4.

6. A pharmaceutical composition comprising two or more different immunogenic complexes and an aluminum salt adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within 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 complexes and an aluminum phosphate adjuvant, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, the sugar antigen being covalently bonded to the carrier polypeptide via unnatural amino acid residues within 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 complexes, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues in the carrier polypeptide, and (ii) the amount of the pharmaceutical composition is 0.25 to 1.25 mL.

9. A pharmaceutical composition comprising two or more different immunogenic complexes and a preservative, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

10. A preservative-free pharmaceutical composition comprising two or more different immunogenic complexes, wherein each immunogenic complex comprises a carrier polypeptide and a sugar antigen, and the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues within the carrier polypeptide.

11. A pharmaceutical composition comprising two or more different immunogenic complexes, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues in the carrier polypeptide, and (ii) the composition has a weight osmolality of 200 to 400 mOsm / kg.

12. A pharmaceutical composition comprising two or more different immunogenic complexes and at least one excipient, wherein (i) each immunogenic complex comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via unnatural amino acid residues in the carrier polypeptide, and (ii) at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80.

13. A pharmaceutical composition containing n different immunogenic complexes (i) Each of the n immunogenic complexes comprises a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via non-natural amino acid residues within the carrier polypeptide; (ii) n is an integer from 3 to 50, and (iii) The total amount of carrier polypeptides in the n immunogenic complexes is 3 n μg or less per dose; (iv) The total concentration of carrier polypeptides in the n immunogenic complexes is 6 n μg / ml or less; (v) The total amount of sugar antigens in the n immunogenic complexes is 3 n μg or less per dose; (vi) The total concentration of sugar antigens in the n immunogenic complexes is 6 n μg / mL or less; (vii) The average amount of carrier polypeptide per complex is 1 to 4 μg per dose; (viiii) The average concentration of the carrier polypeptide per complex is 2 to 8 μg / mL; (ix) The average amount of sugar antigen per complex is 1 to 4 μg per dose; (x) The average concentration of sugar antigen per complex is 2 to 8 μg / mL; (xi) The composition does not contain a carrier polypeptide in an unbound form; (xi) The composition contains a carrier polypeptide in an unbound form, the mass of the carrier polypeptide in the composition being less than 10% of the mass of the carrier polypeptide in the n immunogenic complexes; (xiii) The composition does not contain sugar antigens in an unbound form; and / or (xiv) A pharmaceutical composition comprising at least one sugar antigen in an unbound form, wherein the total mass of the sugar antigen in the unbound form in the composition is less than 10% of the total mass of the sugar antigen in the n-type immunogenic complex.

14. A method for preparing multiple unit doses of pharmaceutical compositions, wherein (i) the pharmaceutical composition comprises an immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein the sugar antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the method comprises the steps of preparing a bulk composition comprising the immunogenic complex and packaging individual unit doses from the bulk composition into a plurality of separate containers.

15. A method for preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises two or more different immunogenic complexes and an aluminum salt adjuvant, (i) each of the immunogenic complexes comprises a carrier polypeptide and a sugar antigen, and (ii) the sugar antigen is covalently bonded to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide, and the method comprises (A) the step of separately adsorbing each of the immunogenic complexes onto an aluminum salt adjuvant and then mixing the individually adsorbed complexes together, or (B) the step of sequentially adsorbing each of the immunogenic complexes onto the aluminum salt adjuvant.

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

17. (i) having at least 80% sequence identity with SEQ ID NO: 1; and (ii) a carrier polypeptide comprising an amino acid sequence in which one or more of the following amino acid residues (numbered according to SEQ ID NO: 1) have an nnAA substitution: 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; Glun 252; Glun 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; 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; 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 according to claim 16 or 17, wherein Arg-193 in SEQ ID NO: 1 is substituted with a different amino acid, such as Asn.

19. An immunogenic complex comprising the carrier polypeptide according to claim 16, claim 17, or claim 18, which is bound to the antigen via an nnAA residue within the carrier polypeptide.

20. An immunogenic complex comprising a carrier polypeptide and a sugar antigen, wherein (i) the carrier polypeptide comprises the amino acid sequence of SEQ ID NO: 4, and (ii) the sugar 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 as described in claim 20.

22. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 21, wherein the carrier polypeptide comprises 4 to 9 nnAA residues.

23. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 22, wherein the lysine in the natural sequence of the carrier polypeptide is substituted with at least one nnAA.

24. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 23, wherein the carrier polypeptide has at least 90% sequence identity with respect to SEQ ID NO:

1.

25. A container, device, composition, method, polypeptide, or complex according to claim 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. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 25, wherein the carrier polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

27. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 26, wherein the nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

28. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 27, wherein the antigen has an alkyne group that binds to nnAA via an azide group.

29. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 28, wherein the antigen is a bacterial capsular sugar, for example, a capsular sugar from a bacterium selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Group A Streptococcus, Group B Streptococcus, and Porphyromonas gingivalis.

30. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 29, wherein the antigen is a capsular sugar of a serotype of Streptococcus 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. A container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 30, wherein the ratio (w / w) of sugars to the carrier polypeptide in the complex is greater than 1.

32. The container, device, composition, method, polypeptide, or complex according to any one of claims 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 aforementioned complex is a container, device, composition, method, polypeptide, or complex according to any one of claims 1 to 32, wherein the complex has a molecular weight of 900 kDa to 5 MDa.

34. Pharmaceutical compositions, A complex of capsular sugars from two or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 14 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 15 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from more than 20 different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 21 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 24 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; Capsular sugar complexes from 25 or more different serotypes of Streptococcus pneumoniae selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A complex of capsular sugars 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 according to any one of claims 1 to 15 or 21 to 33, comprising a complex of capsular sugars from two or more different serotypes of P. gingivalis selected from the group consisting of serotypes K1, K2, K3, K4, K5, and K6.

35. A container, device, composition, method, polypeptide, or complex according to any one of claims 30 to 34, wherein serotype 20 is serotype 20B.

36. A container, device, composition, method, polypeptide, or complex according to any one of claims 30 to 34, wherein serotype 20 is serotype 20A.

37. A method for eliciting an immunoprotective antibody response to a certain antigen in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 6 to 13 or 21 to 34, or an immunogenic complex according to any one of claims 19 to 33, in an excipient suitable for parenteral administration.

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