Improvements in immunogenic conjugates

Abstract

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

Description

[0001] This application is a divisional application of Chinese patent application No. 2019800441698, entitled "Improvement of Immunogenic Conjugates", filed on July 1, 2019 (PCT application No. PCT / US2019 / 040131). Cross-reference to related applications

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

[0003] Merging of electronic text files submitted with this document The contents of the text file submitted electronically with this article are incorporated herein by reference in their entirety: a computer-readable copy of the sequence list (filename: STRO_005_01WO_SeqList_ST25.txt, date of record: July 1, 2019, file size: approximately 23 kilobytes). Background Technology

[0004] An immune response to a "weak" sugar antigen can be achieved through interactions with antigens such as diphtheria toxoid, tetanus toxoid, and Haemophilus influenzae. H. influenzae Enhancement is achieved by conjugating known “strong” carrier peptide antigens such as protein D or CRM197. WO 2018 / 126229 (SutroVax, Inc., Foster City, California) discloses methods, compositions, and techniques for generating conjugated vaccine antigens using carrier peptides containing non-natural amino acids (nnAAs). Orthogonal linking chemistry of nnAAs allows the antigen to conjugate with the carrier peptide to produce an immunogenic conjugate that can be used for immunization.

[0005] The object of this invention is to provide variations and improvements of such methods, compositions, and techniques. The variations and improvements described below can be applied to or combined with any of the methods, compositions, or techniques disclosed in WO 2018 / 126229 or U.S. Provisional Patent Application Serial Nos. 62 / 693,978 and 62 / 693,981 (both filed July 4, 2018). The foregoing patent applications are incorporated herein by reference in their entirety. Summary of the Invention

[0006] In one embodiment, a sterile container (e.g., a vial) is provided containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. The container may contain a unit dose of the pharmaceutical composition. A sterile glass container is preferred.

[0007] In another embodiment, a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.) is provided, the delivery device containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. The delivery device may contain a unit dose of the pharmaceutical composition. The delivery device may be used to administer the pharmaceutical composition to a mammalian subject.

[0008] In another embodiment, an airtight container is provided containing a pharmaceutical composition comprising an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. Suitable containers for sealing include, for example, vials. When airtight, the contents are preferably sterile.

[0009] In another embodiment, a syringe is provided containing 0.25-0.75 mL (e.g., 0.3-0.75 mL, preferably 0.5 mL) of a pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0010] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) the aluminum salt adjuvant is aluminum hydroxide or aluminum phosphate adjuvant; and (iii) the volume of the pharmaceutical composition is 0.25-0.75 mL (e.g., 0.3-0.75 mL, preferably 0.5 mL).

[0011] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of aluminum ions in the composition is <300 µg / mL (e.g., between 100 and 300 µg / mL). Ideally, the concentration of aluminum ions... < 1.7 mg / mL. The conjugates within 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 conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) the carrier polypeptide does not include SEQ ID NO: 3; and (iii) the concentration of aluminum ions in the composition is <2.5 mg / mL. Ideally, the concentration of aluminum ions is... < 1.7 mg / mL. The conjugates within the composition can be adsorbed onto the aluminum phosphate adjuvant.

[0013] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the volume of the pharmaceutical composition is 0.25-1.25 mL (e.g., 0.3-0.7 mL, preferably 0.5 mL). This composition may contain an aluminum phosphate adjuvant, and the conjugates within the composition may be adsorbed onto the aluminum phosphate adjuvant.

[0014] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and a preservative, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

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

[0016] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide; and (ii) the composition has a permeability of 200-400 mOsm / kg.

[0017] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates and at least one excipient, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80. The pharmaceutical composition may also contain an aluminum salt adjuvant. This composition may contain both sodium chloride and polysorbate 80 as excipients.

[0018] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer from 3 to 50; and (iii) states n The total amount of carrier polypeptide in each immunogenic conjugate is less than or equal to that in each dose of the pharmaceutical composition. 3n µg.

[0019] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer from 3 to 50; and (iii) in the pharmaceutical composition, the n The total concentration of the carrier polypeptide in the immunogenic conjugate is less than or equal to 6n µg / mL.

[0020] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... nEach of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer from 3 to 50; and (iii) states n The total amount of glycoantigen in each immunogenic conjugate is less than or equal to that in each dose of the pharmaceutical composition. 3n µg.

[0021] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer from 3 to 50; and (iii) in the pharmaceutical composition, the n The total concentration of glycoantigen in the immunogenic conjugate is less than or equal to 6n µg / mL.

[0022] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the average amount of the carrier polypeptide in each conjugate is 1-4 µg per dose of the pharmaceutical composition.

[0023] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) in the pharmaceutical composition, the average concentration of the carrier polypeptide of each conjugate is 2-8 µg / mL.

[0024] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the average amount of the glycoantigen in each conjugate is 1-4 µg per dose of the pharmaceutical composition. In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) in the pharmaceutical composition, the average concentration of the glycoantigen in each conjugate is 2-8 µg / mL.

[0025] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer between 3 and 50; and (iii) the composition does not contain one or more unconjugated forms of the carrier polypeptide or (iv) the composition contains one or more unconjugated forms of the carrier polypeptide, wherein the mass of the one or more unconjugated forms of the carrier polypeptide in the composition is less than that of the... n The mass of the carrier polypeptide in the immunogenic conjugate is 10%.

[0026] In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising: n Different immunogenic conjugates, wherein: (i) the... n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n It is an integer between 3 and 50; and (iii) the composition does not contain the unconjugated form of the glycoantigen or (iv) the composition contains at least one of the unconjugated forms of the glycoantigen, wherein the total mass of the unconjugated forms of the glycoantigen in the composition is less than the mass of the glycoantigen. n 40% of the total mass of the glycoantigen in the immunogenic conjugate (e.g., < 30% < 20% or < 10%.

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

[0028] In another embodiment, a pharmaceutical composition is provided comprising 14 or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of the carrier polypeptide per dose... < 80 µg / mL.

[0029] In another embodiment, a method for preparing multiple unit doses of a pharmaceutical composition is provided, wherein (i) the pharmaceutical composition comprises an immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide, and (ii) the method includes the steps of preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into multiple individual containers. This method is ideally performed aseptically. The individual containers can be sealed after the unit doses have been packaged into them. The individual containers are ideally syringes.

[0030] In another embodiment, a pharmaceutical composition is provided comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the composition is lyophilized.

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

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

[0033] In another embodiment, a modified CRM197 carrier polypeptide is provided, the modified CRM197 carrier polypeptide comprising an amino acid sequence that (i) has at least 80% sequence identity with SEQ ID NO: 1 and (ii) contains an nnAA substitution at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): 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;Ly s-218; Lys-221; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-49 8; Lys-516; Lys-522; Lys-534; Arg-377; Arg-407; Arg-455; Arg-460; Arg-462; Arg-472; A rg-493;Ser-198;Ser-200;Ser-231;Ser-233;Ser-239;Ser-261;Ser-374;Ser-381;Ser- 297; Ser-397; Ser-451; Ser-475; Ser-494; Ser-495; Ser-496; Ser-501; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and / or Thr-517. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., glycoantigens) via one or more of the nnAA residues in the carrier polypeptide.

[0034] In another embodiment, a modified CRM197 carrier polypeptide is provided, the modified CRM197 carrier polypeptide comprising: an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO: 1; (ii) lacking an Arg-Arg dipeptide sequence; and (iii) containing an nnAA substitution at one or more of the following amino acid residues (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; G lu-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-2 97; Ser-397; Ser-451; Ser-475; Ser-494; Ser-495; Ser-496; Ser-501; Ser-505; Thr-253; Thr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and / or Thr-517. The modified CRM197 carrier polypeptide can be used to prepare immunogenic conjugates (e.g., glycoantigens) via one or more of the nnAA residues in the carrier polypeptide.

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

[0036] In another embodiment, a syringe is provided containing a pharmaceutical composition comprising two or more different immunogenic conjugates, each immunogenic conjugate comprising a carrier polypeptide and a pneumococcal glycoantigen, wherein the syringe is a non-silicone syringe. The pharmaceutical composition in the non-silicone syringe ideally has 13 or more different pneumococcal conjugates, and the carrier polypeptide optionally comprises nnAA, but may instead be, for example, CRM197. Further details of the non-silicone syringe are given below. Attached Figure Description

[0037] Figure 1 The geometric mean titer of each of the 32 indicated serotypes in the 32-valent vaccine of the present invention relative to the polysaccharide / alum formulation and Prevnar-13™ is provided as described in the examples. Detailed Implementation

[0038] International Patent Publication No. WO2018 / 126229 discloses various details of methods, compositions and techniques for generating conjugated antigens, the entire contents of which are incorporated herein by reference.

[0039] Immunogenic conjugates This invention generally relates to immunogenic conjugates. These conjugates comprise a carrier polypeptide covalently linked to an antigen. This linkage can convert T-cell-independent immunogens (such as sugars) into T-cell-dependent immunogens, thereby enhancing the induced immune response (especially in children). The conjugates used herein contain covalent bonds formed between the antigen and non-natural amino acid ('nnAA') residues within the carrier polypeptide. These nnAA residues can provide functional groups that promote reactivity with the antigen of interest.

[0040] A single carrier polypeptide is typically linked to multiple antigen molecules. The antigen may have a single linker group (e.g., a reducing end of a sugar) for linking to the carrier polypeptide, or it may have multiple linker groups (e.g., multiple aldehyde or cyanate groups). In the case of antigen molecules having multiple linker groups, this typically results in the formation of high molecular weight crosslinks or lattice conjugates, involving the linking of the antigen between multiple carrier polypeptides. Crosslinked conjugates are preferred herein (particularly for pneumococci), and therefore antigens having multiple linker groups are also preferred.

[0041] A covalent bond is formed between the antigen and the nnAA residues in the carrier polypeptide. Preferably, the antigen does not conjugate with lysine residues in the carrier polypeptide; more preferably, the antigen does not conjugate with native amino acid residues in the carrier polypeptide.

[0042] Useful carrier peptides contain T-cell epitopes. Various such carrier peptides are known in the art, and their use in approved vaccines is known to involve diphtheria toxoids (derived from Corynebacterium diphtheriae). Corynebacterium diphtheriae Chemically processed toxins; 'Dt'), tetanus toxoid (from Clostridium tetani) Clostridium tetani Chemically treated tetanus toxin; 'Tt', protein D ("PD" or "HiD") from Haemophilus influenzae, outer membrane protein complex ('OMPC') from group B meningococcus, and CRM197 mutant diphtheria toxin.

[0043] The preferred carrier polypeptide upon which the carrier of the present invention is based is CRM197. CRM197 is well known in the art (e.g., see Bröker et al. 2011, Biologics). Biologicals (39:195-204) and has the following amino acid sequence (SEQ ID NO: 1), wherein the underlined residue (Glu-52) is different from the natural diphtheria toxin, and the substitution of Gly→Glu leads to the loss of toxic enzyme activity in the protein: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK EFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGK RGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSET ADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGES GHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS This invention does not use natural CRM197. Instead, it uses a modified amino acid sequence containing at least one nnAA, rather than CRM197 including SEQ ID NO: 1. These modified CRM197 carrier peptides are described in more detail below.

[0044] Besides CRM197, other detoxified mutant forms of diphtheria toxin can be used. For example, the non-toxic K51E / E148K double mutant has also been used as a carrier peptide in conjugates (Pecetta et al. 2016, Vaccines). Vaccine (34:1405-11), and nnAA residues can be incorporated into the sequence of this double mutant in the same manner as in CRM197.

[0045] Another carrier polypeptide of interest is PD from Haemophilus influenzae, which naturally has the following amino acid sequence (SEQ ID NO: 5): CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAA ETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK Instead of using the native PD, a modified amino acid sequence containing at least one nnAA is used. For example, one or more Lys residues in SEQ ID NO: 5 can be replaced with nnAA. SEQ ID NO: 5 contains 36 Lys residues, so several residues can be replaced with nnAA and then used for conjugation. This has been demonstrated by Hua et al. (2016) in Clinical and Vaccine Immunology (Clinical and Vaccine Immunology). Clin Vaccine Immunol The article 23:155-61 reported on the prediction and identification of T cell epitopes in PD.

[0046] More generally, any polypeptide containing a T-cell epitope can be used as a carrier polypeptide. T-cell epitopes can bind to MHC class II and interact with T-cell receptors on the surface of CD4+ T cells, thereby enhancing antibody responses against antigens or haptens conjugated to them (e.g., see Costantino et al. 2011, Expert Opinions on Drug Discovery). Expert Opin Drug Discov (6:1045-66). Micoli et al. (2018) "Molecular ( Molecules The journal *Vaccines*, 23, 1451, reviewed various carrier peptides and their selection criteria. Tontini et al. (2016) in *Vaccines*, 34:4235-42, discussed preclinical studies of 28 carrier peptides, including tests of their ability to induce antibodies against glycoantigens. Multiepitope carrier peptides containing multiple broadly reactive (i.e., immunogenic in most MHC class II molecules) human CD4+ T cell epitopes from various pathogen-derived antigens have been designed, such as N19 and others, as described in *European Journal of Immunology*, 23, 1451. Eur J Immunol)》 31:3816-24, Baraldo et al. (2004) "Infection and Immunity" Infect Immun Other peptides disclosed in US Patents 6,855,321 and 7,867,498, and 72:4884-7, and US Patent 6,855,321 and 7,867,498. The ability to design these multi-epitope vectors demonstrates the ability of those skilled in the art to identify suitable T-cell epitopes from diverse sources and to use them to design effective vector peptides. See also patent application US2016-0101187. T-cell epitopes present within known vectors (e.g., Tt, PD, CRM197) can be used. Various detoxified bacterial toxins have been successfully used as vectors, such as Tt, Dt, and Pseudomonas aeruginosa. P. aeruginosa Exotoxins, Clostridium difficile ( C. diifficile Toxins A and B, etc. Many different carrier peptides have been used for pneumococcal sugars, such as CRM197 in Prevnar™, PD, Tt, and Dt in Synflorix™, and various epitopes in Velasco et al. (1995) *Infectious Diseases and Immunity* 63:961-8. The present invention can use any of these many carrier peptides modified to contain at least one nnAA to enhance the immunogenicity of the antigen of interest.

[0047] The nnAA-containing carrier polypeptides used in conjunction with this invention can generally be prepared using the techniques disclosed in Section 6 (“Methods for Carrier Protein Production”) of WO2018 / 126229. Preferred carriers contain nnAA in addition to at least one T-cell epitope of the carrier. If the T-cell epitope region of the carrier is unknown, the epitope can be identified using standard techniques, for example, see Reece et al. (1993), *International Journal of Immunology* 151:6175-84, and Beissbarth et al. (2005), *Bioinformatics*. Bioinformatics )》 21 Supplement 1: i29-37, Maciel Jr et al. (2008) Virology ( Virol )》 378:105-17, Fridman et al. (2012) Tumor Immunology ( Oncoimmunol(Including empirical and / or predictive methods) 1:1258-70 etc. It can also be confirmed that any specific modification of the vector polypeptide sequence does not eliminate the expected T cell response to conjugated antigens (such as sugars in this text). A preferred group of vectors does not contain any modifications within the T cell epitopes, including the insertion or substitution of nnAAs. Particularly preferred vectors contain at least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs. Particularly preferred vectors may also have up to 10, 9, 8, 7, or 6 nnAAs. A particularly preferred range for nnAAs in the vector polypeptide is 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 4-10, 4-9, 4-8, 4-7, and 4-6 nnAAs.

[0048] The immunogenic conjugates used in this article may contain a variety of antigens. Antigens are typically sugars. The term "sugar" includes polysaccharides having 50 or more repeating units and oligosaccharides having fewer than 50 repeating units. Polysaccharides typically have about 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 repeating units up to about 2,000 (sometimes more) repeating units, and optionally about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1,000 repeating units up to about 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800 or 1,900 repeating units. Oligosaccharides typically have about 6, 7, 8, 9 or 10 repeating units to about 15, 20, 25, 30 or 35 to about 40 or 45 repeating units.

[0049] Sugars that can be incorporated into immunogenic conjugates include sugars present in bacteria. These sugars can be non-capsulated sugars (such as extracellular polysaccharides, for example, Staphylococcus aureus). S.aureus (Extracellular polysaccharides), but preferably bacterial capsular sugars.

[0050] Bacterial capsular sugars are high-molecular-weight sugars present in the capsules of Gram-positive or Gram-negative bacteria, and can be used as vaccine antigens. These capsular sugars are typically prepared from whole-cell lysates or culture supernatants of the corresponding bacteria by methods involving percolation, protein removal, ethanol precipitation, nucleic acid removal, and freeze-drying. Bacterial sugars used in conjunction with this invention can be whole sugars as present in bacteria or fragments obtained from whole sugars, for example, by hydrolysis of sugars purified from bacteria.

[0051] The carbohydrate antigens of particular interest include, but are not limited to: -Streptococcus pneumoniae ( S. pneumoniae Capsular sugars: Further details are given below regarding pneumococcal capsular sugars that can be used as antigens for carrying out the present invention.

[0052] - Streptococcus pyogenes ( Streptococcus pyogenes ) sugar The antigen may be a sugar derived from Streptococcus pyogenes. In one embodiment, the antigen is a capsular sugar of Streptococcus pyogenes, the capsular sugar being composed of the high molecular weight polymer hyaluronic acid, wherein the repeating unit has the following structure: [→4)-β-D-GlcUA p -(1→3)- β -D-Glc p NAc-(→) The structure appears to be invariant across Streptococcus pyogenes serotypes. In another embodiment, the antigen is a non-capsulated sugar derived from Streptococcus pyogenes, such as a group A stroma cell wall sugar comprising a backbone of poly-L-pyranoside units linked by alternating α-L-(1→3) and α-L-(1→2) bonds, with an N-acetyl-β-D-glucosamine residue linked to the bond at position 3 of the rhamnose backbone.

[0053] - agalactococcus ( Streptococcus agalactiae Capsular sugars The antigen can be a capsular sugar from agalactiae (group B streptococci or GBS). There are at least 10 GBS serotypes with different capsular sugar repeat units (Ia, Ib, II–IX), but only a few serotypes are commonly the cause of the disease. These serotypes include serotypes Ia, Ib, II, III, and V, and conjugates derived from the capsular sugars of these serotypes can be prepared.

[0054] - Capsule of Haemophilus influenzae The antigen can be a capsular sugar derived from Haemophilus influenzae. At least six Haemophilus influenzae serotypes have different capsular sugar chemical structures (af types). However, only types a and b are considered "highly virulent" strains, and the preferred type of Haemophilus influenzae capsular sugar used in conjunction with this invention is type b (Hib).

[0055] - Neisseria meningitidis ( Neisseria meningitidis Capsular sugars The antigen can be a capsular sugar derived from Neisseria meningitidis. At least 13 Neisseria meningitidis serogroups have different capsular sugar chemical structures (serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z'), but only six (A, B, C, W-135, X, and Y) are considered life-threatening. The sugar antigen is usefully derived from any one of serogroups A, C, W135, X, or Y.

[0056] - Porphyromonas gingivalis ( Porphyromonas gingivalis Capsular sugars The antigen can be a capsular sugar derived from one of the six serotypes of Porphyromonas gingivalis, K1, K2, K3, K4, K5, and K6.

[0057] - Salmonella typhi ( Salmonella typhi Capsular sugars The antigen can be Vi sugar. Vi is Salmonella typhi (Enterocera) (Salmonella enterica). S.enterica The capsular sugar of typhoid serum variants. Vi sugar is aminohexuronic acid α1,4- N A linear homopolymer of acetylgalactose-aminouronic acid, which is acetylated by 60-90% at the C-3 position.

[0058] - Sugars of Staphylococcus aureus The antigen can be a sugar derived from Staphylococcus aureus. The sugar can be an extracellular polysaccharide of Staphylococcus aureus or a capsular sugar of Staphylococcus aureus, wherein the extracellular polysaccharide is poly-N-acetylglucosamine (PNAG), and the capsular sugar can be, for example, serotype 5, serotype 8, or serotype 336.

[0059] - surface sugars of Clostridium difficile The antigen can be a surface glycan from Clostridium difficile, such as PS-I or PS-II.

[0060] - beta-glucan Antigens can be dextrans containing β-1,3-bonds and / or β-1,6-bonds. These conjugated dextrans can be used to generate antifungal immune responses, such as against Candida albicans. Candida albicans ).

[0061] Further details about these glycoantigens can be found in WO2018 / 126229.

[0062] Antigens typically do not inherently contain suitable or ideal functional groups for conjugation. Therefore, antigens may require functionalization before conjugation with nnAA. Further details of such functionalization are given below.

[0063] pneumococcal capsula The preferred antigen used in conjunction with this invention is the capsular sugar of *Streptococcus pneumoniae*. *Streptococcus pneumoniae* is a capsular Gram-positive bacterium that can cause pneumonia, bacteremia, and meningitis. At least 90 different documented *Streptococcus pneumoniae* serotypes are available (see, for example, Kalin, M., *Pleural Cavity*). Thorax ( ) 1998; 53:159-162) carries capsular sugars with serotype-specific repeating unit structures. As those skilled in the art will understand, it has been proposed that Streptococcus pneumoniae serotype 20 is actually composed of two closely related serotypes, whose capsular polysaccharides are largely cross-protected (Calix et al. 2012, Journal of Biochemistry). J Biol Chem(287:27885-94). Therefore, as those skilled in the art will further understand, serotype 20 refers to a sugar that would previously be classified as serotype 20 in the art and therefore may structurally be 20A or 20B (from a strain that would previously be classified as serotype 20 in the art, but may genotype 20A or 20B), as disclosed by Calix et al. For example, the strain used to produce the serotype 20 polysaccharide in Pneumovax™ (Merck) is now considered to be serotype 20A. In some cases, 20A may be preferred. In other cases, 20B may be preferred. The prevalence in the target population may be the basis for selection among these serotypes. Nevertheless, since strains classified as 20, 20A, and 20B are serologically similar, the strains have a high degree of cross-protection in vaccines and the selection between strains may not be important.

[0064] The antigen used in conjunction with this invention may be a capsular sugar derived from any of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 1 9F, 19A, 19B, 19C, 20, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A or 48 (Henrichsen, *Journal of Clinical Microbiology*) J Clin Microbiol(See 1995; 33:2759-2762). However, only a subset of these serotypes are often the cause of clinically significant bacterial infections, therefore the antigen can be a capsular sugar from any of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F. Serotypes 6C, 7C, 15A, 15C, 16F, 20A, 20B, 23A, 23B, 24B, 31, 34, 35B, 35F, 37, and 38 have also raised clinical concern, therefore the antigen can be a capsular sugar from one of these Streptococcus pneumoniae serotypes.

[0065] When the present invention uses conjugates derived from different pneumococcal serotypes, it preferably contains sugars derived from at least 14 different Streptococcus pneumoniae serotypes (e.g., from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more). When the composition contains 14 or more serotypes, these serotypes preferably include 13 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In addition to these 13 Streptococcus pneumoniae serotypes, the composition preferably contains one or more of serotypes: 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20 (or alternatively, 20A or 20B), 22F, and / or 33F. Alternatively, in addition to the 13 serotypes mentioned above, the composition preferably includes one or more Streptococcus pneumoniae serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38. Useful combinations of 15 or more (e.g., 16 or more) serotypes include Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6... Each of the following Streptococcus pneumoniae serotypes is included: A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and may also include serotype 8. Useful combinations of 20 or more (e.g., 21 or more) Streptococcus pneumoniae serotypes include each of the following Streptococcus pneumoniae serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. Useful combinations of 24 or more serotypes include each of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0066] The structure of the capsular sugar repeat unit of common pneumococcal serotypes was described by Jones et al. (Jones C et al., Proceedings of the Brazilian Academy of Sciences). An Acad Bras Ciênc. (June 2005; 77(2):293-324) Type 1 [→3)-D-AAT-α-Gal p -(1→4)-α-D-Gal p A(2 / 3OAc)-(1→3)-α-D-Gal p A-(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→] Background3 [→3)-β-D-GlcA-(1→4)-β-D-Glc p -(1→] Settings4 [→3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp2,3(S)Py-(1→] Section5 [→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→] Section 6B [→2)-α-D-Galp-(1→3)-α-D-Glcp-(1→3)-α-L-Rhap-(1→4)-D-Rib-ol-(5→P→] Background 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→] Rating 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→] Range 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-(→] Section 14 [→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→] Temperature 18C [→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→] Type 19F [→4)-β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap-(1→P→] Type 23F [→4)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→] In Geno et al. (2015) Clinical Microbiology Reviews Clin. Microbiol. Rev. A broader discussion of sugars can be found in 28:871-99, where Table 1 shows the structures of 97 known serotypes. This table also discloses the proportion of acetylated sugar residues when acetylation is incomplete.

[0067] Capsular sugars can be O-acetylated. In some embodiments, capsular sugars from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F include sugars with O-acetylation levels between 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 75% and 100%, 80% and 100%, 90% and 100%, 50% and 90%, 60% and 90%, 70% and 90%, or 80% and 90%. In other embodiments, the degree of O-acetylation is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or about 100%. The degree of O-acetylation of sugars can be determined by proton NMR (see, for example, Lemercinier and Jones (1996) *Carbohydrate Research*). Carbohydrate Research )》 296:83-96; Jones et al. (2002) Journal of Pharmaceutical and Biomedical Analysis ( J. Pharmaceutical and Biomedical Analysis(See 30:1233-1247). The sugars used to prepare the conjugates will typically retain at least 50% (e.g., 75% or even 100%) of the O-acetylation level observed in the starting capsular sugar purified from bacteria.

[0068] Streptococcus pneumoniae capsular sugar can be obtained directly from bacteria using isolation methods known to those skilled in the art (see, for example, methods disclosed in: U.S. Patent Application Publications Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498, and WO 2008 / 118752). Alternatively, Streptococcus pneumoniae capsular sugar can be obtained from commercial sources (e.g., ATCC).

[0069] The molecular weight of the pneumococcal capsular glycoantigen used in conjunction with the present invention can be usefully between 10 kDa and 4,000 kDa, for example between 50 kDa and 3,000 kDa or between 100 kDa and 2,000 kDa. For example, molecular weight can be between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 4,000 kDa; between 200 kDa and 3,500 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; 200 Between 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500 kDa. Further details and guidance regarding molecular weight are available in U.S. Serial No. 62 / 693,978, which is incorporated herein by reference.

[0070] Capsular sugars are optionally chemically modified relative to naturally occurring capsular sugars. For example, the sugars are optionally de-acetylated (partially or completely), de-N-acetylated (partially or completely), N-propionic acid-modified (partially or completely), etc. Deacetylation optionally occurs before, during, or after activation, derivatization, or conjugation, but usually occurs before conjugation.

[0071] Some embodiments of the present invention involve the use of two or more different conjugates. Regarding pneumococcal capsular sugar conjugates, this means (when a single type of carrier polypeptide is used for each conjugate) that each 'different' conjugate has sugars derived from different pneumococcal serotypes.

[0072] Multivalent conjugates Preferred compositions of the present invention involve, for example, the use of two or more different conjugates within a single pharmaceutical composition. These examples are also referred to as multivalent. When any two conjugates are described as 'different' or provide different valences in a 'multivalent' composition, this refers to the difference between the combination of carrier polypeptide and antigen in the two conjugates. For example, when a single type of modified CRM197 (e.g., SEQ ID NO: 4) is conjugated with a capsular sugar from a single serotype of pneumococcus, the reaction product will contain many different types of molecules (different molecular weights, different bond patterns within each molecule, etc.), but is considered herein as a single conjugate. Those skilled in the art are familiar with this heterogeneity at the molecular level and similarly define individual conjugates of vaccines by antigen-carrier combinations of specific conjugates, where other properties (such as molecular weight) are averages within the conjugate composition. Two 'different' conjugates have different carrier polypeptides (i.e., different amino acid sequences) and / or different antigens (i.e., different antigenic structures).

[0073] For example, capsular sugar antigens can be purified from two different serotypes of Streptococcus pneumoniae. These two different capsular sugars can be conjugated separately to a carrier polypeptide (which may be the same or different) to provide two different conjugates. Therefore, regarding bacterial capsular sugar conjugates, the difference between two 'different' conjugates will generally be that one contains a capsular sugar from a first serotype or serogroup of the bacterial species, while the other contains a capsular sugar from a second serotype or serogroup of the said bacterial species, for example, capsular sugars from different Streptococcus pneumoniae serotypes or from different Neisseria meningitidis serogroups. If the two conjugates contain capsular sugars different from antigens from multiple bacterial species, for example, a Hib sugar conjugate and a meningococcal sugar conjugate, then the two conjugates are also 'different'.

[0074] The preferred multivalent composition of the present invention comprises n Different immunogenic glycoconjugates, among which n The glycoantigen of each immunogenic conjugate differs from that of the others. n-1 A glycoantigen of an immunogenic conjugate. For example, if the composition contains an antigen from a single bacterial species, there may be antigens from said species. n Different serotypes or n Capsular sugars from different serum groups.

[0075] This nomenclature associated with 'distinct' conjugates is used in the field of conjugate vaccines. For example, Glesby et al. (2015) in the *Journal of Infectious Diseases* (…). J InfectDis Reference 212:18-27 mentions that the Prevnar™ PCV13 vaccine contains '13 different conjugates' because the vaccine contains glycoantigens from 13 different pneumococcal serotypes, each conjugated to CRM197. Similarly, EP-A-2932979 refers to 'an immunogenic composition comprising 13 different polysaccharide-protein conjugates'.

[0076] Therefore, the PCV7 Prevnar™ vaccine has 7 different conjugates, the PCV13 Prevnar™ vaccine has 13 different conjugates, the Menveo™ vaccine has 4 different conjugates, the Menactra™ vaccine has 4 different conjugates, the Nimenrix™ vaccine has 4 different conjugates, the Menitorix™ vaccine has 2 different conjugates, the Menhibrix™ vaccine has 3 different conjugates, the Synflorix™ vaccine has 10 different conjugates, and so on.

[0077] The multivalent compositions of pneumococcal conjugates preferably contain more than 13 different conjugates, such as 14, 15, 20, 21, 24, 25 or more. Suitable selection of serotypes for these >13-valent compositions has been discussed above.

[0078] For high-priced vaccines (e.g., those with more than 13 different conjugates), it may sometimes be preferable to use more than one carrier peptide to reduce the likelihood of carrier inhibition (e.g., see WO98 / 51339 and WO2011 / 110241). For example, in vaccines including n Among multivalent vaccines with different conjugates, the first vector polypeptide and ny The second polypeptide carrier is conjugated with different antigens (e.g., capsular sugars from different bacterial serotypes or serogroups), and the remaining polypeptide carrier is conjugated with the remaining... y Antigen conjugation. Similarly, three, four, or more vectors can be used, among which... n The antigen is distributed among these carriers. When more than one carrier is used, at least the first carrier is a carrier polypeptide containing nnAA according to the invention. In a preferred embodiment, at least the first and second carriers are carrier polypeptides containing nnAA according to the invention.

[0079] Non-natural amino acids As mentioned above, the conjugates used in this paper contain covalent bonds between functional groups within nnAA residues of the antigen and the carrier polypeptide. The side chains of the nnAA residues can provide reactive functional groups that can be used to conjugate the antigen to discrete sites in the carrier polypeptide.

[0080] Generally, nnAA can be any amino acid that can be incorporated into a polypeptide during translation, but it is not one of the 20 common amino acids. nnAA can be conveniently incorporated into a polypeptide by transforming tRNA molecules so that their codons incorporate nnAA instead of the natural homologous amino acid. One technique for achieving this involves using a "suppression codon," a nucleotide triplet, which is introduced into the desired position of the coding sequence and is recognized by a specific tRNA that can recognize natural stop codons (e.g., amber, ochre, or opal stop codons) but allows translation to continue, in which nnAA is incorporated (thus suppressing the natural stop codon).

[0081] The nnAA residue can be any of the nnAA residues described herein or any other residue that has been identified as compatible with cell-based or cell-free protein synthesis (see, for example, Schultz et al., Annals of Biochemistry). Annu Rev Biochem ( ) 2010; 79:413-44, specifically pp. 418-420; and Chin et al., Annals of Biochemistry 2014; 83:5.1-5.30, which are incorporated herein by reference. Ideally, nnAA is not naturally produced intracellularly through modification of one of the 20 common amino acids (e.g., pyrrolidone, selenocysteine, phosphotyrosine, formyl-methionine, etc.).

[0082] The particularly preferred nnAA used herein is an nnAA with a side chain that can be incorporated during translation (in cellular or cell-free systems), said side chain providing a functional group not present in the side chains of any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into polypeptides are known, for example see Young and Schultz (2010) *Journal of Biochemistry* 285:11039-44, Maza et al. (2015) *Bioconjugation Chemistry* (… Bioconjugate ChemSee also 26:1884-9 and Zimmerman et al. (2014) *Bioconjugation Chemistry* 25:351-61. WO2018 / 126229 discloses in detail how to incorporate nnAA residues into a carrier polypeptide, for example, using cell-free expression mixtures, orthogonal tRNA / aminoacyl-tRNA synthetase pairs specific for nnAA, repressive codons, etc. See also US Patent Application US-2017 / 0267637.

[0083] nnAA may contain chemical groups suitable for a "click" chemistry reaction with a corresponding group on the antigen of interest. Chemical groups suitable for "click" chemistry include, but are not limited to, azide (-N3), acetylene (-C≡C-), alkene (-C=C-), and 1,2,4,5-tetraazine (...). ) and phosphine (e.g., - P(Ph)2) group.

[0084] nnAA can be any of the following: 2-amino-3-(4-azidophenyl)propionic acid (p-azido-L-phenylalanine or pAF), 2-amino-3-(4-(azidomethyl)phenyl)propionic acid (p-azidomethyl-L-phenylalanine or pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propionic acid, or 2-amino-5-azidopentanoic acid.

[0085] The most preferred nnAA used in this paper is pAMF: pAMF provides a very favorable reaction kinetic for the generation of conjugates (e.g., much faster than pAF when reacting with alkyne-containing carbohydrate antigens using the SPAAC method).

[0086] nnAA can be a 2,3-disubstituted propionic acid carrying: an amino substituent at the 2-position; and an azide-containing substituent, a 1,2,4,5-tetraazine-containing substituent, or an ethynyl-containing substituent at the 3-position. Preferably, the substituent at the 3-position is an azide-containing substituent, particularly an azide-containing substituent including a terminal azide group linked to the carbon atom at the 3-position by a linking group. For example, the linking group can include an arylene moiety that is optionally substituted and optionally contains heteroatoms. For example, the linking group can include a 5- or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 non-hydrogen ring substituents.

[0087] nnAA can have the structure of formula XII: (XII) Wherein: Ar includes a 5- or 6-membered aromatic ring optionally containing at least one heteroatom; W 5 Selected from C1-C 10 Alkylene, -NH-, -O-, and -S-; Q1 is zero or 1; and W 6 Ar is selected from azide, optionally substituted with a lower alkyl C-, 1,2,4,5-tetraazine, and ethynyl. In some embodiments, Ar does not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene (i.e., Ar is -C6H4-). In other embodiments, Ar contains a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described below, and Ar can be, for example, pyridine or pyridazine. In a particularly preferred embodiment, Q1 is 1, W 5 It is a lower alkylene group, and W 6 It is an azide group.

[0088] nnAA can be nnAA containing an azide group, such as nnAA in Formula I: Wherein: D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-; each of W1, W2, and W3 is independently a single bond or a lower alkylene group; each X1 is independently -NH-, -O-, or -S-; each Y1 is independently a single bond, -NH-, or –O-; each Y2 is independently a single bond, -NH-, -O-, or N-linked or C-linked pyrroleylene group; Ar is... , , , , , or Furthermore, one of Z1, Z2, and Z3 is -N-, and the other terms in Z1, Z2, and Z3 are independently -CH-.

[0089] In other embodiments, nnAA has Equation II: Where W4 is C1-C 10 Alkylene.

[0090] The preparation of azide-containing amino acids according to Formulas I and II can be found, for example, in Stafford et al., US2014-0066598A1, specifically in paragraphs

[0331] -

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

[0091] nnAA can be nnAA containing 1,2,4,5-tetraazine. For example, Formula III: Where: Ar is , , , , , or V is a single bond, a lower alkylene group, or -W1-W2-; one of W1 and W2 is absent or a lower alkylene group, while the other is -NH-, -O-, or -S-; each of Z1, Z2, and Z3 is independently -CH- or -N-; and X1 is independently -NH-, -O-, or -S-; and R is a lower alkyl group.

[0092] The preparation of amino acids containing 1,2,4,5-tetraazine according to Formula III can be found, for example, in Yang et al., US2016 / 0251336, specifically in paragraphs

[0341] -

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

[0093] nnAA can be an alkyne-containing nnAA. In one embodiment, this is propargyl. Various propargyl-containing amino acids (including their synthesis) can be found in: Beatty et al., *Applied Chemistry International Edition* (…). Angew.Chem. Int. Ed. )》 2006, 45, 7364-7; Beatty et al., Journal of the American Chemical Society ( J. Am. Chem. Soc. )》 2005(127): 14150-1; Nguyen et al.《 JACS 》 2009 (131):8720-1. This propargyl-containing amino acid is suitable for incorporation into proteins using a cell-based system. In some embodiments, the propargyl-containing nnAA is selected from the group consisting of homopropargylglycine, etynylphenylalanine, and N6-[(2-propargyloxy)carbonyl]-L-lysine.

[0094] The nnAA used in this article is typically an α-amino acid with a chiral center at the α-carbon, and is preferably an L-stereoisomer.

[0095] The polypeptide carrier used in conjunction with the present invention comprises at least one nnAA residue. Preferably, the carrier polypeptide should contain a plurality of nnAA residues, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues (or sometimes more). Carrier polypeptides having fewer than 10 nnAA residues are preferred. Thus, the polypeptide may contain 2-9 nnAA residues and preferably 4-6 nnAA residues.

[0096] When the carrier polypeptide contains multiple nnAA residues, it is preferable to contain only a single species of nnAA (e.g., the only nnAA in the carrier is pAMF). This allows the same conjugation chemistry to be used simultaneously at each nnAA. If it is desired to link two different antigens to a single carrier molecule, this can be achieved by using different nnAA species within a single carrier and conjugating each antigen to a different nnAA, but conjugation to a single species of nnAA in the carrier is preferred. Furthermore, in cases where multiple different conjugates are used (e.g., different pneumococcal serotypes), it is sometimes preferable that each conjugate contains the same single species of nnAA. Additionally, in cases where the composition contains multiple different conjugates (e.g., different pneumococcal serotypes), it is sometimes preferable that each conjugate contains the same carrier polypeptide.

[0097] nnAA can be incorporated into the carrier polypeptide by substitution or by insertion (or by C-terminal or N-terminal extension). In one embodiment, one or more nnAA residues are incorporated by substitution. Conveniently, nnAA can replace lysine residues in the native polypeptide. For example, in CRM197, substitution can occur at one or more of the following positions in SEQ ID NO: 1 or 2: K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522, and K526. Preferably, nnAA (e.g., pAMF) is substituted at each of K33, K212, K244, K264, K385, and K526 (and in one embodiment, not at the other positions).

[0098] However, the substitutions used for incorporating nnAA are not limited to the lysine position, and other amino acids such as Phe, Asp, Asn, Glu, Gln, Arg, Ser and / or Thr can also be substituted with nnAA.

[0099] The nnAAs within the carrier peptide are ideally surface-accessible residues. This can be assessed using the 3D structure of the peptide or by performing a complete substitution of native amino acids with nnAAs followed by a conjugation test to evaluate the utility of each site.

[0100] To maintain the functionality of the carrier peptide, it is preferable not to incorporate nnAA into the T-cell activation epitope of the carrier peptide. The use of nnAA allows for selective placement of conjugation sites and thus avoids using the T-cell activation epitope of the carrier peptide as a site for antigen conjugation. As mentioned above, these epitopes are easily identifiable. For example, studies on CRM197 by Raju et al., Bixler et al., Leonard et al., and Pillai et al. (e.g., *European Journal of Immunology*) Eur J Immunol (WO89 / 06974) December 1995; 25(12):3207-14, WO89 / 06974) has identified various T cell epitopes, such as residues P271-D290, V321-G383, and Q411-I457. Therefore, it is preferable to avoid introducing nnAA in these regions of SEQ ID NO: 1.

[0101] Adhesion Conjugation involves forming a covalent bond between an nnAA residue and an antigen. This requires reactive functional groups in both the nnAA and the antigen. The nnAA of a carrier polypeptide is often chosen because it already possesses suitable functional groups (e.g., the azide group in pAMF), but antigens typically do not inherently contain suitable or ideal functional groups for conjugation. Therefore, the antigen may need to be functionalized before conjugation with the nnAA.

[0102] Detailed technical information on conjugation can be found in "Bioconjugation Technology" (…). Bioconjugate Techniques The information can be found in (Greg T Hermanson, 3rd edition, 2013). WO2018 / 126229 discloses in detail how antigens are functionalized and then conjugated with nnAA. As noted above, useful nnAAs contain functional groups (e.g., azide groups) suitable for “click” chemistry with functional groups on the antigen. Therefore, functionalized antigens ideally contain groups suitable for such “click” reactions.

[0103] Generally, conjugation is therefore performed by a method comprising the following three steps: (a) activating the antigen; (b) optionally derivatizing the activated antigen (e.g., with a linker or nucleophilic group) to introduce reactive functional groups not normally present in the antigen; and (c) conjugating the antigen to a carrier polypeptide by means of the group introduced in step (a) or (if present) step (b). In some embodiments, step (a) includes a first step of removing blocking groups on the antigen to make certain functional groups (e.g., hydroxyl, amine, thiol) more readily activated. Sometimes, steps (a)-(c) can occur substantially simultaneously (e.g., in the case of adding a reactive moiety such as N-hydroxysuccinimide to the antigen), but in other embodiments, two or more steps in steps (a)-(c) are discrete, with purification optionally occurring between steps.

[0104] As noted above, cross-linked conjugates are preferred, and therefore, the introduction of multiple reactive functional groups into each antigen molecule is also preferred. For example, multiple aldehyde or cyanate groups can be introduced when activating a sugar molecule. These groups can then be derivatized, for example, to introduce reactive cyclooctyne that can then react with the azide group in nnAA.

[0105] Various chemically activated antigens can be used, including but not limited to: periodate oxidation (e.g., for oxidizing hydroxyl groups on adjacent carbon atoms to produce reactive aldehyde groups), such as those disclosed in WO2011 / 110531; cyanohydrination, for example, using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP); activation with a 1,1'-carbonyldiimidazole (CDI) hydroxyl group followed by nucleophilic addition; or exposure of the intrinsic aldehyde (e.g., the reducing end of a sugar).

[0106] Periodate oxidation and CDAP cyanation are two preferred activation techniques. Periodate oxidation has been shown to be particularly effective for activating pneumococcal serotypes 1, 2, 3, 7F, 8, 9N, and 11A. CDAP cyanation has been shown to be particularly effective for activating pneumococcal serotypes 3, 7F, and 10A.

[0107] Activated antigens can be directly conjugated to nnAA, but the activated group is usually derivatized to introduce a functional group that exhibits better reactivity with nnAA. For example, an alkynyl group can be introduced. Bifunctional reagents having both amino and alkynyl groups can react with the aldehyde group already introduced into the antigen (e.g., by reductive amination), thereby leaving a side alkyne that can react with nnAA. For example, bifunctional reagents containing both amino and DBCO functional groups can be used.

[0108] In one embodiment, nnAA reacts with an alkynyl group (e.g., propargyl) in the antigen. The alkynyl group in the antigen is ideally suited for reaction with the alkynyl group in nnAA using reactions known in the art, such as copper-catalyzed azido-alkynyl cycloaddition (CuAAC), ruthenium-catalyzed azido-alkynyl cycloaddition (RuAAC), or Huisgen azido-alkynyl 1,3-dipolar cycloaddition. The alkynyl group may have a molecular environment that increases its reactivity; for example, it may be intracyclic. For example, an alkylene group may be within a cyclooctyne ring (optionally containing heteroatoms) such as a diaryl-strained cyclooctyne ring (e.g., DBCO). This reaction can be a cycloaddition [3+2] known in the art as a strain-promoted azido-alkynyl cycloaddition reaction (SPAAC). For these reactions, reagents based on DIFO and DBCO are readily available.

[0109] Useful alkyne rings in the SPAAC reaction include difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne. These can be obtained, for example, using any of the following reagents, utilizing side functional groups for attachment to the activated antigen (e.g., side amino groups for attachment to aldehydes or cyanates): DBCO-PEGn-NH2 DBCO-PEGn-NH2 DBCO carboxylic acid DBCO-NH2 DIFO carboxylic acid The value of 'n' in 'PEGn' represents the number of ethylene oxide repeating units. The value of n is in the range of 1-20, for example, in the range of 2-18, 3-16, or 4-14. Therefore, n can be any one of, for example, 4, 5, 11, 12, or 13.

[0110] Other click chemistry reactions that can be used to conjugate antigens with nnAA include, but are not limited to, tetrazine-olefin linkages and Staudinger linkages between phosphine and azides.

[0111] The molecular weight of the conjugates of the present invention can be at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa or higher. In some embodiments, the molecular weight of the conjugate is between about 750 kDa and about 5,000 kDa. In some embodiments, the molecular weight of the conjugate is between about 800 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 850 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 900 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 950 kDa and about 2,800 kDa. In some embodiments, the molecular weight of the conjugate is between about 1,000 kDa and about 2,800 kDa. The molecular weight of the conjugate is calculated by size exclusion chromatography (SEC) combined with multi-angle laser scattering (MALS).

[0112] The conjugates of the present invention comprise an antigen (e.g., a sugar) and a carrier polypeptide, and the weight ratio of these two components can be used as a parameter defining the conjugate. A higher antigen-carrier weight ratio in the sugar-carrier conjugate allows for the delivery of a greater amount of sugar antigen with a smaller amount of carrier polypeptide. For pneumococcal conjugate vaccines, this ratio is typically in the range of 0.3–3.0, but this can vary depending on serotype and aspects of the conjugation chemistry (Appendix 2: Recommendations for the production and control of pneumococcal conjugate vaccines). Annex 2: Recommendations for the production and control of pneumococcal conjugate vaccines (WHO Technical Report Series, No. 927, 2005). The ratio of the commercial vaccine Prevnar-13™ is 0.9. For compositions containing conjugates of multiple pneumococcal serotypes (e.g., more than 13 serotypes), the ratio of the whole composition is ideally greater than 1.0 (i.e., an excess of pneumococcal glycoantigen by weight) and preferably 1.5 or higher (e.g., in the range of 1.5-3.0 or preferably 1.5-2.0).

[0113] Modified CRM197 carrier peptide As mentioned above, the carrier polypeptide of primary interest herein is a modified form of CRM197. Therefore, preferred carrier polypeptides for use with this invention comprise those having at least 80% sequence identity with SEQ ID NO: 1 (e.g., > 85%, > 90%, > 95% > 96%, > 97% or preferably >The amino acid sequence (98%). For example, in addition to the presence of up to 10 nnAAs, the carrier polypeptide may include the amino acid sequence SEQ ID NO: 1, as discussed above.

[0114] SEQ ID NO: 1 contains the Arg-Arg dipeptide sequence at positions 192-193. In some cases, this sequence can be cleaved by proteolytic hydrolysis. If desired, this site can be modified to prevent cleavage and improve yield. Therefore, in some embodiments, the modified CRM197 carrier polypeptide used herein does not contain the Arg-Arg dipeptide sequence. For example, Arg-192 and / or Arg-193 of SEQ ID NO: 1 may be omitted or may be substituted with different amino acids. Therefore, a preferred carrier polypeptide comprises an amino acid sequence that: (i) has at least 80% (e.g., ...) of SEQ ID NO: 1. > 85%, > 90%, > 95% > 96%, > 97% or preferably > (ii) has 98% sequence identity; (iii) does not contain an Arg-Arg dipeptide sequence; and (iv) contains at least one (e.g., at least two, and preferably more, as discussed above) nnAA residue.

[0115] One such amino acid sequence is SEQ ID NO: 2, which differs from SEQ ID NO: 1 in that it has an Arg→Asn substitution at position 193: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVR NSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESI INLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS Any embodiment described herein or in WO2018 / 126229 by reference to SEQ ID NO: 1 may instead be effective using SEQ ID NO: 2.

[0116] Therefore, a carrier polypeptide comprising the amino acid sequence SEQ ID NO: 2 is provided, wherein SEQ ID NO: 2 has been modified to contain 1-10 (e.g., 3-9, 2-8, 2-6, 3-6, or 4-6) nnAA residues. These nnAA residue modifications can be incorporated into SEQ ID NO: 2 as insertions and / or substitutions (e.g., SEQ ID NO: 4, which contains 6 Lys→nnAA substitutions). The residue Asn-193 of SEQ ID NO: 2 is preferably not substituted with nnAA. This carrier polypeptide can be used to prepare (e.g., glycoantigens) immunogenic conjugates via one or more nnAA residues in the carrier polypeptide.

[0117] In some embodiments, these carrier peptides comprise an amino acid sequence upstream and / or downstream of SEQ ID NO: 1 or 2. Thus, for example, these carrier peptides may comprise a methionine residue upstream of the N-terminal amino acid residue of SEQ ID NO: 1 or 2. This methionine residue may be formylated. A methionine residue is not present at this position in wild-type CRM197, but it may be included here to initiate translation (e.g., in a cell-free peptide synthesis system) without requiring the entire native leader sequence. In some embodiments, the carrier peptide does not comprise (i) an amino acid upstream of the N-terminus of SEQ ID NO: 1 or 2, except optionally a methionine, and (ii) an amino acid downstream of the C-terminus of SEQ ID NO: 1 or 2.

[0118] Preferably, at least one Lys residue in SEQ ID NO: 1 or 2 is substituted with an nnAA residue. It is preferable to substitute more than one residue in SEQ ID NO: 1 or 2 with nnAA, and ideally, residues of only one species in SEQ ID NO: 1 are substituted with nnAA, for example, only the Lys residue is substituted. When more than one residue in SEQ ID NO: 1 is substituted with nnAA, it is preferable to use the same nnAA at each position, for example, pAMF at each substitution position. As noted above, in some embodiments, residues other than Lys are substituted.

[0119] Vector polypeptides, including those with 2-9 nnAA residue substitutions (e.g., Lys→nnAA substitution, preferably Lys→pAMF) and ideally having 2-8, 2-6, 3-8, 3-6, 4-9, 4-8, or 4-6 nnAA substitutions, such as amino acid sequences with 4, 5, or 6 nnAA residues, are preferred. This allows for a more extensive binding of the antigen to the vector compared to using a single nnAA, thereby increasing the antigen:carrier ratio while avoiding excessive destruction of the native sequence and structure that could lead to insolubility.

[0120] Structural studies of CRM197 revealed two general 3D regions within SEQ ID NO: 1 or 2: a first region extending from the N-terminus to Asn-373; and a second region extending from Ser-374 to the C-terminus. The first region roughly corresponds to the 'C' and 'T' (catalytic and transmembrane) domains, and the second region corresponds to the 'R' (receptor binding) domain. Ideally, the carrier polypeptide contains at least one nnAA in the first region and at least one nnAA in the second region, for example, at least two nnAAs in each region or at least three nnAAs in each region. This allows the conjugated antigen to spatially separate upon attachment to the carrier. Carriers with three nnAAs in both the first and second regions are useful.

[0121] The first region contains 27 Lys residues, and the second region contains 12 Lys residues. Therefore, one or more (e.g., 3) Lys residues within the N-terminal 374 amino acids and one or more (e.g., 3) Lys residues within the C-terminal 162 amino acids of SEQ ID No: 1 or 2 can be substituted with nnAA, for example, within pAMF.

[0122] Preferred embodiments of the CRM197-based nnAA-containing vector have the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2, in which one or more of the following residues are replaced by nnAA (such as pAMF): K24, K33, K37, K39, K212, K214, K227, K264, K385, K522, and K526. One such sequence is SEQ ID NO:3, where each X represents an nnAA (preferably the same nnAA, such as pAMF): MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO: 3) Another such sequence is SEQ ID NO: 4, where each X represents nnAA (preferably the same nnAA, such as pAMF): MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ XGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS (SEQ ID NO: 4) SEQ ID NO: 3 and 4 can be adequately expressed in cell-free protein synthesis systems while maintaining good solubility and providing a good immunogenic response upon conjugation with pneumococcal capsular sugars. SEQ ID NO: 4 lacks the native Arg-Arg dipeptide.

[0123] The polypeptide consisting of SEQ ID NO: 4, in which each X is pAMF, is another preferred carrier polypeptide for use with the present invention.

[0124] WO2018 / 126229 describes several amino acid residues suitable for nnAA substitution (e.g., Lys-24, Lys-33, Lys-37, Lys-39, Lys-212, Lys-214, Lys-227, Lys-244, Lys-264, Lys-385, Lys-522, Lys-526, Phe-12, Phe-53, Phe-123, Phe-127, Phe-140, Phe-167, Phe-250, Phe-389, Phe-530 or Phe-531, as specified in SEQ ID NO: 1). Other residues that can be substituted are: Asp-211; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp-507; Asp-519; Asn-296; Asn-359; Asn-399; Asn-481; Asn-486; Asn-502; Asn-524; Glu-240; Glu-248 ;Glu-249;Glu-256;Glu-259;Glu-292;Glu-362;Gln-252;Gln-287;Lys-212;Lys-218;L ys-221; Lys-229; Lys-236; Lys-264; Lys-299; Lys-385; Lys-456; Lys-474; Lys-498; Lys -516; Lys-522; Lys-534; Arg-377; Arg-407; Arg-455; Arg-460; Arg-462; Arg-472; Arg-4 93;Ser-198;Ser-200;Ser-231;Ser-233;Ser-239;Ser-261;Ser-374;Ser-381;Ser-297 ;Ser-397;Ser-451;Ser-475;Ser-494;Ser-495;Ser-496;Ser-501;Ser-505;Thr-253;T hr-265; Thr-267; Thr-269; Thr-293; Thr-386; Thr-400; Thr-408; Thr-469; and / or Thr-517.

[0125] Also provided are polypeptides comprising an amino acid sequence, said amino acid sequence: (i) having at least 80% (e.g., SEQ ID NO: 1) > 85%, > 90%, > 95% > 96%, > 97% or preferably >(ii) has 98% sequence identity; (iii) does not contain an Arg-Arg dipeptide sequence; and (iii) contains at least one nnAA residue; and wherein the polypeptide has an N-terminal methionine and / or is in monomeric form.

[0126] These CRM197-derived carrier peptides can be used for conjugation in the same manner as CRM197 has been used in the prior art (e.g., see Bröker et al. 2011, ibid., WO2015 / 117093, etc.), but with the improvement of allowing site-specific conjugation via one or more nnAA residues. These carrier peptides will generally be used as monomers rather than associated with other CRM197 or CRM197-derived subunits to form peptide multimers. Similarly, these carrier peptides will generally contain at least one disulfide bridge, for example, between Cys-186 and Cys-201 (numbered according to SEQ ID NO: 1) and optionally between Cys-461 and Cys-471.

[0127] Immunogenic conjugates are also provided, comprising any of these various carrier polypeptides conjugated to a glycoantigen via at least one of its nnAA residues. The carrier polypeptides are particularly useful for conjugating pneumococcal capsular sugars via one or more nnAA residues in the carrier polypeptide. Immunogenic conjugates prepared in this manner can be combined to form multivalent compositions as discussed elsewhere herein.

[0128] Therefore, immunogenic conjugates comprising a carrier polypeptide and a glycoantigen are provided, wherein (i) the carrier polypeptide has an amino acid sequence SEQ ID NO: 4, for example, wherein each X is pAMF; and (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4. Multivalent pharmaceutical compositions comprising two or more of these immunogenic conjugates are also provided.

[0129] Therefore, a pharmaceutical composition comprising a variety of different conjugates (e.g., different pneumococcal serotypes) is provided, wherein each conjugate comprises a carrier polypeptide having the amino acid sequence SEQ ID NO: 4.

[0130] Immunogenic conjugates comprising a carrier polypeptide and a glycoantigen are also provided, wherein (i) the carrier polypeptide has the amino acid sequence SEQ ID NO: 4; (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4; and (iii) the glycoantigen is a capsular sugar from any of the following pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. These individual conjugates can be combined to prepare the multivalent pharmaceutical compositions of the present invention.

[0131] Also provided are polynucleotides encoding the vector polypeptides described herein. In another embodiment, this disclosure provides an expression vector comprising a polynucleotide. In yet another embodiment, this disclosure provides a host cell comprising an expression vector.

[0132] adjuvant The pharmaceutical compositions of the present invention may contain aluminum salt adjuvants. Adjuvants can enhance the immunogenicity of conjugates within the pharmaceutical composition. Conjugates within the composition can be adsorbed onto the aluminum salt adjuvant.

[0133] Useful aluminum salt adjuvants include, but are not limited to, aluminum hydroxide adjuvants and aluminum phosphate adjuvants. These adjuvants are described, for example, in Chapters 8 and 9, of the Vaccine Design (Vaccine Design) series. Vaccine Design ) … (1995) Powell and Newman, eds. ISBN: 030644867X. Plenum Corporation.

[0134] The adjuvant commonly referred to as "aluminum hydroxide" is typically an aluminum hydroxide salt, which is usually at least partially crystalline. Aluminum hydroxide, which can be represented by the formula AlO(OH), can be visualized by infrared (IR) spectroscopy, particularly at 1070 cm⁻¹. –1 The adsorption band at 3090-3100 cm⁻¹ –1The presence of a strong shoulder at the site distinguishes it from other aluminum compounds such as Al(OH)3 (Powell and Newman, Chapter 9). The crystallinity of aluminum hydroxide adjuvants is reflected by the half-maximum diffraction band width (WHH), where poorly crystallized particles exhibit greater line broadening due to their smaller crystallite size. Surface area increases with increasing WHH, and adjuvants with higher WHH values ​​have been considered to have greater antigen adsorption capacity. For example, aluminum hydroxide adjuvants with needle-like particles of about 2 nm in diameter are often in fibrous form (e.g., as seen in transmission electron microscopy). The pI of aluminum hydroxide adjuvants is typically about 11, meaning that the adjuvant itself has a positive surface charge at physiological pH. Adsorption capacity of aluminum hydroxide adjuvants at pH 7.4 has been reported to be between [value missing] mg Al. +++ Between 1.8 and 2.6 mg of protein.

[0135] The adjuvant commonly referred to as "aluminum phosphate" is typically aluminum hydroxyphosphate, which usually also contains a small amount of sulfate (i.e., aluminum hydroxyphosphate sulfate). This adjuvant can be obtained by precipitation, and the reaction conditions and concentration during precipitation affect the degree to which the phosphate ester substitutes for the hydroxyl group in the salt. The PO4 / Al molar ratio of the hydroxyphosphate is typically between 0.3 and 1.2. The hydroxyphosphate can be distinguished from strictly AlPO4 by the presence of hydroxyl groups. For example, 3164 cm⁻¹ -1 The IR spectral bands at a point (e.g., when heated to 200 °C) indicate the presence of structural hydroxyl groups (Chapter 9 of Powell and Newman).

[0136] PO4 / Al of aluminum phosphate adjuvant 3+ The molar ratio will typically be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably between 0.95 and 0.1. Aluminum phosphate will generally be amorphous, especially for hydroxyphosphate. A typical adjuvant is 0.6 mg Al. 3+ Aluminum hydroxyphosphate is an amorphous form of aluminum hydroxyphosphate, contained within a PO4 / Al molar ratio between 0.84 and 0.92. Aluminum phosphate is typically in particulate form (e.g., a sheet-like morphology as seen in transmission electron microscopy, where primary particles range in size from 50 nm). After any antigen adsorption, the typical diameter of the particles ranges from 0.5 to 20 μm (e.g., about 5 to 10 μm). Aluminum phosphate adjuvant has been reported to adsorb between 0.7 and 1.5 mg of protein per mg Al+++ at pH 7.4.

[0137] The point of zero charge (PZC) of aluminum phosphate is inversely proportional to the degree to which the hydroxyl groups of the phosphate ester are substituted, and this degree of substitution can vary depending on the reaction conditions and the concentration of the reactants used to prepare the salt by precipitation. The PZC also changes by altering the concentration of free phosphate ions in the solution (more phosphate ester = more acidic PZC) or by adding a buffer such as histidine buffer (making the PZC more basic). The PZC of aluminum phosphate used according to the invention is typically between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example, about 5.7.

[0138] The concentration of aluminum ions in the composition used for administration to patients is preferably less than 2.5 mg / ml, for example, < 2 mg / ml < 1 mg / ml, etc. The preferred maximum concentration is... < 1.7 mg / mL. The Al in the compositions of this invention +++ The dosage range can be 0.3-1 mg / ml or 0.3-0.5 mg / ml. The maximum dose is preferably 0.85 mg / dose.

[0139] In solution, both aluminum phosphate adjuvant and aluminum hydroxide adjuvant tend to form stable porous aggregates with a diameter of 1-10 μm. The composition may contain a mixture of both aluminum hydroxide adjuvant and aluminum phosphate adjuvant.

[0140] In compositions comprising multiple conjugates, each of which is adsorbed onto an aluminum salt adjuvant, each conjugate may be adsorbed onto the aluminum salt individually and then mixed, or the conjugates may be added sequentially to the aluminum salt, thereby forming a mixed conjugate composition. Both methods of mixing can also be used.

[0141] Excipients for pharmaceutical compositions The pharmaceutical compositions of the present invention will generally contain one or more pharmaceutically acceptable excipients. A full discussion of such excipients can be found in the Handbook of Pharmaceutical Excipients (…). Handbook of Pharmaceutical Excipients ) (edited by Rowe et al.), 6th edition, 2009.

[0142] The pharmaceutical composition is preferably in an aqueous form, especially when applied, but it may also be in a dry form (e.g., as a lyophilized product), which can be converted into an aqueous form for application.

[0143] The pharmaceutical composition may contain a buffer or a pH adjuster. The buffer may be selected from the group consisting of phosphate buffers, acetate buffers, histidine buffers, citrate buffers, succinate buffers, Tris buffers, HEPES buffers, etc. The buffer salt is typically contained in the range of 5-20 mM.

[0144] Pharmaceutical compositions may contain physiological salts such as sodium salts, for example, to control stress. Sodium chloride (NaCl) is typical, and it may be present at concentrations of 1-20 mg / ml, for example, 10+2 mg / ml or 9 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium dehydrated phosphate, magnesium chloride, calcium chloride, etc. Other useful salts may have sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions.

[0145] The pharmaceutical composition may contain organic acids, such as acetic acid or succinic acid. This can be part of a buffer system.

[0146] The pharmaceutical composition may contain sugar alcohols, such as mannitol or sorbitol. The pharmaceutical composition may contain sugars, such as sucrose or glucose.

[0147] The pharmaceutical composition may contain a surfactant. Suitable surfactants include, but are not limited to, polysorbate 20, polysorbate 80, and sodium dodecyl sulfate (SDS). In some embodiments, the surfactant is present at a concentration of 0.0003% to 0.3% (w / w), for example, 0.01% to 0.03%. Polysorbate 80 is a preferred surfactant.

[0148] Pharmaceutical compositions may contain preservatives such as thimerosal or 2-phenoxyethanol. Preferably, the composition should be substantially free of (e.g., <10 μg / ml) mercury-free materials, such as thimerosal-free. Mercury-free compositions are more preferred. The inclusion of preservatives can be particularly useful when the composition contains aluminum salt adjuvants, as their insolubility means the composition is typically a suspension with a turbid appearance that can mask the growth of contaminating bacteria. Preservatives are also particularly useful if the composition is intended for use more than once, for example, in multi-dose vials. However, pharmaceutical compositions may generally be preservative-free.

[0149] The permeability of the pharmaceutical composition can be from 200 mOsm / kg to 400 mOsm / kg, for example, 240-360 mOsm / kg or 290-310 mOsm / kg.

[0150] The pH of the pharmaceutical composition is typically 5.0 to 9.5, for example, 5.0 to 8.0 or 6.0 to 8.0.

[0151] The pharmaceutical composition is preferably pyrogen-free, for example, containing <1 Eu (endotoxin unit, standard measure) per dose and preferably <0.1 EU per dose.

[0152] The permeability of the pharmaceutical composition can be 200-400 mOsm / kg, for example, 240-360 mOsm / kg or 280-320 mOsm / kg.

[0153] The pharmaceutical composition is preferably gluten-free.

[0154] The pharmaceutical composition is suitable for administration to animal (and especially human) patients and therefore comprises both human and veterinary uses.

[0155] The pharmaceutical composition can be prepared in unit dose form. In some embodiments, the volume of a unit dose can be 0.1-1.0 ml, for example about 0.25 mL or preferably about 0.5 ml. Such a volume is ideal for human injection.

[0156] Conjugate Level Pharmaceutical compositions may comprise multiple immunogenic conjugates. Currently licensed meningococcal conjugate vaccines comprise capsular sugars from four different serotypes, and licensed pneumococcal conjugate vaccines comprise capsular sugars from seven, ten, or thirteen different serotypes. Therefore, the compositions of the present invention may comprise, for example, three to fifty different conjugates (e.g., 14, 15, 20, 21, 24, 25, or more). For example, each of these conjugates may comprise capsular sugars from different serotypes or serogroups of the same bacterial species (e.g., multiple meningococcal serogroups or multiple pneumococcal serotypes).

[0157] In the pharmaceutical composition containing n In the case of different immunogenic conjugates, this n The total amount of carrier polypeptide in the conjugate can be less than or equal to that per dose. 3n µg. In other words, the average amount of the carrier polypeptide in each conjugate is less than 3 µg. The total amount can be, for example, per dose. n -2.5 n µg.

[0158] In the pharmaceutical composition containing n In the case of different immunogenic conjugates, this n The total amount of glycoantigen in the conjugate can be less than or equal to that per dose. 4.4n µg. In other words, the average amount of sugar in each conjugate is less than 4.4 µg. The total amount can be, for example, 0.4 µg per dose. n -4.4 n µg, for example, 1.1 n -2.2 n .

[0159] In the pharmaceutical composition containing n In the case of different immunogenic conjugates, thisn The total concentration of the carrier polypeptide in the conjugate can be less than or equal to 6n µg / mL. In other words, the average concentration of the carrier peptide for each conjugate is less than 6 µg / mL. The total concentration can be, for example, µg / mL. n -4 n µg / mL.

[0160] In the pharmaceutical composition containing n In the case of different immunogenic conjugates, this n The total concentration of the glycoantigen in the conjugate can be less than or equal to 8.8n µg / mL. In other words, the average sugar concentration of each conjugate is less than 8.8 µg / mL. The total concentration can be, for example, 0.8 µg / mL. n -8.8 n µg / mL, for example, 2.2 n -4.4 n µg / mL.

[0161] In some embodiments, the total amount of conjugated carrier peptide in a unit dose of the multivalent pharmaceutical composition of the present invention may be 4-128 µg, for example, 8-64 µg or 16-48 µg. The concentration of the conjugated carrier peptide in the multivalent pharmaceutical composition of the present invention may be 8-256 µg / mL, for example, 16-128 µg / mL or 32-96 µg / mL.

[0162] In some embodiments, the total amount of conjugated glycoantigen in a unit dose of the multivalent pharmaceutical composition of the present invention may be 10-120 µg, for example, 20-90 µg or 30-60 µg. The concentration of conjugated glycoantigen in the multivalent pharmaceutical composition of the present invention may be 20-240 µg / mL, for example, 40-180 µg / mL or 60-120 µg / mL.

[0163] Unconjugated components As noted above, the pharmaceutical composition may contain a variety of immunogenic conjugates, for example, 3 to 50 different conjugates (e.g., 14, 15, 20, 21, 24, 25 or more). For example, each of these conjugates may contain capsular sugars from different serotypes or serogroups of the same bacterial species.

[0164] In some embodiments, the composition does not contain one or more unconjugated carrier polypeptides. In other embodiments, a lower level of one or more unconjugated carrier polypeptides is present, provided that the mass of one or more unconjugated carrier polypeptides in the composition is less than that of the composition as a whole. nThe mass of one or more carrier peptides in the immunogenic conjugate is 10% (e.g., <5% or <2%).

[0165] In some embodiments, the composition does not contain the unconjugated form of the sugar. In other embodiments, a lower level of unconjugated sugar is present, provided that the mass of unconjugated sugar in the composition is less than that of the composition. n The total mass of sugars in the immunogenic conjugate is 10% (e.g., <5% or <2%).

[0166] Containers, delivery devices, etc. Pharmaceutical compositions containing immunogenic conjugates can be packaged in sterile containers, delivery devices, etc. Sterility can be maintained by using airtight, airtight containers. Suitable containers include, but are not limited to, vials, syringes, nebulizers, sprayers, inhalers, skin patches, etc. Vials and syringes are preferred.

[0167] The vials typically contain an immunogenic composition. The vials are preferably made of plastic or, more preferably, glass. The vials are sealed after filling, and the seal can be broken during use. Preferably, the vials are sterilized before the composition is added and then sealed. To avoid problems for patients sensitive to latex, the vials can be sealed with latex-free stoppers, and preferably, all packaging materials are latex-free. Ideally, the vials contain a single unit dose of the composition, but sometimes more than one dose ('multi-dose' vials), such as 10 doses, may be contained. The vials are preferably made of colorless glass.

[0168] The vial may have a cap (e.g., a Luer lock) adapted to allow a syringe to be inserted into the cap for transferring material between the vial and the syringe (in both directions). After the syringe is removed from the vial, a needle can then be attached, and the composition can be administered to the subject. The cap is preferably located inside a seal or cap, such that the seal or cap must be removed before access to the cap is possible. The vial may have a cap that allows for aseptic removal of the vial's contents, particularly for multi-dose vials.

[0169] The delivery device may contain a composition prepared for administration to a subject. The composition may be transferred into the delivery device at the time of use (e.g., from a vial), or the composition may be placed into the delivery device during the manufacturing stage (e.g., in the form of a pre-filled syringe).

[0170] The syringe used in conjunction with this invention can be made of glass or plastic (e.g., of a cyclic olefin polymer or cyclic olefin copolymer). The syringe (particularly a glass syringe) can be a siliconized syringe. Non-silicone syringes can also be used, such as the i-Coating™ system from Terumo (available in its PLAJEX™ syringes), the Daikyo CZ™ syringe with an ethylene-tetrafluoroethylene (ETFE) copolymer, or the TriboGlide™ syringe with perfluoropolyether (PFPE). A carbon film can be used instead of siliconization (e.g., see JP2001190665). Silicone-free syringes are also disclosed in JP2011212183. Non-silicone syringes comprising a plunger stopper, as disclosed in EP-A-0375778, can be used, i.e., wherein the stopper has a thermoplastic elastomer layer at least partially covered by a thermoplastic resin layer with a low dynamic coefficient of friction.

[0171] When the syringe contains a composition, it may have a needle attached thereto for injecting the contents of the syringe into a subject or container. A pre-attached needle may be supplied to the syringe. If the needle is not attached, the needle may be supplied separately with the syringe for assembly and use, or it may be supplied alone. Such needles should be sterile during use and may be sheathed. Safety needles may be used. 1-inch 23, 1-inch 25, and 5 / 8-inch 25 needles are typical. ½-1½-inch 22-25 needles may also be used. If the syringe and needle are packaged separately, the needle is preferably fitted with a butyl rubber sheath.

[0172] Syringes may be equipped with peel-off labels, which may be printed with the batch number and expiration date of the contents to facilitate record keeping. The plunger in the syringe may have a stopper to prevent accidental removal of the plunger during aspiration. Syringes may have latex rubber caps and / or plungers, but latex-free rubbers may be used, such as latex-free chlorobutyl rubber or latex-free isoprene bromobutyl rubber. Syringes will typically have a tip cap for sealing the tip before connecting the needle, and the tip cap is preferably made of butyl rubber, such as latex-free isoprene bromobutyl rubber. Useful syringes are, for example, those sold under the trade name "Tip-Lok"™.

[0173] Containers may be labeled to indicate a half-dose volume, for example, to facilitate delivery to children. For instance, a syringe containing a 0.5 ml dose may have a label indicating a 0.25 ml volume. The syringe itself may have a volume larger than the dose; for example, a 1 ml syringe may be used to hold a 0.5 ml dose of the pharmaceutical composition. Disposable or pre-filled syringes typically contain a single dose of vaccine.

[0174] When using glass containers (e.g., syringes or vials), the glass containers are preferably made of borosilicate glass rather than soda-lime glass.

[0175] Containers may be packaged (e.g., in the same box) with leaflets containing vaccine details such as administration instructions, details of the antigens contained in the vaccine, etc. The instructions may also contain warnings, such as ensuring the availability of epinephrine solution in case of an allergic reaction after vaccination. Multiple containers may be packaged together, for example, in the same box.

[0176] Pharmaceutical compositions can be presented in unit dose form, where each container (e.g., each syringe or each vial) has a single dose. Instead of manufacturing each unit dose individually, a large volume composition is prepared and unit doses are extracted and individually packaged into their containers. Thus, for example, multiple unit doses are extracted from the large volume composition and each unit dose is placed in a separate container, such as a syringe or vial.

[0177] Produce an immune response Immunogenic conjugates can be administered to mammalian subjects to elicit a protective immune response against antigens in the conjugate. The immunogenic conjugates are administered in the form of a pharmaceutical composition. The composition may contain multiple immunogenic conjugates as described elsewhere herein, thus enabling the simultaneous elicit of protective immune responses against numerous antigens.

[0178] Therefore, a method is provided for inducing a protective antibody response against one or more antigens in a mammalian subject by administering a conjugate of one or more antigens to the subject.

[0179] Conjugates, as disclosed herein, are also provided for use in inducing protective antibody responses.

[0180] The use of conjugates, as disclosed herein, in the manufacture of drugs for inducing protective antibody responses is also provided.

[0181] Also provided are (i) a method for inducing a protective antibody response against one or more antigens of a mammalian subject by administering the multivalent composition of the invention to the subject; (ii) the multivalent composition of the invention for use in inducing a protective antibody response; and (iii) the use of the various conjugates disclosed herein in the manufacture of multivalent pharmaceutical compositions for inducing a protective antibody response against a variety of antigens.

[0182] The ability to elicit a protective immune response means that the conjugate can be used, for example, to provide active immunization to prevent invasive diseases caused by Streptococcus pneumoniae, to prevent otitis media caused by Streptococcus pneumoniae, to prevent pneumonia caused by Streptococcus pneumoniae, and to provide active immunization to prevent invasive diseases in subjects at risk of exposure to Neisseria meningitidis.

[0183] Pharmaceutical compositions can be prepared in various forms. For example, the composition can be prepared as an injectable preparation, such as a liquid solution or suspension. Injectable preparations for intramuscular administration are typical. An injection volume of about 0.5 ml is preferred for humans. Therefore, a preferred unit dose volume is about 0.5 ml. Intramuscular injection is typical for example, into the anterolateral thigh of infants or into the deltoid muscle of the upper arm in young children and adults.

[0184] Conjugates are typically administered according to a multiple-dose regimen. Multiple doses may be used in primary immunization regimens and / or booster immunization regimens. Administration of more than one dose (usually two doses) is particularly useful for patients with an immune blank. Multiple doses are typically administered at intervals of at least 1 week (e.g., approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, etc.).

[0185] Overview The term "comprising" encompasses both "including" and "consisting of," for example, a composition "comprising" X may consist of only X or may contain additional substances, such as X + Y.

[0186] With numerical values x The related term "about" is optional and means, for example... x + 10%.

[0187] The term "substantially" does not exclude "completely," for example, a composition that is "substantially free of" Y can be completely free of Y. Where necessary, the term "substantially" can be omitted from the definition of this invention.

[0188] In the context of two amino acid sequences, the term "sequence identity" refers to two sequences that, when compared and aligned against the largest corresponding sequence within a comparison window, have the same or the same specific percentage of amino acid residues, as measured using a sequence comparison algorithm (e.g., BLASTP). The identity percentage is determined on a full-length reference sequence disclosed herein, such as the one shown in SEQ ID NO: 1 or 2. The method for calculating sequence identity, as provided herein, is the BLASTP procedure, which is defaulted to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, 1989, Proceedings of the National Academy of Sciences). Proc Natl Acad Sci USA (89:10915). See, for example, available on the WWW. blast.ncbi.nlm.nih.gov / Blast.cgi BLAST comparison tools obtained from other sources.

[0189] As used herein and unless otherwise stated, the term "lower alkyl" refers to a saturated straight-chain or branched hydrocarbon having one to six carbon atoms, i.e., a C1 to C6 alkyl group. In some embodiments, the lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted portions. See also US-2014 / 0066598. The term "lower alkylene" refers to an alkylene of a lower alkyl group.

[0190] Unless otherwise defined, all technical and scientific terms used herein have their commonly understood meanings. Practitioners should refer in particular to Green and Sambrook (eds.) Molecular Cloning: Laboratory Manual ( Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012) and Ausubel, FM et al. , *Current Protocols in Molecular Biology* (Supplement 99), John Wiley & Sons, New York (2012) and Plotkin, SA, Orenstein, WA, and Offit, PA, *Vaccines*. , 6th edition, Elsevier, London (2013).

[0191] Methods for cell-free synthesis are described in Spirin and Swartz (2008) "Cell-Free Protein Synthesis (… Cell-free Protein Synthesis ) , Wiley-VCH GmbH, Weinheim, Germany. A method for incorporating non-natural amino acids into proteins using cell-free synthesis is described in Shimizu et al. (2006) FEBS Journal ( FEBS Journal (See also: ) , 273, 4133-4140 and Chong (2014) Molecular Biology Laboratory Guide 108:16.30.1-11.

[0192] In some embodiments, the present invention does not cover compositions in which SEQ ID NO: 3 is used as a carrier polypeptide for conjugates of each of the following 24 pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F (as illustrated by example in WO2018 / 126229). More generally, in some embodiments, the present invention does not cover compositions in which SEQ ID NO: 3 is used as a carrier polypeptide for each conjugate in a multivalent composition.

[0193] Listed Examples Example I-1. A sterile container containing a pharmaceutical composition comprising an immunogenic conjugate, said immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein said glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0194] Example 1-2. An airtight container containing a pharmaceutical composition comprising an immunogenic conjugate, said immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein said glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide. Suitable containers for airtight sealing include, for example, vials. When airtight, the contents are preferably sterile.

[0195] Example I-3. The container according to Example I-1 or I-2 is a sterile glass container such as a vial.

[0196] Example 1-4. A delivery device comprising a pharmaceutical composition including an immunogenic conjugate, the immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0197] Example I-5. The container according to Example I-1 or I-2 or the delivery device according to Example I-4, wherein the container or the delivery device is a syringe.

[0198] Example I-6. A pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the aluminum salt adjuvant is aluminum hydroxide or aluminum phosphate adjuvant.

[0199] Example 1-7. A pharmaceutical composition comprising two or more different immunogenic conjugates and an aluminum phosphate adjuvant, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the concentration of aluminum ions in the composition. < 2.5 mg / mL.

[0200] Example 1-8. A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the volume of the pharmaceutical composition is 0.25-1.25 mL.

[0201] Examples I-9. A pharmaceutical composition comprising two or more different immunogenic conjugates and a preservative, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0202] Example I-10. A preservative-free pharmaceutical composition comprising two or more different immunogenic conjugates, wherein each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide.

[0203] Example I-11. A pharmaceutical composition comprising two or more different immunogenic conjugates, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the composition has a permeability of 200-400 mOsm / kg.

[0204] Example I-12. A pharmaceutical composition comprising two or more different immunogenic conjugates and at least one excipient, wherein: (i) each immunogenic conjugate comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and (ii) the at least one excipient is selected from the group consisting of sodium chloride, succinic acid, and polysorbate 80.

[0205] Example I-13. A pharmaceutical composition comprising n Different immunogenic conjugates, among which: (i) The above n Each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; (ii) n is an integer from 3 to 50; and (iii) The total amount of carrier polypeptide in the n immunogenic conjugates is less than or equal to 3 per dose. n µg; (iv) The total concentration of the carrier polypeptide in the n immunogenic conjugates is less than or equal to 6. n µg / ml; (v) The total amount of glycoantigen in the n immunogenic conjugates is less than or equal to 3 per dose. n µg; (vi) The total concentration of glycoantigens in the n immunogenic conjugates is less than or equal to 6. n µg / mL; (vii) The average amount of the carrier polypeptide for each conjugate is 1-4 µg per dose; (viii) The average concentration of the carrier peptide for each conjugate is 2-8 µg / mL; (ix) The average amount of glycoantigen in each conjugate is 1-4 µg per dose; (x) The average concentration of the glycoantigen in each conjugate is 2-8 µg / mL; (xi) The composition does not contain one or more unconjugated forms of the carrier polypeptide; (xii) The composition contains one or more unconjugated forms of the carrier polypeptide, wherein the mass of the one or more unconjugated forms of the carrier polypeptide in the composition is less than that of the... n 10% by mass of the carrier polypeptide in the immunogenic conjugate; (xiii) The composition does not contain the unconjugated form of the glycoantigen; and / or (xiv) The composition contains at least one of the unconjugated forms of the glycoantigen, wherein the total mass of the unconjugated forms of the glycoantigen in the composition is less than that of the composition. n 10% of the total mass of the glycoantigen in the immunogenic conjugate.

[0206] Example I-14. A method for preparing a plurality of unit doses of a pharmaceutical composition, wherein (i) the pharmaceutical composition comprises an immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein the glycoantigen is covalently bound to the carrier polypeptide via non-natural amino acid residues in the carrier polypeptide, and (ii) the method comprises the steps of preparing a bulk composition comprising the immunogenic conjugate and packaging individual unit doses from the bulk composition into a plurality of individual containers.

[0207] Example I-15. A method for preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises two or more different immunogenic conjugates and an aluminum salt adjuvant, wherein (i) each of the immunogenic conjugates comprises a carrier polypeptide and a glycoantigen, and (ii) the glycoantigen is covalently bound to the carrier polypeptide via a non-natural amino acid residue in the carrier polypeptide; and the method comprises (A) adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant and then mixing the individually adsorbed conjugates together, or (B) sequentially adsorbing each of the immunogenic conjugates onto the aluminum salt adjuvant.

[0208] Example I-16. A carrier polypeptide comprising an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO: 1; (ii) lacking an Arg-Arg dipeptide sequence; and (iii) containing at least one nnAA residue.

[0209] Example I-17. A carrier polypeptide comprising an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO: 1 and (ii) containing nnAA substitutions at one or more of the following amino acid residues (numbered according to SEQ ID NO: 1): Asp-211; Asp-295; Asp-352; Asp-392; Asp-465; Asp-467; Asp 507; Asp 519; Asn 296; Asn 359; Asn 399; Asn 481; Asn 486; Asn 502; Asn 524; Glu 240; Glu 248; Glu 249; Glu 256; Glu 259; Glu 292; Glu 362; Gln 252; Gln 287; Lys212; 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; Arg472; 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; Thr253; Thr 265; Thr 267; Thr 269; Thr 293; Thr 386; Thr 400; Thr 408; Thr-469; and / or Thr517.

[0210] Example I-18. The carrier polypeptide according to Example I-16 or I-17, wherein Arg-193 of SEQ ID NO: 1 is replaced with different amino acids such as Asn.

[0211] Example I-19. An immunogenic conjugate comprising a carrier polypeptide according to Example I-16, I-17, or I-18, wherein the carrier polypeptide is conjugated to an antigen via an nnAA residue in the carrier polypeptide.

[0212] Example I-20. An immunogenic conjugate comprising a carrier polypeptide and a glycoantigen, wherein (i) the carrier polypeptide comprises the amino acid sequence SEQ ID NO: 4; and (ii) the glycoantigen is covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO: 4.

[0213] Example 1-21. A pharmaceutical composition comprising two or more different immunogenic conjugates according to Example 1-20.

[0214] Examples I-22. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises 4 to 9 nnAA residues.

[0215] Examples I-23. A container, apparatus, composition, method, polypeptide or conjugate according to any of the foregoing examples, wherein at least one nnAA replaces a lysine residue in the natural sequence of the carrier polypeptide.

[0216] Examples I-24. Containers, devices, compositions, methods, polypeptides or conjugates according to any of the foregoing examples, wherein the carrier polypeptide has at least 90% sequence identity with SEQ ID NO: 1.

[0217] Examples I-25. Containers, devices, compositions, methods, polypeptides or conjugates according to Examples I-24, wherein at least one nnAA replaces K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and / or K526 in SEQ ID NO: 1 or 2.

[0218] Examples I-26. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises the amino acid sequence SEQ ID NO: 14.

[0219] Examples I-27. Containers, devices, compositions, methods, polypeptides or conjugates according to any of the foregoing examples, wherein the nnAA is 2-amino-3-(4-(azidomethyl)phenyl)propionic acid.

[0220] Examples I-28. Containers, devices, compositions, methods, polypeptides, or conjugates according to any of the foregoing examples, wherein the antigen has an alkynyl group conjugated to the nnAA via an azide group.

[0221] Examples I-29. A container, apparatus, composition, method, polypeptide, or conjugate according to any of the foregoing examples, wherein the antigen is a bacterial capsular sugar; for example, a capsular sugar from bacteria selected from the group consisting of: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis.

[0222] Examples I-30. Containers, devices, compositions, methods, polypeptides, or conjugates according to any of the foregoing examples, wherein the antigen is a capsular sugar of a Streptococcus pneumoniae serotype selected from the group consisting of: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F.

[0223] Example I-31. A container, apparatus, composition, method, polypeptide or conjugate according to any of the foregoing examples, wherein the ratio (w / w) of sugar to carrier polypeptide in one or more of the conjugates is greater than 1.

[0224] Examples 1-32. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the carrier peptide comprises three or more nnAA residues, and the conjugate has a molecular weight of at least 500 kDa.

[0225] Examples I-33. Containers, devices, compositions, methods, peptides or conjugates according to any of the foregoing examples, wherein the molecular weight of the conjugate is between 900 kDa and 5 MDa.

[0226] Example I-34. A container, device, composition, or method according to any one of Examples I-1 to I-15 or Examples I-21 to I-33, wherein the pharmaceutical composition comprises: A conjugate of capsular sugars from two or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 14 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 15 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 20 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 21 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 24 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 25 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from four or more different serogroups of meningococci, wherein the different serogroups of meningococci are selected from the group consisting of: A, C, W135, X, and Y; or A conjugate of capsular sugars from two or more different serotypes of Porphyromonas gingivalis, wherein the different serotypes of Porphyromonas gingivalis are selected from the group consisting of the following serotypes: K1, K2, K3, K4, K5 and K6.

[0227] Example I-35. A method for inducing an immune protective antibody response against an antigen in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of Examples I-6 to I-13 or Examples I-21 to I-34 or an immunogenic conjugate according to any one of Examples I-19 to I-33 in an excipient suitable for parenteral administration.

[0228] Example The invention is illustrated in the following examples. The materials, methods, and examples are merely illustrative and not intended to be limiting. Various variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. Unless otherwise described in detail, the examples are performed using techniques and conventional methods well known to those skilled in the art.

[0229] Example from WO2018 / 126229 The examples in WO2018 / 126229 describe in detail the synthesis of unit site eCRM moieties (e.g., K11TAG). These moieties are expressed in cell-free protein synthesis (CFPS) extracts and are incorporated with pAMF instead of native Lys.

[0230] Variants of CRM containing multiple nnAAs per polypeptide were also expressed, with each protein having various numbers of Lys→pAMF substitutions. Generally, the more substitutions found, the higher the resulting vector-induced MW conjugate, but the lower the vector solubility. Vectors with six pAMF residues typically provide both good solubility (>>50 mg / mL) and immunogenicity. The high solubility is surprising because replacing charged Lys residues in the native sequence with hydrophobic pAMF residues increases the hydrophobicity of CRM197, a protein whose hydrophobicity has been reported to affect its solubility. This demonstrates that the same linker sites (i.e., Lys residues) already used in known CRM197 conjugates can be maintained without insoluble matter when charged residues are lost.

[0231] A particularly useful set of six Lys→pAMF substitutions was observed using K34, K213, K245, K265, K386, and K527 (numbered according to SEQ ID NO: 3). This combination of pAMF substitution sites was surprisingly effective, especially since individual substitutions at positions K245 and K527 resulted in relatively poor expression levels.

[0232] This set of six substitutions can be combined with the disruption (RR→RN) of the Arg-Arg dipeptide at residues 192-193 of SEQ ID NO: 1 to provide SEQ ID NO: 4, where each X is pAMF.

[0233] Examples in WO2018 / 126229 further describe general protocols for sugar activation with sodium metaperiodate, for periodate oxidation of polysaccharides with DBCO, for sugar activation with CDAP, and for sugar-DBCO conjugation with eCRM. See also U.S. Serial No. 62 / 693,978, previously incorporated by reference.

[0234] Multivalent immunogenic compositions Combinations of conjugates for each of the 24 pneumococcal serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) were prepared using a CRM197 derivative SEQ ID NO: 4 (where X = pAMF) as the carrier polypeptide in each conjugate. The immunogenicity of this multivalent composition was confirmed by intramuscular injection of 0.25 mL of vaccine in a group of seven rabbits using a three-dose regimen. Each dose contained 24 µg of sugar (1 µg per serotype), yielding a concentration of 96 µg / mL.

[0235] Then, combinations of conjugates for each of the 32 pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B were prepared, and immunogenicity was confirmed in a similar manner.

[0236] For comparative purposes, a 13-valent Prevnar™ conjugate vaccine and a 24-valent unconjugate vaccine made from a 23-valent Pneumovax™ vaccine supplemented with unconjugated serotype 6A polysaccharide were also tested. All three compositions contained equivalent polysaccharide doses for each serotype (except 6B, where Prevnar™ contained a double dose), which involved diluting Prevnar™ and Pneumovax™. All three compositions contained aluminum phosphate adjuvant (60 µg Al per dose). +++ This involves adding an adjuvant to Pneumovax™. The composition is preservative-free.

[0237] The 24-valent conjugate composition contains a lower amount of carrier peptide than the approved Prevnar-13™ vaccine, although the composition also contains capsular sugars from 11 additional serotypes. The total weight ratio of capsular sugars to carrier peptides in the 24-valent conjugate composition is approximately twice that observed in Prevnar™.

[0238] IgG and OPA responses in rabbits were measured. Following the third dose, these responses were significantly greater in rabbits receiving both conjugated vaccines than in rabbits receiving the unconjugated vaccine. Furthermore, the IgG and OPA responses using the 24-valent composition were comparable to those achieved using Prevnar™ in the 13 serotypes covered by the approved vaccines, but also superior to those in the 11 serotypes not included in Prevnar™. Surprisingly, there was no evidence that the vector used in the 24-valent composition induced epitope inhibition.

[0239] Figure 1 The geometric mean titer of each of the 32 serotypes in the 32-valent conjugate composition relative to the polysaccharide / alum formulation and Prevnar-13™ is provided.

[0240] Multivalent conjugate compositions can be usefully packaged into pre-filled sterile syringes, allowing them to be easily dispensed in unit dose form and then administered at the time of use without the need to transfer the contents of the vial into a syringe for injection, etc.

[0241] Replaceable positions in CRM197 Based on the work disclosed in WO2018 / 126229, various Asp, Asn, Glu, Gln, Lys, Arg, Ser, and Thr residues in the natural CRM197 sequence (SEQ ID NO: 1) were replaced with pAMF by mutating their codons to TAG and expressing the protein in a cell-free system at 25°C, where this codon is recognized by tRNA incorporating nnAA. The mutant polypeptide was expressed using an N-terminal methionine and a downstream six-histidine tag linked via a Gly-Ser-Gly tripeptide linker. Residues in Asn270-Ile289, Ala320-Glu349, and Phe410-His-449 were avoided due to the T-cell epitopes recognized in these regions (see above).

[0242] By inspection 14C-Leu incorporation into mutant proteins and examination of both total and soluble protein levels are used to assess expression efficiency. Generally, mutations in the catalytic domain of CRM197 result in lower expression levels relative to the unmodified CRM197 sequence, and mutants with optimal expression levels typically involve substitutions downstream of Arg-193, which can be used to delineate the ends of the catalytic domain.

[0243] The optimal 72 mutants showed increased expression levels of both total and soluble protein and had substitutions at the following residues numbered according to SEQ ID NO: 1: Ser-198, Ser-200, Asp-211, Lys-212, Lys-218, Lys-221, Lys-229, Ser-231, Ser-233, Lys-236, Ser-239, Glu-240, Glu-248, Glu-249, Gln-252, Thr-253, Glu- 256, Glu-259, Ser-261, Lys-264, Thr-265, Thr-267, Thr-269, Gln-287, Glu-292, Thr-29 3. 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, Se r-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-50 1. Asn-502, Ser-505, Asp-507, Lys-516, Thr-517, Asp-519, Lys-522, Asn-524 and Lys-534.

[0244] The embodiments described herein are provided by way of example only, and various alternatives to the embodiments are not excluded when practicing them.

[0245] sequence list SEQ ID NO: 1 (Natural CRM197) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS SEQ ID NO: 2 (CRM197 with Arg-Asn substitution) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS SEQ ID NO: 3 (CRM197 with 6 preferred nnAA sites and N-terminal Met) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSEPNKTVSEEKA X QYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS SEQ ID NO: 4 (with Arg-Asn substitution) n Six preferred nnAA sites and CRM197 of N-terminal Met) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQ X GIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRD X TKTKIESLKEHGPIKNKMSESPNKTVSEEKA X QYLEEFHQTALEHPELSEL X TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGH X TQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS X LSLFFEIKS SEQ ID NO: 5 (Haemophilus influenzae protein D) CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

Claims

1. A carrier polypeptide comprising an amino acid sequence (i) having at least 80% sequence identity with SEQ ID NO: 1; (ii) lacking an Arg-Arg dipeptide sequence; and (iii) comprising 4-9 nnAA residues that substitute for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and / or K526 in SEQ ID NO: 1 or 2.

2. The carrier polypeptide according to claim 1, wherein Arg-193 of SEQ ID NO: 1 is replaced with different amino acids such as Asn.

3. The carrier polypeptide according to claim 1, wherein the carrier polypeptide has at least 90% sequence identity with SEQ ID NO:

1.

4. The carrier polypeptide according to claim 1, wherein the carrier polypeptide comprises the amino acid sequence SEQ ID NO:

4.

5. The carrier polypeptide according to claim 1, wherein the nnAA residue is 2-amino-3-(4-(azidomethyl)phenyl)propionic acid.

6. An immunogenic conjugate comprising a carrier polypeptide according to any one of claims 1-5, wherein the carrier polypeptide is conjugated to an antigen via an nnAA residue in the carrier polypeptide.

7. The immunogenic conjugate according to claim 6, wherein the antigen has an alkynyl group conjugated to the nnAA via an azide group.

8. The immunogenic conjugate according to claim 6, comprising a carrier polypeptide and a glycoantigen, wherein (i) the carrier polypeptide comprises the amino acid sequence SEQ ID NO: 4; and (ii) the antigen is a glycoantigen covalently bound to the carrier polypeptide via at least one nnAA residue in SEQ ID NO:

4.

9. The immunogenic conjugate according to claim 8, wherein the antigen has an alkynyl group conjugated to the nnAA via an azide group.

10. The immunogenic conjugate according to claim 6, wherein the antigen is a bacterial capsular sugar; for example, a capsular sugar from bacteria selected from the group consisting of: Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae ), Neisseria meningitidis ( Neisseria meningitidis Haemophilus influenzae ( ) Haemophilus influenzae ), Streptococcus pyogenes ( Streptococcus pyogenes ), agalactococcus ( Streptococcus agalactiae ) and Porphyromonas gingivalis ( Porphyromonas gingivalis ).

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

12. The immunogenic conjugate of claim 6, wherein the carrier polypeptide comprises three or more nnAA residues, and the conjugate has a molecular weight of at least 500 kDa.

13. The immunogenic conjugate according to claim 11, wherein the molecular weight of the conjugate is between 900 kDa and 5 MDa.

14. A pharmaceutical composition comprising two or more different immunogenic conjugates according to claim 6.

15. The pharmaceutical composition of claim 14, wherein the ratio (w / w) of sugar to carrier polypeptide in the composition is greater than 1.

16. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition comprises: A conjugate of capsular sugars from two or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 14 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 15 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 20 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 21 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 24 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 25 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from four or more different serogroups of meningococci, wherein the different serogroups of meningococci are selected from the group consisting of: A, C, W135, X, and Y; or A conjugate of capsular sugars from two or more different serotypes of Porphyromonas gingivalis, wherein the different serotypes of Porphyromonas gingivalis are selected from the group consisting of the following serotypes: K1, K2, K3, K4, K5 and K6.

17. The immunogenic conjugate according to claim 7 or 8, wherein the antigen is a bacterial capsular sugar; for example, a capsular sugar from bacteria selected from the group consisting of: Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae ), Neisseria meningitidis ( Neisseria meningitidis Haemophilus influenzae ( ) Haemophilus influenzae ), Streptococcus pyogenes ( Streptococcus pyogenes ), agalactococcus ( Streptococcus agalactiae ) and Porphyromonas gingivalis ( Porphyromonas gingivalis ).

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

19. The immunogenic conjugate according to claim 7 or 8, wherein the carrier polypeptide comprises three or more nnAA residues, and the conjugate has a molecular weight of at least 500 kDa.

20. The immunogenic conjugate of claim 19, wherein the molecular weight of the conjugate is between 900 kDa and 5 MDa.

21. A pharmaceutical composition comprising two or more different immunogenic conjugates according to any one of claims 7-13.

22. The pharmaceutical composition according to claim 21, wherein the ratio (w / w) of sugar to carrier polypeptide in the composition is greater than 1.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises: A conjugate of capsular sugars from two or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 14 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 15 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 20 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 21 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 24 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from 25 or more different pneumococcal serotypes, wherein the different pneumococcal serotypes are selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; A conjugate of capsular sugars from four or more different serogroups of meningococci, wherein the different serogroups of meningococci are selected from the group consisting of: A, C, W135, X, and Y; or A conjugate of capsular sugars from two or more different serotypes of Porphyromonas gingivalis, wherein the different serotypes of Porphyromonas gingivalis are selected from the group consisting of the following serotypes: K1, K2, K3, K4, K5 and K6.

24. Use of the polypeptide carrier according to any one of claims 1-5, the immunogenic conjugate according to any one of claims 6-13, 17-20, the pharmaceutical composition according to any one of claims 14-16, and the pharmaceutical composition according to any one of claims 21-23 in the preparation of a medicament that induces an immune protective antibody response against an antigen in a subject.

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