Antigenic synthetic proteins containing a tetrazine-amino acid

Abstract

The present invention relates to synthetic protein comprising at least one synthetic amino acid bearing a diene moiety and at least one sulfide linkage, to method for producing said synthetic protein and to the use of the synthetic protein in the production of a pharmaceutical composition.

Description

ANTIGENIC SYNTHETIC PROTEINS CONTAINING A TETRAZINE-AMINO ACID Description Field of the Invention

[0001] The present invention relates to the field of novel synthetic proteins containing one or more sulfide linkages and synthetic amino acids, specifically to antigenic carrier proteins bearing a tetrazine moiety, and to a process for soluble cytoplasmic expression of such proteins and to the use of the novel synthetic proteins. Background Art

[0002] The production of synthetic proteins plays an important role in business and science. By incorporating artificial amino acids into proteins, their existing properties can be specifically improved.

[0003] Conjugate vaccines are an established class of vaccines. They typically have a bacterial or viral antigen conjugated to a carrier protein. The carrier protein is usually a protein with several T-cell epitopes which can trigger and activate the immune system. By conjugating an antigen to these carrier proteins an immune response is triggered to the antigen as well, providing the vaccinated person with protection against the pathogen.

[0004] Chemical conjugation of a protein, e.g., a carrier protein to an antigen, e.g., a polysaccharide or peptide, is the basis for many highly effective vaccines including ones for Hemophilus influenzae b, Neisseria meningitis, Typhi, Group B strep, and Streptococcus pneumoniae. Prominent examples of these types of vaccines are Hemophilus Influenza Conjugate Vaccine (Hib) and Pneumococcal Conjugate Vaccine (Prevnar®).

[0005] Also peptides are conjugated to carrier proteins to make vaccines against pathogens and tumorous growths (Yong T, Chang KK, Wang YS, Ma C. Active Humoral Response Reverts Tumorigenicity through Disruption of Key Signaling Pathway. Vaccines (Basel).2022 Jan 21;10(2):163. and Peptide-Based Vaccines: Current Progress and Future Challenges Ryan J. Malonis, Jonathan R. Lai, and Olivia Vergnolle Chemical Reviews 2020120 (6), 3210-3229)

[0006] A variety of chemical approaches have been employed to link a carrier protein to a polysaccharide (Vaccine Development: From Concept to Clinic, Krishna Prasad, Wiley, 2022, ISBN 178801877X). CDAP, a cyanylating reagent, is a widely used method for derivatizing polysaccharides to facilitate conjugation (Vaccine, 14, 190,1996). CDAP can be used to aminate polysaccharide, which can be subsequently reacted with an NHS ester of trans-cyclooctene (TCO). CDAP can be used to directly derivatize polysaccharides with TCO. The polysaccharide is activated with CDAP as described (Vaccines 2020, 8, 777; doi:10.3390 / vaccines8040777) and reacted with an TCO-amine reagent such as TCO-PEG3-NH2 (BroadPharm).

[0007] WO2018126229A2 discloses conjugates wherein an antigen is conjugated to a non-natural amino acid via a triazole linking moiety. Preferably para-methylazido-L- phenylalanine (pAMF) is used as non-natural amino acid.

[0008] Azide-DBCO chemistry has several drawbacks like its slow speed and significant hydrophobicity of the resulting conjugate structure which may lead to product aggregation.

[0009] Usually, lysine conjugation is used to manufacture this type of conjugates, leading to a mixture of different bioconjugate species with different numbers of antigen attached to the carrier protein. This is non-ideal since the drug is not precisely defined and leads to challenges and costs in manufacturing and regulatory approval.

[0010] E.coli is a widely used host to produce recombinant proteins for research and therapeutic purposes. Recombinant proteins can be expressed in the cytoplasm or periplasm of E.coli. WO2021 / 188379A discloses a method of producing soluble recombinant peptides and proteins in bacterial cells wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes.

[0011] Therefore, there is still the need for site-specific conjugations of antigens to carrier protein for creating a defined product with reduced regulatory and production risks. Summary of invention

[0012] It is the object of the present invention to provide a synthetic protein comprising at least one synthetic amino acid and at least one sulfide linkage. The object is solved by the subject matter of the present invention.

[0013] According to the invention, there is provided a carrier protein comprising at least one synthetic amino acid bearing a diene moiety of general formula (I) and at least one sulfide linkage,wherein R1is selected from the group consisting of, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa; R2is denotes a group, optionally substituted by one or more, identical or different Rband / or Rc, selected from among C1-6alkyl, C3-10cycloalkyl, C6-10aryl, C7-16arylalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; Rais hydrogen or C1-6alkyl,

[0014] each Rbis a suitable substituent and is selected in each case independently of one another from among =O, –ORc, C1-3haloalkyloxy, –OCF3, =S, -SRc, =NRc, =NORc, =NNRcRc, =NN(Rg)C(O)NRcRc, –NRcRc, –ONRcRc, –N(ORc)Rc, –N(Rg)NRcRc, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Rc, –S(O)ORc, –S(O)2Rc, –S(O)2ORc, –S(O)NRcRc, –S(O)2NRcRc, –OS(O)Rc, –OS(O)2Rc, –OS(O)2ORc, –OS(O)NRcRc, –OS(O)2NRcRc, –C(O)Rc, –C(O)ORc, –C(O)SRc, –C(O)NRcRc, –C(O)N(Rg)NRcRc, –C(O)N(Rg)ORc, –C(O)N(Rg)S(O)2Rc, –C(NRg)NRcRc, –C(NOH)Rc, –C(NOH)NRcRc, –OC(O)Rc, –OC(O)ORc, –OC(O)SRc, –OC(O)NRcRc, –OC(NRg)NRcRc, –SC(O)Rc, –SC(O)ORc, –SC(O)NRcRc, –SC(NRg)NRcRc, –N(Rg)C(O)Rc, –N[C(O)Rc]2, –N(ORg)C(O)Rc, –N(Rg)C(NRg)Rc, –N(Rg)N(Rg)C(O)Rc, –N[C(O)Rc]NRcRc, –N(Rg)C(S)Rc, –N(Rg)S(O)Rc, –N(Rg)S(O)ORc, –N(Rg)S(O)2Rc, –N[S(O)2Rc]2, –N(Rg)S(O)2ORc, –N(Rg)S(O)2NRcRc, –N(Rg)[S(O)2]2Rc, –N(Rg)C(O)ORc, –N(Rg)C(O)SRc, –N(Rg)C(O)NRcRc, –N(Rg)C(O)NRgNRcRc, –N(Rg)N(Rg)C(O)NRcRc, –N(Rg)C(S)NRcRc, –[N(Rg)C(O)]2Rc, –N(Rg)[C(O)]2Rc, –N{[C(O)]2Rc}2, –N(Rg)[C(O)]2ORc, –N(Rg)[C(O)]2NRcRc, –N{[C(O)]2ORc}2, –N{[C(O)]2NRcRc}2, –[N(Rg)C(O)]2ORc, –N(Rg)C(NRg)ORc, –N(Rg)C(NOH)Rc, –N(Rg)C(NRg)SRcand –N(Rg)C(NRg)NRcRc; each Rcindependently of one another denotes hydrogen or a group, optionally substituted by one or more, identical or different Rdand / or Re, selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C4-16cycloalkylalkyl,C6-10aryl, C7-16arylalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; each Rddenotes a suitable substitutent and is selected in each case independently of one another from among =O, –ORe, C1-3haloalkyloxy,–OCF3, =S, –SRe, =NRe, =NORe, =NNReRe, =NN(Rg)C(O)NReRe, –NReRe, –ONReRe, –N(ORe)Re, –N(Rg)NReRe, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Re, –S(O)ORe, –S(O)2Re, –S(O)2ORe, –S(O)NReRe, –S(O)2NReRe, –OS(O)Re, –OS(O)2Re, –OS(O)2ORe, –OS(O)NReRe, –OS(O)2NReRe, –C(O)Re, –C(O)ORe, –C(O)SRe, –C(O)NReRe, –C(O)N(Rg)NReRe, –C(O)N(Rg)ORe, –C(NRg)NReRe, –C(NOH)Re, –C(NOH)NReRe, –OC(O)Re, –OC(O)ORe, –OC(O)SRe, –OC(O)NReRe, –OC(NRg)NReRe, –SC(O)Re, –SC(O)ORe, –SC(O)NReRe, –SC(NRg)NReRe, –N(Rg)C(O)Re, –N[C(O)Re]2, –N(ORg)C(O)Re, –N(Rg)C(NRg)Re, –N(Rg)N(Rg)C(O)Re, –N[C(O)Re]NReRe, –N(Rg)C(S)Re, –N(Rg)S(O)Re, –N(Rg)S(O)ORe, –N(Rg)S(O)2Re, –N[S(O)2Re]2, –N(Rg)S(O)2ORe, –N(Rg)S(O)2NReRe, –N(Rg)[S(O)2]2Re, –N(Rg)C(O)ORe, –N(Rg)C(O)SRe, –N(Rg)C(O)NReRe, –N(Rg)C(O)NRgNReRe, –N(Rg)N(Rg)C(O)NReRe, –N(Rg)C(S)NReRe, –[N(Rg)C(O)]2Re, –N(Rg)[C(O)]2Re, –N{[C(O)]2Re}2, –N(Rg)[C(O)]2ORe, –N(Rg)[C(O)]2NReRe, –N{[C(O)]2ORe}2, –N{[C(O)]2NReRe}2, –[N(Rg)C(O)]2ORe, –N(Rg)C(NRg)ORe, –N(Rg)C(NOH)Re, –N(Rg)C(NRg)SReand –N(Rg)C(NRg)NReRe; and each Reindependently of one another denotes hydrogen or a group selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, and 5-12 membered heteroaryl; and each Rgindependently of one another denotes hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, or 5-12 membered heteroaryl; and wherein the compound of formula (I) is linked to an amino acid residue which is comprised in the carrier protein.

[0015] One embodiment of the invention relates to a method for producing a carrier protein as described herein, comprising: a. providing a recombinant bacterial cell having a reduced activity of one or more disulfide reductase enzymes; b. expression of the carrier protein in a soluble form from a vector in said bacterial cell; andc. recovering the carrier protein.

[0016] A further embodiment relates to a use of the carrier protein as described herein for the production of a pharmaceutical composition, such as a vaccine. Brief description of drawings

[0017] Fig.1: A and B: Variants of CRM197 with 1 natural amino acid substituted with a synAA C, D and E: Variants of CRM197 with 3 natural amino acid substituted with a synAA, Natural CRM197: CRM197 containing only natural amino acids, Inducers: no: no inducers +I: +IPTG +synAA

[0018] Fig.2 shows the binding of natural and modified CRM197 to HB-EGF.

[0019] Fig.3 shows the elution profile of TCO-dex, CRM197, and CRM197 comprising one synthetic amino acid and TCO-dex conjugate.

[0020] Fig.4 shows the elution profile of TCO-dex, CRM197, and CRM197 comprising three synthetic amino acid and TCO-dex conjugate.

[0021] Fig.5 depicts scFv-antibody fragment containing synthetic amino acids is produced soluble in the cytosol using oxidative cytoplasm strain Escherichia coli BL21(DE3) with a mutation in the gorA gene.

[0022] Fig.6 depicts Fab-antibody fragment containing synthetic amino acids is produced soluble in the cytosol using oxidative cytoplasm strain Escherichia coli BL21(DE3) with a mutation in the gorA gene. Description of Embodiments.

[0023] The present invention relates to a modified carrier protein comprising at least one diene moiety. This at least diene moiety enables site-specific conjugation the accordingly modified carrier protein to antigens. This further allows the creation of well- defined pharmaceutical products with reduced regulatory and production risks.

[0024] One embodiment of the invention relates to a carrier protein comprising at least one synthetic amino acid bearing a diene moiety of general formula (I),wherein R1is selected from the group consisting of, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa;R2is denotes a group, optionally substituted by one or more, identical or different Rband / or Rc, selected from among C1-6alkyl, C3-10cycloalkyl, C6-10aryl, C7-16arylalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; Rais hydrogen or C1-6alkyl, each Rbis a suitable substituent and is selected in each case independently of one another from among =O, –ORc, C1-3haloalkyloxy, –OCF3, =S, -SRc, =NRc, =NORc, =NNRcRc, =NN(Rg)C(O)NRcRc, –NRcRc, –ONRcRc, –N(ORc)Rc, –N(Rg)NRcRc, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Rc, –S(O)ORc, –S(O)2Rc, –S(O)2ORc, –S(O)NRcRc, –S(O)2NRcRc, –OS(O)Rc, –OS(O)2Rc, –OS(O)2ORc, –OS(O)NRcRc, –OS(O)2NRcRc, –C(O)Rc, –C(O)ORc, –C(O)SRc, –C(O)NRcRc, –C(O)N(Rg)NRcRc, –C(O)N(Rg)ORc, –C(O)N(Rg)S(O)2Rc, –C(NRg)NRcRc, –C(NOH)Rc, –C(NOH)NRcRc, –OC(O)Rc, –OC(O)ORc, –OC(O)SRc, –OC(O)NRcRc, –OC(NRg)NRcRc, –SC(O)Rc, –SC(O)ORc, –SC(O)NRcRc, –SC(NRg)NRcRc, –N(Rg)C(O)Rc, –N[C(O)Rc]2, –N(ORg)C(O)Rc, –N(Rg)C(NRg)Rc, –N(Rg)N(Rg)C(O)Rc, –N[C(O)Rc]NRcRc, –N(Rg)C(S)Rc, –N(Rg)S(O)Rc, –N(Rg)S(O)ORc, –N(Rg)S(O)2Rc, –N[S(O)2Rc]2, –N(Rg)S(O)2ORc, –N(Rg)S(O)2NRcRc, –N(Rg)[S(O)2]2Rc, –N(Rg)C(O)ORc, –N(Rg)C(O)SRc, –N(Rg)C(O)NRcRc, –N(Rg)C(O)NRgNRcRc, –N(Rg)N(Rg)C(O)NRcRc, –N(Rg)C(S)NRcRc, –[N(Rg)C(O)]2Rc, –N(Rg)[C(O)]2Rc, –N{[C(O)]2Rc}2, –N(Rg)[C(O)]2ORc, –N(Rg)[C(O)]2NRcRc, –N{[C(O)]2ORc}2, –N{[C(O)]2NRcRc}2, –[N(Rg)C(O)]2ORc, –N(Rg)C(NRg)ORc, –N(Rg)C(NOH)Rc, –N(Rg)C(NRg)SRcand –N(Rg)C(NRg)NRcRc; each Rcindependently of one another denotes hydrogen or a group, optionally substituted by one or more, identical or different Rdand / or Re, selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C4-16cycloalkylalkyl, C6-10aryl, C7-16arylalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; each Rddenotes a suitable substitutent and is selected in each case independently of one another from among =O, –ORe, C1-3haloalkyloxy,–OCF3, =S, –SRe, =NRe, =NORe, =NNReRe, =NN(Rg)C(O)NReRe, –NReRe, –ONReRe, –N(ORe)Re, –N(Rg)NReRe, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Re, –S(O)ORe, –S(O)2Re, –S(O)2ORe, –S(O)NReRe, –S(O)2NReRe,–OS(O)Re, –OS(O)2Re, –OS(O)2ORe, –OS(O)NReRe, –OS(O)2NReRe, –C(O)Re, –C(O)ORe, –C(O)SRe, –C(O)NReRe, –C(O)N(Rg)NReRe, –C(O)N(Rg)ORe, –C(NRg)NReRe, –C(NOH)Re, –C(NOH)NReRe, –OC(O)Re, –OC(O)ORe, –OC(O)SRe, –OC(O)NReRe, –OC(NRg)NReRe, –SC(O)Re, –SC(O)ORe, –SC(O)NReRe, –SC(NRg)NReRe, –N(Rg)C(O)Re, –N[C(O)Re]2, –N(ORg)C(O)Re, –N(Rg)C(NRg)Re, –N(Rg)N(Rg)C(O)Re, –N[C(O)Re]NReRe, –N(Rg)C(S)Re, –N(Rg)S(O)Re, –N(Rg)S(O)ORe, –N(Rg)S(O)2Re, –N[S(O)2Re]2, –N(Rg)S(O)2ORe, –N(Rg)S(O)2NReRe, –N(Rg)[S(O)2]2Re, –N(Rg)C(O)ORe, –N(Rg)C(O)SRe, –N(Rg)C(O)NReRe, –N(Rg)C(O)NRgNReRe, –N(Rg)N(Rg)C(O)NReRe, –N(Rg)C(S)NReRe, –[N(Rg)C(O)]2Re, –N(Rg)[C(O)]2Re, –N{[C(O)]2Re}2, –N(Rg)[C(O)]2ORe, –N(Rg)[C(O)]2NReRe, –N{[C(O)]2ORe}2, –N{[C(O)]2NReRe}2, –[N(Rg)C(O)]2ORe, –N(Rg)C(NRg)ORe, –N(Rg)C(NOH)Re, –N(Rg)C(NRg)SReand –N(Rg)C(NRg)NReRe; and each Reindependently of one another denotes hydrogen or a group selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, and 5-12 membered heteroaryl; and each Rgindependently of one another denotes hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, or 5-12 membered heteroaryl; and wherein the compound of formula (I) is linked to an amino acid residue which is comprised in the synthetic protein.

[0025] As used herein, the following definitions apply, unless stated otherwise.

[0026] Unless specified otherwise, the term “alkyl”, when used alone or in combination with other groups or atoms, refers to a saturated straight or branched chain consisting solely of 1 to 6 hydrogen-substituted carbon atoms, and includes methyl, ethyl, propyl, isopropyl, n-butyl, 1-methylpropyl, isobutyl, t-butyl, 2,2- dimethylbutyl, 2,2-dimethyl-propyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, n-hexyl and the like.

[0027] Unless specified otherwise, the term “alkenyl” refers to a partially unsaturated straight or branched chain consisting solely of 2 to 6 hydrogen-substituted carbon atoms that contains at least one double bond, and includes vinyl, allyl, 2-methylprop-1- enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, penta-1,3-dienyl, penta-2,4- dienyl, 2-methylbut-1-enyl, 2-methylpent-1-enyl, 4-methylpent-1-enyl, 4-methylpent-2- enyl, 2-methylpent-2-enyl, 4-methylpenta-1,3-dienyl, hexen-1-yl and the like.

[0028] Unless specified otherwise, the term “alkynyl” refers to a partially unsaturated straight or branched chain consisting solely of 2 to 6 hydrogen-substituted carbon atoms that contains at least one triple bond, and includes ethynyl, 1-propynyl, 2-propynyl, 2-methylprop-1-ynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1,3-butadiynyl, 3-methylbut-1-ynyl, 4-methylbut-ynyl, 4-methylbut-2-ynyl, 2-methylbut-1-ynyl, 1- pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 3- methylpent-1-ynyl, 4-methylpent-2-ynyl, 4-methylpent-2-ynyl, 1-hexynyl, and the like.

[0029] Unless specified otherwise, the term “cycloalkyl”, when used alone or in combination with other groups or atoms, refers to monocyclic hydrocarbon rings, bicyclic hydrocarbon rings or spirohydrocarbon rings, which each may be either saturated or unsaturated (cycloalkenyl). The term unsaturated means that in the ring system in question there is at least one double bond, but no aromatic system is formed. In bicyclic hydrocarbon rings two rings are linked such that they have at least two carbon atoms in common. In spirohydrocarbon rings one carbon atom (spiroatom) is shared by two rings. If a cycloalkyl is substituted, the substitution may be mono- or polysubstitution in each case, at all the hydrogen-carrying carbon atoms, independently of one another. Cycloalkyl itself may be linked to the molecule as substituent via any suitable position of the ring system.

[0030] Typical examples of individual sub-groups are listed below.

[0031] Monocyclic saturated hydrocarbon rings: cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cycloheptyl, etc.

[0032] Monocyclic unsaturated hydrocarbon rings: cycloprop-1-enyl; cycloprop-2-enyl; cyclobut-1-enyl; cyclobut-2-enyl; cyclopent-1-enyl; cyclopent-2-enyl; cyclopent-3-enyl; cyclohex-1-enyl; cyclohex-2-enyl; cyclohex-3-enyl; cyclohept-1-enyl; cyclohept-2-enyl; cyclohept-3-enyl; cyclohept-4-enyl; cyclobuta-1,3- dienyl; cyclopenta-1,4-dienyl; cyclopenta-1,3-dienyl; cyclopenta-2,4-dienyl; cyclohexa-1,3- dienyl; cyclohexa-1,5- dienyl; cyclohexa-2,4-dienyl; cyclohexa-1 ,4-dienyl; cyclohexa-2,5- dienyl, etc.

[0033] Saturated and unsaturated bicyclic hydrocarbon rings: bicyclo[2.2.0]hexyl; bicyclo[3.2.0]heptyl; bicyclo[3.2.1]octyl; bicyclo[2.2.2]octyl; bicyclo[4.3.0]nonyl (octahydroindenyl); bicyclo[4.4.0]decyl (decahydronaphthalene); bicyclo[2,2,1]heptyl (norbornyl); (bicyclo[2.2.1]hepta-2,5-dienyl (norborna-2,5-dienyl); bicyclo[2,2,1]hept-2- enyl (norbornenyl); bicyclo[4.1.0]heptyl (norcaranyl); bicyclo- [3.1.1]heptyl (pinanyl), etc.

[0034] Saturated and unsaturated spirohydrocarbon rings: spiro[2.5]octyl, spiro[3.3]heptyl, spiro[4.5]dec-2-ene, etc.

[0035] “Cycloalkylalkyl” denotes the combination of the above-defined groups alkyl, alkenyl, alkynyl, and cycloalkyl, in each case in their broadest sense. The alkyl group as substituent is directly linked to the molecule and is in turn substituted by a cycloalkyl group. The alkyl and cycloalkyl may be linked in both groups via any carbon atoms suitable for this purpose. The respective sub-groups of alkyl and cycloalkyl are also included in the combination of the two groups.

[0036] Unless specified otherwise, the term “aryl” refers to an aromatic mono- or bicyclic group containing from 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, that may be optionally fused with a fully or partially saturated or unsaturated carbocyclic ring and may optionally be substituted with one or more, identical or different substituents, suitably one to three substituents. Examples of aryl groups include phenyl, naphthyl, indanyl, and the like.

[0037] “Arylalkyl” denotes the combination of the group alkyl, alkenyl, alkynyl and aryl as hereinbefore defined, in each case in their broadest sense. The alkyl group as substituent is directly linked to the molecule and is in turn substituted by an aryl group. The alkyl and aryl may be linked in both groups via any carbon atoms suitable for this purpose. Typical examples include benzyl, 1-phenylethyl, 2-phenylethyl, phenylvinyl, phenylallyl, etc.

[0038] Unless specified otherwise, the term “heteroaryl” refers to an aromatic mono- or bicyclic group containing from 5 to 14 carbon atoms, preferably 5 to 12 carbon atoms, of which one to five is replaced with a heteroatom selected from N, S and O, that may optionally be reduced to a non-aromatic heterocycle and may optionally be substituted with one or more, identical or different substituents. Examples of heteroaryl groups include pyrrolyl, dihydropyrrolyl, pyrrolidinyl, oxopyrrolidinyl, indolyl, isoindolyl, indolizinyl, imidazolyl, pyrazolyl, benzimidazolyl, imidazo(1,2-a)pyridinyl, indazolyl, purinyl, pyrrolo(2,3-c)pyridinyl, pyrrolo(3,2-c)pyridinyl, pyrrolo(2,3-b)pyridinyl, pyrazolo(1,5-a)pyridinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, oxazolyl, 1,2 oxazolyl, isoxazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,3- thiadiazolyl, furanyl, dihydrofuranyl, tetrahydrofuranyl, benzofuranyl, isobenzofuranyl, thiophenyl, dihydrothiophenyl, tetrahydrothiophenyl, benzothiophenyl,benzoisothiophenyl, pyridyl, piperidinyl, quinolinyl, isoquinolinyl, tetrahydroisoqinolinyl, quinolizinyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyranyl, tetrahydropyranyl, 1,2,3- triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, chromenyl, morpholinyl, diazepinyl, benzodiazepinyl, and the like.

[0039] “Heteroarylalkyl” denotes the combination of the alkyl, alkenyl, alkynyl, and heteroaryl groups defined hereinbefore, in each case in their broadest sense. The alkyl group as substituent is directly linked to the molecule and is in turn substituted by a heteroaryl group. The linking of the alkyl and heteroaryl may be achieved on the alkyl side via any carbon atoms suitable for this purpose and on the heteroaryl side by any carbon or nitrogen atoms suitable for this purpose.

[0040] By the term “heterocycloalkyl” are meant groups which are derived from cycloalkyl as hereinbefore defined if in the hydrocarbon rings one or more of the groups -CH2- are replaced independently of one another by the groups -O-, -S- or -NH- or one or more of the groups =CH- are replaced by the group =N-, while not more than five heteroatoms may be present in total, there must be at least one carbon atom between two oxygen atoms and between two sulphur atoms or between one oxygen and one sulphur atom and the group as a whole must be chemically stable. Heteroatoms may simultaneously be present in all the possible oxidation stages (sulphur -> sulphoxide -SO-, sulphone -SO2-; nitrogen -> N-oxide). It is immediately apparent from the indirect definition / derivation from cycloalkyl that heterocycloalkyl is made up of the sub-groups monocyclic hetero-rings, bicyclic hetero-rings and spirohetero-rings, while each sub-group can also be further subdivided into saturated and unsaturated (heterocycloalkenyl). The term unsaturated means that in the ring system in question there is at least one double bond, but no aromatic system is formed. In bicyclic hetero-rings two rings are linked such that they have at least two atoms in common. In spirohetero-rings one carbon atom (spiroatom) is shared by two rings. If a heterocycloalkyl is substituted, the substitution may be mono- or polysubstitution in each case, at all the hydrogen-carrying carbon and / or nitrogen atoms, independently of one another. Heterocycloalkyl itself as substituent may be linked to the molecule via any suitable position of the ring system.

[0041] The term “heterocyclic group” as used herein refers to a heterocycloalkyl group which optionally may be fused to an aromatic aryl or heteroaryl group.

[0042] Typical examples of individual sub-groups are listed below:

[0043] Monocyclic heterorings (saturated and unsaturated): oxolane, pyrrolidinyl, pyrrolinyl, imidazolidinyl, thiazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, oxiranyl, aziridinyl, azetidinyl, 1,4-dioxanyl, azepanyl, diazepanyl, morpholinyl, thiomorpholinyl, homomorpholinyl, homopiperidinyl, homopiperazinyl, homothiomorpholinyl, thiomorpholinyl-S-oxide, thiomorpholinyl-S,S- dioxide, 1,3-dioxolanyl, oxane, tetrahydrothiopyranyl, 1,4-oxazepanyl, tetrahydrothienyl, homothiomorpholinyl-S,S-dioxide, oxazolidinonyl, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazinyl, dihydropyridyl, dihydro-pyrimidinyl, dihydrofuryl, dihydropyranyl, tetrahydrothienyl-S-oxide, tetrahydrothienyl-S,S-dioxide, homothiomorpholinyl-S-oxide, 2,3-dihydroazet, 2H-pyrrolyl, 4H-pyranyl, 1,4- dihydropyridinyl, etc;

[0044] Bicyclic heterorings (saturated and unsaturated): 8-azabicyclo[3.2.1]octyl,

[0045] 8-azabicyclo[5.1.0]octyl, 2-oxa-5-azabicyclo[2.2.1]heptyl, 8-oxa- 3-aza- bicyclo[3.2.1]octyl, 3,8-diaza-bicyclo[3.2.1]octyl, 2,5-diaza-bicyclo-[2.2.1]heptyl, 1-aza- bicyclo[2.2.2]octyl, 3,8-diaza-bicyclo[3.2.1]octyl, 3,9-diaza-bicyclo[4.2.1]nonyl, 2,6- diaza-bicyclo[3.2.2]nonyl, hexahydro-furo[3,2-b]furyl, etc;

[0046] Spiro-heterorings (saturated and unsaturated): 1,4-dioxa-spiro[4.5]decyl; 1- oxa-3,8-diaza-spiro[4.5]decyl; 2,6-diaza-spiro[3.3]heptyl; 2,7-diaza-spiro[4.4]nonyl; 2,6- diaza-spiro[3.4]octyl; 3,9-diaza-spiro[5.5]undecyl; 2,8-diaza- spiro[4.5]decyl, etc.

[0047] “Heterocycloalkylalkyl” denotes the combination of the alkyl, alkenyl, alkynyl, and heterocycloalkyl groups defined hereinbefore, in each case in their broadest sense. The alkyl group as substituent is directly linked to the molecule and is in turn substituted by a heterocycloalkyl group. The linking of the alkyl and heterocycloalkyl may be achieved on the alkyl side via any carbon atoms suitable for this purpose and on the heterocycloalkyl side by any carbon or nitrogen atoms suitable for this purpose.

[0048] By the term "suitable substituent" is meant a substituent that on the one hand is fitting on account of its valency and on the other hand leads to a system with chemical stability.

[0049] It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds thatare chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.

[0050] A further embodiment relates to the synthetic protein as described herein, wherein said synthetic protein comprises 1, 2, 3, 4, or 5 diene moieties of general formula (I). The synthetic protein may comprise identical or different diene moieties of general formula (I).

[0051] According to one embodiment of the invention, the carrier protein comprise a diene moiety of general formula (I), wherein R2denotes a residue of general formulawherein X denotes NH or O, and optionally substituted by halogen, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa; and R3is an amino acid residue which is comprised in the carrier protein.

[0052] A further embodiment relates to the synthetic protein as described herein, wherein said synthetic protein comprises at least one T-cell activating epitope.

[0053] The term “T-cell activating epitope” refers to a structural unit of molecular structure which is capable of inducing T-cell immunity. The function of carrier proteins which include T-cell activating epitopes is well known and documented for conjugates. Without wishing to be bound by theory, a T-cell activating epitope in the carrier protein enables the covalently-attached antigen to be processed by antigen-presenting cells and presented to CD4+veT cells to induce immunological memory against the antigen.

[0054] As used herein, “carrier protein” refers to a non-toxic or detoxified polypeptide containing a T-cell activating epitope which is able to be attached to an antigen (e.g., a polysaccharide) to enhance the humoral response to the conjugated antigen in a subject. In some embodiments, the carrier protein is Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D (PD, HiD), outer membrane protein complex of serogroup B meningococcus (OMPC), CRM197, malaria ookinete specific surface protein Pfs25, bovine serum albumin (BSA), keyholelimpet hemocyanin, or ovalbumin. An “enhanced carrier protein” has at least one non- natural amino acid replaced for a naturally occurring amino acid in the carrier protein. The terms “carrier protein” and “carrier polypeptide” are used interchangeably herein.

[0055] One exemplary carrier protein upon which to base the carriers of the present invention is CRM197. CRM197 is well-known in the art (e.g., see Broker et al.2011 Biologicals 39:195-204).

[0056] According to one embodiment, the synthetic peptide according to the invention comprises a single synthetic amino acid or multiple synthetic amino acids which bear a tetrazine moiety. The synthetic amino acid is introduced into a natural occurring protein or peptide or into a recombinant protein or peptide at predefined sites. Having amino acids bearing a tetrazine moiety at predefined sites provides the ability to produce a precisely defined synthetic peptide or protein conjugate. Having amino acids bearing a tetrazine moiety avoids problems of undefined, random labelling or incomplete labelling (if a reaction does not go to completion, heterogeneous products can result which can be a problem which is usefully addressed by synthetic amino acids bearing a tetrazine moiety).

[0057] The production of correctly folded, disulfide bonded proteins in high yields remains a technological challenge, especially for the commonly used expression system E. coli. The formation of disulfide bonds in proteins is impaired in E. coli cytoplasm, due to the inherently reducing environment. Thus, recombinant proteins that contain or require disulfide bonds commonly misfold and form inactive inclusion bodies, and the consequent and often tedious denaturation-refolding procedures required to restore the correct folding and obtain an active product limit the application of this expression route.

[0058] Recombinant E. coli cells may be genetically engineered to shift the redox state of the cytoplasm to a more oxidative state such as, for example, by mutation of one or more disulfide reductase genes such as, for example, an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, a glutamate cysteine lyase, a disulfide reductase, a protein reductase, and / or a glutathione reductase. The disulfide reductase genes may be mutated and rendered non-functional or marginally functional such that the redox state of the cytoplasm of the cell is shifted to a more oxidative state as compared to wild type without compromising viability. Oxidative protein foldinginvolves the formation and isomerization of disulfide bridges and plays a key role in the stability and solubility of many proteins and peptides.

[0059] One embodiment of the invention relates to a synthetic protein or peptide comprising at least one sulfide linkage. Thus, the synthetic protein or peptide exhibits at least one disulfide bond.

[0060] Some embodiments of the invention relate to methods of producing a synthetic peptide or protein comprising a single or multiple tetrazine moieties, said methods comprising genetically incorporating a synthetic amino acid comprising a tetrazine moiety into a peptide or protein. Genetically incorporating the tetrazine moiety allows precise construction of a defined peptide or protein conjugate. The location of the tetrazine moieties can be precisely controlled. This advantageously avoids the need to subject the whole peptide or protein to complex reaction steps using chemical functional groups occurring in the natural amino acids.

[0061] As used herein, the term “click reaction” refers to a chemical reaction characterized by a large thermodynamic driving force that usually results in irreversible covalent bond formation. Click reactions can often be conducted under aqueous conditions (e.g., physiological conditions) without producing cytotoxic byproducts. Without intending to be limiting, examples of click reactions include [3+2] cycloadditions, such as the Huisgen 1,3-dipolar cycloaddition reaction of an azide and an alkyne; thiol-ene reactions, such as the Michael addition of a thiol to a maleimide or other unsaturated acceptor; [4+1] cycloaddition reactions between an isonitrile and a tetrazine; the Staudinger ligation between an azide and an ester-functionalized phosphine or an alkanethiol-functionalized phosphine; Diels-Alder reactions (e.g., between a furan and a maleimide); and inverse electron demand Diels-Alder reactions (e.g., between a tetrazine and a dienophile such as a strained trans cyclooctene).

[0062] Suitably the method described for producing the peptide or protein comprises (i) providing a nucleic acid encoding the peptide or protein which nucleic acid comprises an orthogonal codon encoding the amino acids having a tetrazine moiety; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase / tRNA pair capable of recognizing said orthogonal codon and incorporating said amino acid having a tetrazine moiety into the peptide or protein chain. Suitably said orthogonal codon comprises an amber codon (TAG), said tRNA comprisestRNAcuA and said tRNA synthetase comprises PylRS from the organisms Methanosarcina mazei / Methanosarcina Bakeri / Methanomethylophilus alvus.

[0063] In one embodiment, the disclosure provides a carrier protein comprising a synthetic amino acid residue. In another embodiment, the carrier protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 synthetic amino acid residues.

[0064] Where a carrier includes more than one synthetic amino acid residue, it is preferred to include only a single species of synthetic amino acid. This permits the same conjugation chemistry to be used simultaneously at each synthetic amino acid. If it is desired to attach two different antigens to a single earner molecule, this can be achieved by using different synthetic amino acid species within a single carrier and conjugating each antigen to a different synthetic amino acid, but conjugation to a single species of synthetic amino acid in a carrier is preferred. Moreover, where a composition includes multiple different conjugates (e.g., different pneumococcal serotypes) it is preferred that each conjugate includes the same single species of synthetic amino acid. Furthermore, where a composition includes multiple different conjugates (e.g., different pneumococcal serotypes) it is preferred that each conjugate includes the same carrier protein.

[0065] The synthetic amino acid residue optionally comprises any of the non-natural amino acids described in this application, or others that have been identified as compatible with cell-based or cell-free protein synthesis.

[0066] Examples of non-natural amino acids that can be used in the methods of the embodiments include: a non-natural analog of a tyrosine amino acid; a non-natural analog of a glutamine amino acid; a non-natural analog of a phenylalanine amino acid; a non-natural analog of a serine amino acid, a non-natural analog of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxyl amine, keto, or amino substituted amino acid, or any combination thereof, an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal- containing amino acid, a radioactive amino acid; a photocaged and / orphotoisomerizable amino acid; a biotin or biotin-analog containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,a disubstituted amino acid, a β-amino acid; a cyclic amino acid other than proline, etc.

[0067] Particularly preferred synthetic amino acid for use with the invention are those which can be incorporated during translation (in a cellular or a cell-free system) and which provide a functional group which is not found i n any of the 20 naturally occurring amino acids. Various techniques for incorporating such amino acids into polypeptides are known (e.g., WO2019016354A1).

[0068] In one aspect, the polypeptide comprising at least one synthetic amino acid residue is a modified version of a native carrier protein (e.g., eCRM), or a polypeptide comprising one or a plurality of T-cell activating epitopes of a native carrier protein. Carrier proteins suitable for such modification include, but are not limited to, proteins used in conjugate vaccines such as Corynebacterium diphtheriae toxin, Clostridium teiani tetanospasmin, Hemophilus influenzae protein D (PD, HiD), outer membrane protein complex of serogroup B meningococcus (OMPC), or CRM 197. Examples

[0069] The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art. Materials and methods Production and detection of CRM197-synAA

[0070] CRM197 containing synthetic amino acids was produced in an engineered E. coli BL21(DE3) strain carrying a deletion in the gorA gene. The strain harbors the machinery to incorporate synthetic amino acids (synAA) into a protein of choice. Various CRM197-synAA variants were designed and expressed. Each variant containseither a single or multiple substitutions where natural amino acids are replaced by synthetic amino acid(s). The substitutions are non-exhaustive and were chosen to not disturb the natural physicochemical properties of CRM197.

[0071] All CRM197-synAA variants were produced and their expression levels were analyzed via SDS-PAGE (Fig.1, upper pictures). To demonstrate the correct incorporation of synAA into the protein, CRM197-synAA variants were conjugated by click chemistry to a fluorophore. The fluorescent protein bands corresponding to CRM197-synAA clicked to the linker were visualized under blue light (Fig.1, middle pictures). Furthermore, western blotting of all CRM197-synAA variants was performed (Fig.1, lower pictures). In order to characterize the correct folding of various CRM197- synAA variants, a binding assay to Heparin-Binding EGF-Like Growth Factor (HB- EGF) was performed. CRM197-synAA variants binding to HB-EGF was measured by an ELISA-like assay. Binding to HB-EGF indicates correct folding and functionality of the protein (Fig.2). All the assays were performed including as a control unmodified CRM197, which contains only natural amino acids.

[0072] The engineered E. coli BL21(DE3) strain carrying a deletion on the gorA gene was transformed with appropriate plasmids containing the coding sequences of all CRM-synAA variants. Single colonies were inoculated in 1mL MDG media supplemented with 50 µg / mL of kanamycin (Protein production by auto-induction in high-density shaking cultures. F. William Studier. Protein Expression and Purification. Volume 41, Issue 1, May 2005, Pages 207-234.) and grown overnight shaking at 37°C. These precultures were diluted 1:50 into 1mL of fresh MDG media supplemented with 0.5% yeast extract and 50 µg / mL of kanamycin. Cultures were grown at 37°C until they reached OD600 = 0.6-0.8. Expression of CRM197 was induced by adding 0.5 mM IPTG and 0.25 – 5 mM synAA (final concentrations) to the media. Cultures were grown at 22-25°C and shaking overnight. Cells were harvested by centrifugation at 4°C. Cells were lysed using BugBuster®Master Mix (Millipore) following manufacturer’s instructions. Lysates were centrifuged 15 min at 4°C to separate the soluble and insoluble fractions. Insoluble fractions were discarded. Soluble fractions were mixed with 32µM click-reactive BDP FL-PEG4 (BroadPharm) and incubated at room temperature in the dark for 15 min – 48 hrs to allow click reaction to fully complete. Samples were mixed with Laemmli Buffer 2x (Sigma-Aldrich), processed following manufacturer’s instructions, loaded and run on a precast SDS-PAGE protein gel 4–20% Mini-PROTEAN® TGX™ (Bio-Rad). The gel was disassembled and visualized under blue light prior to protein staining. The gel was then stained using a Coomassie blue solution and visualized or used for western blotting. Western blot detection of CRM197 was performed with an antibody anti-CRM197 (0.5 mg / mL in PBST, AIC LLC, Rockville, MD). HB-EFG binding assay

[0073] Binding assay was performed as described elsewhere (Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems. John M. Hickey, et al. Pharmaceutical Biotechnology. Volume 107, Issue 7, July 2018, Pages 1806-1819.). Briefly, 96-well ELISA high binding plates (Millipore) were coated with 0.5 µg / mL HB-EGF (Sigma- Aldrich) in 1xPBS. After blocking the plate with 1%PBS 5% fat-free milk (Carl Roth), the plate was washed with PBST (0.05% Tween 20, Carl Roth). Lysates from cultures expressing different CRM197-synAA variants were processed as described in the previous section. After discarding the insoluble fraction, the soluble fraction was used for the assay. Since every CRM197-synAA variant may be expressed at different yields, one subfraction was used to check CRM197-synAA expression levels in an SDS-PAGE and the other subfraction was used for the binding assay. Protein band intensity analysis (ImageJ) was applied to normalize the amount of soluble fraction that should be loaded in the binding assay. The soluble subfraction of each CRM197- synAA variant and a positive control comprising the soluble fraction of a culture producing natural CRM197 were loaded accordingly. After sample incubation, primary antibody anti-CRM197 (0.5 mg / mL in PBST, AIC LLC, Rockville, MD) was used. As a secondary antibody, goat anti-rabbit IgG HRP-labelled (Fischer Scientific) was used. TMB substrate Kit (Thermo Fisher) was used and the absorbance of each well was measured at 450nm using a fluorescence plate reader. Synthesis of protein-polysaccharide conjugates

[0074] As a model polysaccharide, dextran, a 1,6-glucose polymer was used. Amino dextran (MW 2000kDa) modified with about 300 amines (Product AD2000x300, Fina Biosolutions), was derivatized with trans-cyclooctene (TCO) using TCO-PEG3-NHS (BroadPharm). TCO-dextran was combined with unmodified CRM, CRM containing 1synthetic amino acid (CRM-1synAA) or three synthetic amino acids (CRM-3synAA) at a 1:1 weight ratio in PBS, with a final concentration of 2.2 mg / ml dextran and CRM. After 1 h of reaction at room temperature it was evident that the CRM-synAA solutions were more viscous than the control. The CRM-3synAA solution was more viscous than the CRM-1synAA solution, as would be expected as the former has multiple reactive sites and can crosslink polysaccharide chains. The conjugates were then analyzed by HPLC size exclusion chromatography using a Sepax SEC-200 column using detection at 280nm. On this column, the 2,000 kDa dextran elutes first, at the void volume of the column (about 6 min) whereas the much lower MW CRM elutes later, (about 10 min). Controls of TCO-dextran and the individual proteins were also run. The TCO-dextran had minimal absorbance at 280nm. Therefore, the shift in UV absorbance from the peak at about10 min to the peak at about 6 is an indication of conjugation of the protein to the polysaccharide. This demonstrates that site-specifically modified carrier protein containing synAA can be rapidly and efficiently conjugated to polysaccharides. Cytosolic soluble production of scFv-fragment containing synthetic amino acids:

[0075] Plasmids carrying a kanamycin resistance gene, a p15a origin of replication and under the control of a T5 promoter coding for a cytosolic expression construct of a fluorescein-binding scFv-fragment with one or more stop-codons in frame were transfected into E. coli BL21(DE3) carrying a deletion on the gorA gene containing the tRNA and tRNA-synthetase machinery for synthetic amino acid incorporation. Single colonies were picked of all the constructs. Therewith, 1 mL precultures of every construct were set up in DeLisa + VIT + 0.8% glycerol + Kanamycin in the morning. On the next day, the DeLisa cultures were diluted 1:10 and OD normalized in the morning. For every construct and condition 2 wells were prepared. Afterwards, the cultures were grown for about 6 h and at an OD of 4-5 induced with 1 mM IPTG. On the next day the cell suspensions were spinned down at 6,500 g for 4 min at 4°C. The harvested cells were lysed using the BugBuster Master Mix (50 µL for each sample). The samples were resuspended and incubated for 25 min at RT while shaking. The cell debris and inclusion body fraction were spinned down at full speed for 15 min. The supernatant was taken off and transferred to a fresh tube for soluble fraction analysis.25 µL of the supernatant were clicked with 1 µL TCO-Sulfo-Cy3 for 10 min in the dark (RT). The IBfractions were washed once with 500 µL 1x PBS and subsequently solubilized in 50 µL 100 mM Tris buffer containing 2 % SDS.

[0076] Subsequently, all the samples were cooked in Laemmli buffer and applied to an SDS-PAGE (see Fig.5). Cytosolic soluble production of Fab-fragment containing synthetic amino acids:

[0077] Plasmids carrying a kanamycin resistance gene, a p15a origin of replication and under the control of a T5 promoter coding for a cytosolic expression construct of a HER2 binding Fab-fragment of Trastuzumab with one or more stop-codons in frame were transfected into E. coli BL21(DE3) carrying a deletion on the gorA gene containing the tRNA and tRNA-synthetase machinery for synthetic amino acid incorporation. Single colonies were picked of all the constructs. Therewith, 1 mL precultures of every construct were set up in DeLisa + VIT + 0.8% glycerol + Kanamycin in the morning. On the next day, the DeLisa cultures were diluted 1:10 and OD normalized in the morning. For every construct and condition 2 wells were prepared. Afterwards, the cultures were grown for about 6 h and at an OD of 4-5 induced with 1 mM IPTG. On the next day the cell suspensions were spinned down at 6500 g for 4 min at 4°C. The harvested cells were lysed using the BugBuster Master Mix (50 µL for each sample). The samples were resuspended and incubated for 25 min at RT while shaking. The cell debris and inclusion body fraction were spinned down at full speed for 15 min. The supernatant was taken off and transferred to a fresh tube for soluble fraction analysis.25 µL of the supernatant were clicked with 1 µL TCO-Sulfo- Cy3 for 10 min in the dark (RT). The IB fractions were washed once with 500 µL 1x PBS and subsequently solubilized in 50 µL 100 mM Tris buffer containing 2 % SDS.

[0078] Subsequently, all the samples were cooked in Laemmli buffer and applied to an SDS-PAGE (see Fig.6).

[0079] CRM197 containing synthetic amino acids was reacted with a TCO-derivatized dextran molecule 30 kDa in PBS buffer. The reacted samples were analyzed via SEC- HPLC. Chromatograms of samples and controls are shown in Fig.3 and Fig.4.

Claims

Claims 1) A synthetic protein which comprises at least one sulfide linkage and at least one synthetic amino acid bearing a diene moiety of general formula (I),wherein R1is selected from the group consisting of, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa, −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa; R2is denotes a group, optionally substituted by one or more, identical or different Rband / or Rc, selected from among C1-6alkyl, C3-10cycloalkyl, C6-10aryl, C7-16aryl- alkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; Rais hydrogen or C1-6alkyl; each Rbis a suitable substituent and is selected in each case independently of one another from among =O, –ORc, C1-3haloalkyloxy, –OCF3, =S, -SRc, =NRc, =NORc, =NNRcRc, =NN(Rg)C(O)NRcRc, –NRcRc, –ONRcRc, –N(ORc)Rc, –N(Rg)NRcRc, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Rc, –S(O)ORc, –S(O)2Rc, –S(O)2ORc, –S(O)NRcRc, –S(O)2NRcRc, –OS(O)Rc, –OS(O)2Rc, –OS(O)2ORc, –OS(O)NRcRc, –OS(O)2NRcRc, –C(O)Rc, –C(O)ORc, –C(O)SRc, –C(O)NRcRc, –C(O)N(Rg)NRcRc, –C(O)N(Rg)ORc, –C(O)N(Rg)S(O)2Rc, –C(NRg)NRcRc, –C(NOH)Rc, –C(NOH)NRcRc, –OC(O)Rc, –OC(O)ORc, –OC(O)SRc, –OC(O)NRcRc, –OC(NRg)NRcRc, –SC(O)Rc, –SC(O)ORc, –SC(O)NRcRc, –SC(NRg)NRcRc, –N(Rg)C(O)Rc, –N[C(O)Rc]2, –N(ORg)C(O)Rc, –N(Rg)C(NRg)Rc, –N(Rg)N(Rg)C(O)Rc, –N[C(O)Rc]NRcRc, –N(Rg)C(S)Rc, –N(Rg)S(O)Rc, –N(Rg)S(O)ORc, –N(Rg)S(O)2Rc, –N[S(O)2Rc]2, –N(Rg)S(O)2ORc, –N(Rg)S(O)2NRcRc, –N(Rg)[S(O)2]2Rc, –N(Rg)C(O)ORc, –N(Rg)C(O)SRc, –N(Rg)C(O)NRcRc, –N(Rg)C(O)NRgNRcRc, –N(Rg)N(Rg)C(O)NRcRc, –N(Rg)C(S)NRcRc, –[N(Rg)C(O)]2Rc, –N(Rg)[C(O)]2Rc, –N{[C(O)]2Rc}2, –N(Rg)[C(O)]2ORc, –N(Rg)[C(O)]2NRcRc, –N{[C(O)]2ORc}2, –N{[C(O)]2NRcRc}2, –[N(Rg)C(O)]2ORc, –N(Rg)C(NRg)ORc, –N(Rg)C(NOH)Rc, –N(Rg)C(NRg)SRcand–N(Rg)C(NRg)NRcRc; each Rcindependently of one another denotes hydrogen or a group, optionally substituted by one or more, identical or different Rdand / or Re, selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C4-16cycloalkylalkyl, C6-10aryl, C7-16arylalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl and 6-18 membered heteroarylalkyl; each Rddenotes a suitable substituent and is selected in each case independently of one another from among =O, –ORe, C1-3haloalkyloxy,–OCF3, =S, –SRe, =NRe, =NORe, =NNReRe, =NN(Rg)C(O)NReRe, –NReRe, –ONReRe, –N(ORe)Re, –N(Rg)NReRe, halogen, –CF3, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)Re, –S(O)ORe, –S(O)2Re, –S(O)2ORe, –S(O)NReRe, –S(O)2NReRe, –OS(O)Re, –OS(O)2Re, –OS(O)2ORe, –OS(O)NReRe, –OS(O)2NReRe, –C(O)Re, –C(O)ORe, –C(O)SRe, –C(O)NReRe, –C(O)N(Rg)NReRe, –C(O)N(Rg)ORe, –C(NRg)NReRe, –C(NOH)Re, –C(NOH)NReRe, –OC(O)Re, –OC(O)ORe, –OC(O)SRe, –OC(O)NReRe, –OC(NRg)NReRe, –SC(O)Re, –SC(O)ORe, –SC(O)NReRe, –SC(NRg)NReRe, –N(Rg)C(O)Re, –N[C(O)Re]2, –N(ORg)C(O)Re, –N(Rg)C(NRg)Re, –N(Rg)N(Rg)C(O)Re, –N[C(O)Re]NReRe, –N(Rg)C(S)Re, –N(Rg)S(O)Re, –N(Rg)S(O)ORe, –N(Rg)S(O)2Re, –N[S(O)2Re]2, –N(Rg)S(O)2ORe, –N(Rg)S(O)2NReRe, –N(Rg)[S(O)2]2Re, –N(Rg)C(O)ORe, –N(Rg)C(O)SRe, –N(Rg)C(O)NReRe, –N(Rg)C(O)NRgNReRe, –N(Rg)N(Rg)C(O)NReRe, –N(Rg)C(S)NReRe, –[N(Rg)C(O)]2Re, –N(Rg)[C(O)]2Re, –N{[C(O)]2Re}2, –N(Rg)[C(O)]2ORe, –N(Rg)[C(O)]2NReRe, –N{[C(O)]2ORe}2, –N{[C(O)]2NReRe}2, –[N(Rg)C(O)]2ORe, –N(Rg)C(NRg)ORe, –N(Rg)C(NOH)Re, –N(Rg)C(NRg)SReand –N(Rg)C(NRg)NReRe; and each Reindependently of one another denotes hydrogen or a group selected from among C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, and 5-12 membered heteroaryl; and each Rgindependently of one another denotes hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C6-10aryl, 3-8 membered heterocycloalkyl, or 5-12 membered heteroaryl; and wherein the compound of formula (I) is linked to an amino acid residue which connects to the next residues towards the N- and C- terminus of the carrier protein.2) The synthetic protein of claim 1, wherein said protein comprises 1, 2, 3, 4, or 5 synthetic amino acid(s) bearing a moiety of general formula (I). 3) The synthetic protein of claim 1 or 2, wherein R2denotes a residue of general formula (II),wherein * represents where R2is bound to the diene moiety of formula (I); and ** represents where R2is bound to the protein; X denotes NH or O; R3denotes −ORa, −C(O)Ra, −COORa, −NRaRa, −SRa; n denotes 0, 1, 2, or 3; and Rais the same as defined in claim 1. 4) The synthetic protein of any one of claims 1 to 3, wherein said synthetic protein is a carrier protein. 5) The synthetic protein of claim 4, wherein said carrier protein comprises at least one T-cell activating epitope. 6) The synthetic protein of claim 5, wherein the at least one T-cell activating epitope is selected from the group consisting of Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D, outer membrane protein complex of serogroup B meningococcus, CRM197, malaria ookinete specific surface protein Pfs25, bovine serum albumin (BSA), keyhole limpet hemocyanin, and ovalbumin. 7) A method for producing a synthetic protein of any one of claims 1 to 6, comprising: a) providing a recombinant bacterial cell containing an expression vector that encodes the synthetic protein sequence, and wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes; b) expression of the synthetic protein in a soluble form from said vector in said bacterial cell; and c) recovering the synthetic protein.8) The method according to claim 7, wherein the one or more disulfide reductase enzymes is one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase or a glutathione reductase. 9) The method of claim 8, wherein the recombinant bacterial cell is an E. coli cell or a derivative strain of E. coli. 10) Use of the synthetic protein of any one of claims 1 to 6 for the production of a pharmaceutical composition. 11) The use according to claim 10, wherein the pharmaceutical composition is a vaccine.

Images