WO2013065009A1 - A sortase-click reaction suite for synthesis of multivalent dendrimeric protein assembly - Google Patents

A sortase-click reaction suite for synthesis of multivalent dendrimeric protein assembly Download PDF

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WO2013065009A1
WO2013065009A1 PCT/IB2012/056083 IB2012056083W WO2013065009A1 WO 2013065009 A1 WO2013065009 A1 WO 2013065009A1 IB 2012056083 W IB2012056083 W IB 2012056083W WO 2013065009 A1 WO2013065009 A1 WO 2013065009A1
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protein
multivalent
pspa
sortase
alkyne
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PCT/IB2012/056083
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French (fr)
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Kanchan Gupta
Shikha SINGH
Naeem KHAN
Devinder SEHGAL
Rajendra Prasad Roy
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National Institute Of Immunology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

  • the present invention lies in the broad field of biotechnology and more specifically relates to chemo-enzymatic synthesis of bio-conjugates, multivalent dendrimeric assembly and multivalent vaccines.
  • a bio-conjugate is generally the product of a bio-conjugation or coupling of two bio- molecules.
  • Bio-conjugates have wide ranging applications; they are useful as therapeutic or diagnostic agents.
  • Bio-conjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule.
  • Bio-conjugation is utilized extensively in bio-chemical, immunochemical and molecular biological research. Major applications of bio-conjugation include; detection of gene probes, enzyme-linked immuno solid-phase assay, and monoclonal antibody drug targeting and medical imaging. Bioconjugates are generally classified as either direct or indirect conjugates. Direct conjugates encompass those in which two or more components are joined by direct covalent chemical linkages. Alternatively, indirect conjugates encompass those in which two or more components are joined via an intermediary complex involving a biological molecule.
  • dendrimers are type of bio-conjugates that represent a new class of highly branched polymers whose interior cavities and multiple peripheral groups facilitate potential applications in biomedicine and bio-organic chemistry.
  • Dendrimers possess many advantages including well-defined structure, mono-dispersity, multi-valency and ease of surface functionalization, which make them useful scaffolds for protein mimics.
  • Major advances have been made in the synthesis and study of new carbohydrate, nucleic acid, and peptide dendrimers, as well as their use as magnetic resonance imaging contrast agents, as agents for cellular delivery of nucleic acids, and as scaffolds for bio -mimetic systems.
  • Multivalent display of peptides and proteins on a dendrimeric scaffold can be very useful in a variety of biological settings ranging from delineation of basic mechanisms to applications in biotechnology and medicine like vaccines etc.
  • multiple antigen peptide exploited the availability of two amino groups (a and ⁇ ) in a lysyl residue to generate branch points for iterative elaboration of two, four or eight copies of short antigenic peptide sequences.
  • These multiple antigen peptides contain a lysine dendrimer scaffold holding multiple copies of an antigenic peptide, which enhances its immunogenicity.
  • Dendrimers have conventionally been synthesized following the sequential assembly or iterative approach, but current advances have yielded chemo-selective synthetic approaches that circumvent the need for sequential assembly of the final peptide dendrimeric architecture.
  • Orthogonal reactions have been well utilized in the synthesis of dendrimers. Orthogonality is a well-recognized concept, relating things that are functionally independent of each other. In chemistry, for example, orthogonal protecting groups can be added and removed independently of each other, and therefore are commonly used to control the specificity of reactions in the synthesis of complex macro molecules, including polypeptides and polynucleotides.
  • orthogonal system Several years ago, Baranay and Merrifield defined an orthogonal system as "a set of completely independent classes of protection groups, such that each class can be removed in any order and in the presence of all other classes.”
  • NCL native chemical ligation
  • CuAAC copper-assisted azide-alkyne cycloaddtion
  • oxyamine-ketone ligation have facilitated the synthesis of interesting peptide dendrimers built from monomeric units composed of 50 or less residues.
  • Native Chemical Ligation NCL that usually utilises two peptide components: a peptide C- terminal thio-ester and an N-terminal cysteine containing component, has facilitated the production of proteins.
  • NCL tetravalent green fluorescent protein
  • Meijer's group tetravalent green fluorescent protein
  • Sortases belong to transpeptidase class of prokaryotic enzymes, are utilizable for enzymatic coupling and have an ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile. Sortases modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of (1) the sorting motif LPXTG (Leu-Pro-any-Thr-Gly), (2) a highly hydrophobic transmembrane sequence and (3) a cluster of basic residues such asparginine. Cleavage occurs between the Thr and Gly residues within this pentapeptide sorting motif.
  • the recognition signal consists of (1) the sorting motif LPXTG (Leu-Pro-any-Thr-Gly), (2) a highly hydrophobic transmembrane sequence and (3) a cluster of basic residues such asparginine. Cleavage occurs between the Thr and Gly residue
  • Sortase A is a transpeptidase that attaches surface proteins to the cell wall; cleaves between the Thr and Gly of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl- group of threonine and the amino-group of the cell-wall peptidoglycan as illustrated below: LPXTG-— + GGGGG > LPXTGGGGG + G
  • Transpeptidase sortase A of Staphylococcus aureus has turned out to be a wonderful synthetic catalyst for peptide ligation.
  • the CuAAC in situ click chemistry is Copper(I)-catalyzed ligation and uses the cycloaddition of azides with alkynes as the 'click' mechanism that locks two halves of the desired structure together.
  • two molecules with weak affinity to different structural features of the target enzyme can be linked to produce a high-affinity bivalent inhibitor.
  • Click chemistry based of modular peptide ligation although conceptually extendable to large proteins, in practicality have been applied to the assembly of chemically- defined protein dendrimers only in a limited number of cases. The limited examples of well- defined multivalent protein dendrimers despite their enormous utility is a testimony to the unwieldiness of the current methods.
  • the present application aims to overcome the aforesaid deficiencies of the disclosure in the prior art and provide novel, widely applicable method of synthesis of protein bio-conjugates; well defined, homogeneous protein dendrimer assembly by combination of Sortase and click chemistry.
  • the high yielding Sortase - Click reaction suite of the present invention is applicable to a wide variety of proteins and is utilizable in production of highly effective multivalent vaccines.
  • the bio -orthogonal Sortase-Click reaction suite of the present invention is a simple and generic chemo-enzymatic reaction suite, developed for the assembly of bio-conjugates and well defined multivalent protein dendrimer assembly.
  • the method is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and is easily applicable to more common C-terminus hexahistidine (His 6 )-tagged proteins.
  • His 6 C-terminus hexahistidine
  • lysine dendritic wedges with orthogonal handles are amenable to easy synthesis by standard solid phase peptide chemistry. Both reactions, Sortase labeling as well as CuAAC, produce little or no side products.
  • Sortase-Click approach provides enormous synthetic flexibility for incorporation of diverse proteins in the dendrimer.
  • SrtA-mediated labelling reaction is known to tolerate a wide range of chemically disparate molecules and therefore can generate a variety of orthogonal handles on proteins for conjugation to compatible dendritic scaffold.
  • Sortase-Click strategy thus facilitates the synthesis of a variety of bio-conjugates and protein dendrimers for unprecedented applications in biology and medicine including construction of chemically defined multi-antigenic semisynthetic vaccines.
  • the object of the present invention is to provide a simple, generic, one pot chemo-enzymatic synthetic strategy for synthesis of multivalent protein dendrimer assembly.
  • Another object of the present invention is to provide a novel chemo-enzymatic reaction suite based on sortase-click chemistry, for the assembly of homogenous, well-defined dendrimers from readily accessible His 6 -tagged proteins.
  • Another object of the present invention is to elucidate the mechanism behind sortase- mediated ligation of LPXTG proteins to a glycine-terminated multivalent dendritic scaffold. Yet another object of the present invention is the synthesis of bio-conjugates.
  • Yet another object of the present invention is to provide chemically defined multi-antigenic semisynthetic vaccines based on protein dendrimers.
  • FIGURES AND DRAWINGS Figure 1: Sortase A-catalyzed transpeptidation reaction.
  • Figure 2 Structures of azide/alkyne derivatized lysine dendritic scaffolds and sortase substrates: Structures 1 to 6, 11 and 13 represent azide or an alkyne terminated multivalent dendritic scaffold; Structures 7, 8, 9 and 10 represent orthogonal label (Sortase substrates)
  • Figure 3 SrtA-mediated assembly of GFP dendrimers using Gly-terminated dendritic scaffold 1 and 2, (A) GFP-LPETG-His 6 was incubated with increasing concentrations of divalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37°C for 6 h.
  • FIG. 4 SrtA-mediated assembly of PspA dendrimers using Gly-terminated dendritic scaffold,
  • PspA 203 286 -LPNTG-His 6 (0.5 mM) was incubated with increasing concentrations of divalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37°C for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 1 ;
  • Figure 5 Sortase-Click reaction. His 6 -tagged protein equipped with a LPXTG-recognition motif is labeled with azide or alkyne by SrtA. The labeled protein is purified by Ni-NTA affinity chromatography and conjugated to appropriate dendron using CuAAC reaction.
  • Figure 6 SrtA-mediated installation of alkyne / azide labels on GFP. (A) 0.5 mM of GFP- LPETG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h.
  • the reaction mixture was loaded on to a Ni-NTA agarose column and the unbound alkyne labeled protein (GFP-LPET-alkyne) was collected and analyzed by SDS-PAGE (12%). Lane 1; standard GFP-LPETG-His6, Lane 2; total reaction mixture, Lane 3; unbound Ni-NTA fraction (GFP-LPET-alkyne), Lane 4; Ni-NTA beads (unreacted protein and SrtA) and Lane 5; Pooled and concentrated stock of GFP-LPET-alkyne. Note that the gels are only qualitative.
  • Figure 7 ESI-MS characterization of Ni-NTA purified (A) GFP-LPET-alkyne (B) GFP- LPET-azide.
  • the mass difference of 245 units in each case is attributed to loss of Met and Val from the N-terminus of GFP and a possible modification due to oxidation, adding 16 units extra to the mass of the alkyne/azide labeled GFP.
  • FIG. 8 CuAAC mediated assembly of GFP dendrimers
  • A GFP-LPET-alkyne ligation to azide-terminated dendrons 3 (divalent) and 4 (tetravalent), respectively.
  • GFP-LPET-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS0 4 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%).
  • Lane 1 standard GFP-LPETG- His 6 ; Lane 2; GFP-LPET-alkyne, Lane 3; reaction of GFP-LPET-alkyne with 3 and Lane 4; reaction of GFP-LPET-alkyne with 4.
  • the lower bands in Lanes 3 and 4 are presumably respective monovalent derivatives. The bands indicated by an asterisk may represent trivalent species.
  • B GFP-LPET-azide ligation to alkyne-terminated dendrons 5 (divalent) and 6 (tetravalent), respectively. GFP-LPET-azide was reacted with 5 or 6 under CuAAC conditions as described above. Lane 1 ; GFP-LPETG-His6, Lane 2; GFP-LPET-azide, Lane 3; reaction of GFP-LPET-azide with 5 and Lane 4; reaction of GFP-LPET-azide with 6.
  • Figure 9 SrtA-mediated installation of alkyne / azide labels on PspA 203 286 -LPNTG-His 6 .
  • Alkyne labeling 0.5 mM of PspA 203 286 -LPNTG-His 6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was processed as described in Figure 6 and an aliquot was analyzed by SDS-PAGE (12%). Lane 1; standard
  • PspA " -LPNTG-His 6 Lane 2; total reaction mixture, Lane 3; unbound Ni-NTA fraction (PspA 203_286 -LPNT-alkyne), Lane 4; Ni-NTA beads (unreacted protein and SrtA) and Lane 5;
  • Lane 1 PspA " -LPNTG-His 6 ; Lane 2, total reaction mixture, Lane 3; Ni-NTA flow through (PspA 203 ⁇ 286 -LPNT-azide), Lane 4; Ni-NTA beads (unreacted
  • Figure 10 ESI-MS analysis of (A) PspA 203 ⁇ 286 -LPNT-alkyne and (B) PspA 203 ⁇ 286 -LPNT- azide.
  • Figure 11 CuAAC mediated ligation of PspA 203 286 -LPNT-alkyne to azide-terminated
  • PspA " -LPNT-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS0 4 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%).
  • Lane 1 standard PspA 203 286 -LPNTG- His 6 ; Lane 2; PspA 203"286 -LPNT-alkyne, Lane 3; reaction of PspA 203"286 -LPNT-alkyne with 3 and Lane 4; reaction of PspA 203"286 -LPNT-alkyne with 4.
  • Figure 12 Purification of PspA 203 " 286 dendrimers by size-exclusion chromatography.
  • CuAAC reaction was carried out with PspA " -LPNT-alkyne and (A) dendron 3 and (B) dendron 4. Fractions isolated from Superdex200 column from peak 1 and peak 2 were pooled and analyzed by SDS-PAGE (18%).
  • Figure 13 ESI-MS analysis of purified (A) PspA 203"286 -divalent and (B) PspA 203"286 - tetravalent.
  • Figure 14 CuAAC mediated ligation of PspA 203 286 -LPNT-azide to alkyne-terminated
  • Figure 15 SrtA-mediated installation of alkyne / azide labels on PspA 98"286 .
  • Alkyne labeling 0.5 mM of PspA 98 286 -LPNTG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was processed as described in Figure 6 and an aliquot was analyzed by SDS-PAGE.
  • Lane 1 PspA 98 286 -LPNTG-His6; Lane 2, total reaction mixture, Lane 3; Ni- NTA flow through (PspA 98_286 -LPNT-azide), Lane 4; Ni-NTA beads (unreacted protein and SrtA), and Lane 5; pooled and concentrated PspA 98 286 -LPNT-azide.
  • Figure 16 ESI-MS analysis of Ni-NTA purified (A) PspA 98"286 -alkyne and (B) PspA 98"286 - azide.
  • Figure 17 Synthesis of PspA dendrimers. Purification of PspA 98 " 286 -divalent and PspA 98 " 286 - tetravalent dendrimers respectively by size exclusion chromatography. The inset shows SDS- PAGE of the purified material.
  • Figure 18 ESI-MS analysis of purified and intact (A) PspA 98"286 -divalent and (B) PspA 98"286 - tetravalent.
  • Figure 19 Characterization of PspA 98 " 286 -divalent and PspA 98 " 286 -tetravalent dendrimers by tryptic mapping. A comparison of the tryptic maps of the dendrimers led to the identification of unique peaks labeled A and B in the case of PspA 98 286 -divalent dendrimer and C and D in the case of PspA 98 ⁇ 286 -tetravalent dendrimer, respectively.
  • Figure 20 ESI-MS analysis of peak A, B, C and D obtained from the tryptic digest of PspA- divalent and PspA-tetravalent dendrimer.
  • Figure 21 Analysis of immune response to PspA dendrimers by ELISA.
  • A End-point titer for total IgG were estimated for each group using PspA 98_286 -alkyne as the capture antigen.
  • B PspA-specific antibody titers of various IgG subtype induced after immunization with 3 doses of various preparations of PspA dendrimers.
  • Figure 22 CuAAC mediated ligation of PspA 98_286 -LPNT-azide to alkyne dendrons 5 (divalent) and 6 (tetravalent), respectively.
  • the CuAAC reaction of PspA 98_286 -LPNT-azide with 5 and 6 was carried out as described for PspA 98_286 -LPNT-alkyne.
  • Lane 1 standard PspA 98 286 -LPNTG-His 6 , Lane 2; PspA 98 ⁇ 286 -LPNT-azide, Lane 3; reaction of PspA 98"286 - LPNT-azide with 5 and Lane 4; reaction of Psp A 98 286 -LPNT-azide with 6.
  • Figure 23 CuAAC mediated ligation of PspA " -LPNT-alkyne and per-6-deoxy-6-azido- PCD.
  • a fixed amount of alkyne labelled protein was incubated with increasing concentration of purified azide-derivatized ⁇ -CD in the presence of copper sulphate and sodium ascorbate.
  • Figure 24 Purification of ⁇ -CD-PspA 203 " 286 conjugate by size-exclusion chromatography.
  • Peak 1 corresponds to multivalent ⁇ -CD-PspA " conjugate and peak 2 corresponds to
  • mice immunized with dendron 4/alum showed no detectable immune response.
  • a p value ⁇ 0.05 was considered statistically significant.
  • the present invention relates to synthesis of bio-conjugates and multivalent protein dendrimer assembly by a simple process in which proteins appended with an orthogonal label by facile sortase-mediated ligation are conjugated to a multivalent dendritic scaffold using the versatile copper-catalyzed azide-alkyne cycloaddition reaction.
  • the process mainly involves two steps, wherein protein labeled with alkyne or azide in the first step by the robust action of SrtA is linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction.
  • the unreacted His 6 -tagged protein and sortase are then allowed to bind to Ni-NTA beads and the pure labelled protein is collected in the supernatant.
  • the present invention also provides bio-conjugates generated by the sortase-mediated ligation which comprises of (a) a bio-conjugate comprising LPXTG peptide motif capable of recognition by sortase and (b) an azide or an alkyne terminated multivalent dendritic scaffold.
  • the present invention also provides chemically defined multi-antigenic semisynthetic vaccines. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention has various embodiments relating to synthesis of bio- conjugates and well-defined multivalent protein dendrimers by a general and straightforward chemo-enzymatic method.
  • An embodiment of the present invention relates to a one pot chemo-enzymatic synthetic strategy for synthesis of bio-conjugates and a multivalent protein dendrimer assembly generated by sortase-mediated ligation of LPXTG proteins to a glycine-terminated multivalent dendritic scaffold.
  • the process described in the present invention is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and dendrimers from readily accessible His 6 -tagged proteins.
  • the inventors have shown a novel two step enzymatic approach, a "Sortase- Click Reaction Suite" according to the present invention, in which proteins appended with an orthogonal label by facile sortase-mediated ligation are conjugated to a multivalent dendritic scaffold using the versatile copper-catalyzed azide-alkyne cycloaddition reaction to obtain a bioconjugates and multivalent dendrimers useful for various therapeutic and diagnostic applications.
  • the process mainly involves two steps, wherein protein labeled with alkyne or azide in the first step by the robust action of SrtA is linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction.
  • the unreacted His 6 -tagged protein and sortase are then allowed to bind to Ni-NTA beads and the pure labelled protein is collected in the supernatant.
  • the present invention for the first time presents a bio -orthogonal Sortase-Click reaction suite as depicted above, involving two extremely specific and reliable reactions that occur under aqueous conditions across a wide pH range of 4 to 9.
  • the method is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and is easily applicable to more common C-terminus His 6 -tagged proteins. Unlabelled His 6 -tagged protein and SrtA are easily removed by capture on Ni-NTA beads and pure multivalent proteins, after CuAAC conjugation, are obtained by routine size-exclusion chromatography. Besides, lysine dendritic wedges with orthogonal handles are amenable to easy synthesis by standard solution or solid phase peptide chemistry.
  • SrtA-mediated labelling reaction is known to tolerate a wide range of chemically disparate molecules and therefore can generate a variety of orthogonal handles on proteins for conjugation to compatible dendritic scaffold allowing enormous synthetic flexibility for incorporation of diverse proteins in the scaffold.
  • SrtA-mediated labelling, together with click chemistry described herein is likely to provide synergy in multivalent protein assembly.
  • copper-free click chemistry can also be adopted using cyclooctyne tagged proteins as demonstrated in linear protein- fusions by Witte et al.
  • Sortase-Click strategy developed here in combination with other orthogonal approaches should facilitate the assembly of a variety of protein dendrimers for unprecedented applications in biology and medicine including construction of chemically defined multi-antigenic semisynthetic immunogens as demonstrated with PspA in this report.
  • the one-pot process for the synthesis of multivalent dendrimeric protein assembly comprises of following steps
  • step (c) conjugating labelled protein of step (a) with multivalent dendritic scaffold by incubation in presence of CUSC , ascorbate at room temperature for 1 -2 hours;
  • Another important aspect of the present invention relates to multivalent protein dendrimer assembly comprising
  • orthogonal label is selected from the group comprising structures 7, 8, 9 and 10 and
  • the LPXTG peptide motif is a sortase recognition sequence.
  • Yet another aspect of the present invention relates to bio-conjugates that are homogeneous protein dendrimer assembly.
  • the present disclosure provides a composition comprising the multivalent dendrimeric protein assembly, useful as therapeutic or diagnostic agent.
  • the composition may be dispersed in a pharmaceutically acceptable adjuvant or carrier.
  • Yet another embodiment of the present invention relates to a multivalent vaccine, wherein the vaccine comprises the multivalent dendrimeric protein assembly as claimed in any of the aforesaid claims, wherein the vaccine is effective in preventing or treating various infectious diseases (e.g., viral infections, bacterial infections, fungal infections).
  • various infectious diseases e.g., viral infections, bacterial infections, fungal infections.
  • viruses of the Flaviviridae family such as, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, and Bovine Viral Diarrhea Virus
  • viruses of the Hepadnaviridae family such as, for example, Hepatitis B Virus
  • viruses of the Picornaviridae family such as, for example, Encephalomyocarditis Virus, Human Rhinovirus, and Hepatitis A Virus
  • viruses of the Retroviridae family such as, for example, Human Immunodeficiency Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and Rous Sarcoma Virus
  • viruses of the Coronaviridae family such as, for example, SARS coronavirus
  • viruses of the Rhabdoviridae family such as, for example, Rabies Virus and Vesicular Stomatit
  • Exemplary bacteria and fungi include those falling within the following groups Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionella, Leptospires Listeria, Mycoplasmatales, Neisseriaceae (e.g., Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anth
  • kits useful for one-pot synthesis of multivalent dendrimeric protein assembly comprising:
  • orthogonal label is selected from the group comprising structures 7, 8, 9 and 10
  • Example 1 SrtA-mediated assembly of GFP and PspA dendrimers using Gly- terminated dendritic scaffold
  • Green fluorescent protein was employed to demonstrate the propensity of SrtA to catalyze protein dendrimer assembly and engineered a sortase recognition LPXTG sequence preceding the His 6 tag at the C-terminus of GFP.
  • GFP-LPETG-H1S6 0.5 mM was incubated with increasing concentrations of divalent/ tetravalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37oC for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%).
  • the Sortase Click reaction of the instant invention is a two step bioorthogonal process wherein a protein labelled with alkyne or azide in the first step by the facile action of SrtA can be linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction ( Figure 5).
  • Standard CuAAC reaction conditions were employed for conjugating the azide or alkyne labelled GFP to the respective orthogonally derivatized dendritic scaffold.
  • GFP-LPET-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuSO 4 /20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h.
  • An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%).
  • Incubation of 2 equivalents of GFP-alkyne with divalent dendron 3 in the presence of 10 mM CuS0 4 and 20 mM ascorbate for 1 h at RT produced respective divalent proteins in >80% yield (Figure 8A).
  • Example 3 SrtA-mediated installation of alkyne/azide labels on PspA 203 286 -LPNTG- HiSfi
  • Sortase-Click strategy a 84 amino acid long fragment corresponding to residues 203-286 of pneumococcal surface protein A (PspA) from Streptococcus pneumoniae was chosen.
  • PspA pneumococcal surface protein A
  • PspA -LPNTG-His 6 was labelled with 7 and 8 in the presence of SrtA ( Figure 9A and 9B).
  • PspA 203 ⁇ 286 -LPNT-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS0 4 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h.
  • An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). SrtA- mediated ligations produced about 50-60%) yields suggesting that the labelling reaction was as efficient as that seen with GFP.
  • Example 5 Application of protein dendrimers obtained by Sortase-Click reaction: For this, we chose a longer sequence (188 residues) encompassing the surface exposed immunogenic domain of PspA (PspA 98 286 ) from Streptococcus pneumoniae. The choice of PspA was inspired by the fact that it is a lead candidate for a protein-based pneumococcal vaccine. PspA thus can be a good model antigen to construct a protein dendrimer for evaluating the effect of multivalency on immunogenicity. PspA 98"286 was engineered in the
  • Example 6 Demonstration of the high potential of Sortase-Click method in the construction of homogeneous multivalent vaccines; Effect of multivalency on immunogenicity:
  • mice Three groups of eight mice (female, BALB/c) were immunized subcutaneously thrice at 2-
  • PspA 98 " 286 -tetravalent in alum A fourth group was administered PspA 98 " 286 -tetravalent dendrimer (without alum) to gauge the intrinsic effect of multivalency on immunogenicity.
  • the serum anti-PspA end point titre was determined for individual mice following three immunizations with various PspA dendrimer preparations using ELISA with PspA 98"286 - LPNT-alkyne as the capture antigen.
  • the median PspA 98"286 -specific total IgG titre revealed a hierarchy of immunogenicity as tetravalent > divalent > monovalent ( Figure 21A and Table
  • Synthetic peptide scaffolds are complemented by various naturally occurring sugar based polymeric scaffolds for macromolecular assemblage of multivalent bioconjugates.
  • Cyclodextrin (CD) a cyclic polymer of D-glucose, is one such example (Fig. 1).
  • 6- aminosugars are effective nucleophilic substrates in sortase-catalyzed ligation reaction. 6- aminohexoses or appendages thereof, can be efficiently ligated to peptides and proteins encoded with a LPXTG sortase recognition sequence.
  • Bioorthogonal ligation was carried out with an alkyne labelled protein and per-6-deoxy-6-
  • LPNT-alkyne was prepared by sortase mediated ligation of PspA " -LPNTG-His 6 with peptide 9 using the procedure described above. Under similar CuAAC conditions, incubation of a fixed amount of PspA 203 " 286 -LPNT-alkyne with varying concentration of azide- derivatized ⁇ -CD (compound 13, figure 2) produced a high molecular weight band appearing at ⁇ 250 KDa indicating the formation of multivalent product ( Figure 23). About 70-80 % of monovalent protein was converted into multivalent PspA 203 " 286 - ⁇ -CD conjugate. Importantly, no other protein band was observed between monovalent and the high molecular weight specie indicating the homogeneity of the multivalent protein conjugate.
  • SDS-PAGE depicted an abnormal mobility of multivalent PspA 203 " 286 conjugated to ⁇ -CD.
  • a bulk reaction reaction was set up and multivalent ⁇ -CD-PspA 203 " 286 conjugate was isolated by size-exclusion chromatography

Abstract

The present invention relates to synthesis of bio-conjugates and well-defined multivalent protein dendrimer assembly by a general and straightforward chemo- enzymatic method. The method involves a simple two-step process in which proteins appended with an orthogonal label by facile sortase-mediated ligation are conjugated to a multivalent dendritic scaffold using the versatile copper-catalyzed azide-alkyne cycloaddition reaction. The 'Sortase-Click' strategy produces satisfactory yield of dendrimers under mild conditions from readily available His6 -tagged proteins and serves as a powerful general strategy for covalent assembly of bio-conjugates and protein dendrimers.

Description

"A Sortase-Click Reaction Suite for Synthesis of Multivalent Dendrimeric
Protein Assembly"
FIELD OF INVENTION
The present invention lies in the broad field of biotechnology and more specifically relates to chemo-enzymatic synthesis of bio-conjugates, multivalent dendrimeric assembly and multivalent vaccines.
BACKGROUND OF THE INVENTION
A bio-conjugate is generally the product of a bio-conjugation or coupling of two bio- molecules. Bio-conjugates have wide ranging applications; they are useful as therapeutic or diagnostic agents. Bio-conjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule. This includes, but is not limited to conjugation of proteins, peptides, polypeptides, polysaccharides, hormones, nucleic acids, and liposomes with each other or with any other molecular species that add useful properties, including, but not limited to, drugs, radionuclides, toxins, haptens, inhibitors, chromophores, fluorophores, ligands etc. Bio-conjugation is utilized extensively in bio-chemical, immunochemical and molecular biological research. Major applications of bio-conjugation include; detection of gene probes, enzyme-linked immuno solid-phase assay, and monoclonal antibody drug targeting and medical imaging. Bioconjugates are generally classified as either direct or indirect conjugates. Direct conjugates encompass those in which two or more components are joined by direct covalent chemical linkages. Alternatively, indirect conjugates encompass those in which two or more components are joined via an intermediary complex involving a biological molecule.
A person skilled in art will acknowledge that dendrimers are type of bio-conjugates that represent a new class of highly branched polymers whose interior cavities and multiple peripheral groups facilitate potential applications in biomedicine and bio-organic chemistry. Dendrimers possess many advantages including well-defined structure, mono-dispersity, multi-valency and ease of surface functionalization, which make them useful scaffolds for protein mimics. Major advances have been made in the synthesis and study of new carbohydrate, nucleic acid, and peptide dendrimers, as well as their use as magnetic resonance imaging contrast agents, as agents for cellular delivery of nucleic acids, and as scaffolds for bio -mimetic systems. Multivalent display of peptides and proteins on a dendrimeric scaffold can be very useful in a variety of biological settings ranging from delineation of basic mechanisms to applications in biotechnology and medicine like vaccines etc.
However, till date, assembly of chemically defined and homogeneous protein dendrimers remains a synthetic challenge. The erstwhile solid phase synthesis of peptide dendrimers by Tarn's group, referred to as multiple antigen peptide (MAP), exploited the availability of two amino groups (a and ε) in a lysyl residue to generate branch points for iterative elaboration of two, four or eight copies of short antigenic peptide sequences. These multiple antigen peptides (MAPS) contain a lysine dendrimer scaffold holding multiple copies of an antigenic peptide, which enhances its immunogenicity.
Dendrimers have conventionally been synthesized following the sequential assembly or iterative approach, but current advances have yielded chemo-selective synthetic approaches that circumvent the need for sequential assembly of the final peptide dendrimeric architecture. Orthogonal reactions have been well utilized in the synthesis of dendrimers. Orthogonality is a well-recognized concept, relating things that are functionally independent of each other. In chemistry, for example, orthogonal protecting groups can be added and removed independently of each other, and therefore are commonly used to control the specificity of reactions in the synthesis of complex macro molecules, including polypeptides and polynucleotides. Several years ago, Baranay and Merrifield defined an orthogonal system as "a set of completely independent classes of protection groups, such that each class can be removed in any order and in the presence of all other classes."
Current advances have yielded chemo-selective synthetic approaches that circumvent the need for sequential assembly of the final peptide dendrimeric architecture. Orthogonal protecting group strategies have found widespread use in peptide chemistry. The applications of orthogonal reactions, such as native chemical ligation (NCL), copper-assisted azide-alkyne cycloaddtion (CuAAC), or oxyamine-ketone ligation have facilitated the synthesis of interesting peptide dendrimers built from monomeric units composed of 50 or less residues. Native Chemical Ligation (NCL) that usually utilises two peptide components: a peptide C- terminal thio-ester and an N-terminal cysteine containing component, has facilitated the production of proteins. In NCL, the two species combine to ultimately form a native peptide bond at the site of ligation. The constructions of tetravalent green fluorescent protein (GFP) and collagen binding protein respectively by Meijer's group represent noteworthy examples of protein dendrimers prepared employing NCL.
On the other hand, the assembly of multivalent proteins by NCL and intein-based technology to produce an orthogonal thioester-cysteine coupling pair is somewhat stymied by synthetic and purification difficulties. Intein expression system for recombinant proteins is not as routinely followed as His-tagged proteins. Besides, NCL reaction in multivalent assembly of proteins proceed with slower rates and yield a gamut of oligomers that are difficult to purify. In contrast, azide-alkyne cycloaddition reactions are robust and irreversible under physiological conditions but have rarely been employed for synthesis of multivalent protein dendrimers.
Sortases belong to transpeptidase class of prokaryotic enzymes, are utilizable for enzymatic coupling and have an ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile. Sortases modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of (1) the sorting motif LPXTG (Leu-Pro-any-Thr-Gly), (2) a highly hydrophobic transmembrane sequence and (3) a cluster of basic residues such asparginine. Cleavage occurs between the Thr and Gly residues within this pentapeptide sorting motif. Like other sortases, Staphylococcus aureus sortase, Sortase A (SrtA) is a transpeptidase that attaches surface proteins to the cell wall; cleaves between the Thr and Gly of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl- group of threonine and the amino-group of the cell-wall peptidoglycan as illustrated below: LPXTG-— + GGGGG > LPXTGGGGG + G
Transpeptidase sortase A (SrtA) of Staphylococcus aureus has turned out to be a wonderful synthetic catalyst for peptide ligation. The propensity of SrtA to catalyze in vitro ligation of LPXTG sequence embedded polypeptide fragments to an aminoglycine-derivatized molecule through exchange of the Gly residue of the T-G peptide bond has enabled several applications in chemistry and biology (Figure 1). Because of the ease with which SrtA is known to install a Gly-appended moiety at the C- terminus of proteins, it has been effectively used to append a variety of labels including fluorophores, lipids, carbohydrates and peptides. However, it has limitations such as lower yield of dendrimers, which is presumably related to steric constraints originating from substrate multivalency and reversibility of sortase-catalyzed transpeptidation reaction.
Thus Native sortase ligation, although useful for one pot synthesis of divalent proteins, is not good enough for the synthesis of higher generation dendrimers. US Patent application 20090088372, by the same group of inventors, which relates to Sortase mediated reactions is incorporated in entirety as a reference in this application.
On the other hand, the CuAAC in situ click chemistry is Copper(I)-catalyzed ligation and uses the cycloaddition of azides with alkynes as the 'click' mechanism that locks two halves of the desired structure together. Thus, two molecules with weak affinity to different structural features of the target enzyme can be linked to produce a high-affinity bivalent inhibitor. Click chemistry based of modular peptide ligation, although conceptually extendable to large proteins, in practicality have been applied to the assembly of chemically- defined protein dendrimers only in a limited number of cases. The limited examples of well- defined multivalent protein dendrimers despite their enormous utility is a testimony to the unwieldiness of the current methods.
The present application aims to overcome the aforesaid deficiencies of the disclosure in the prior art and provide novel, widely applicable method of synthesis of protein bio-conjugates; well defined, homogeneous protein dendrimer assembly by combination of Sortase and click chemistry. The high yielding Sortase - Click reaction suite of the present invention is applicable to a wide variety of proteins and is utilizable in production of highly effective multivalent vaccines.
ADVANTAGES
The bio -orthogonal Sortase-Click reaction suite of the present invention is a simple and generic chemo-enzymatic reaction suite, developed for the assembly of bio-conjugates and well defined multivalent protein dendrimer assembly. The method is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and is easily applicable to more common C-terminus hexahistidine (His6)-tagged proteins. Besides, lysine dendritic wedges with orthogonal handles are amenable to easy synthesis by standard solid phase peptide chemistry. Both reactions, Sortase labeling as well as CuAAC, produce little or no side products. His6-tagged unlabeled protein and SrtA are easily removed by capture on Ni-NTA beads and pure protein dendrimers, after the click reaction, are obtained by routine size exclusion chromatography. Additionally, Sortase-Click approach provides enormous synthetic flexibility for incorporation of diverse proteins in the dendrimer. SrtA-mediated labelling reaction is known to tolerate a wide range of chemically disparate molecules and therefore can generate a variety of orthogonal handles on proteins for conjugation to compatible dendritic scaffold. Sortase-Click strategy thus facilitates the synthesis of a variety of bio-conjugates and protein dendrimers for unprecedented applications in biology and medicine including construction of chemically defined multi-antigenic semisynthetic vaccines.
OBJECT OF THE INVENTION
The object of the present invention is to provide a simple, generic, one pot chemo-enzymatic synthetic strategy for synthesis of multivalent protein dendrimer assembly.
Another object of the present invention is to provide a novel chemo-enzymatic reaction suite based on sortase-click chemistry, for synthesis of multivalent protein dendrimer assembly. Another object of the present invention is to provide a novel chemo-enzymatic reaction suite based on sortase-click chemistry that is accessible to all proteins equipped with a LPXTG sortase-recognition sequence.
Another object of the present invention is to provide a novel chemo-enzymatic reaction suite based on sortase-click chemistry, for the assembly of homogenous, well-defined dendrimers from readily accessible His6 -tagged proteins.
Another object of the present invention is to elucidate the mechanism behind sortase- mediated ligation of LPXTG proteins to a glycine-terminated multivalent dendritic scaffold. Yet another object of the present invention is the synthesis of bio-conjugates.
Yet another object of the present invention is to provide chemically defined multi-antigenic semisynthetic vaccines based on protein dendrimers.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS Figure 1: Sortase A-catalyzed transpeptidation reaction. Figure 2: Structures of azide/alkyne derivatized lysine dendritic scaffolds and sortase substrates: Structures 1 to 6, 11 and 13 represent azide or an alkyne terminated multivalent dendritic scaffold; Structures 7, 8, 9 and 10 represent orthogonal label (Sortase substrates) Figure 3: SrtA-mediated assembly of GFP dendrimers using Gly-terminated dendritic scaffold 1 and 2, (A) GFP-LPETG-His6 was incubated with increasing concentrations of divalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37°C for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 2; standard GFP-LPETG- His6, Lanes 3 through 7 represent reaction carried out in the presence of 0.0625 mM, 0.125 mM, 0.25 mM 0.5 mM and 1 mM of dendron 1, respectively. (B) GFP-LPETG-His6 (0.5 mM) was incubated with varying concentrations of tetravalent Gly-terminated dendron 2 in the presence of 50 M SrtA at 37°C for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 2; standard GFP-LPETG-His6, Lane 3; GFP-LPETG-His6 reacted with SrtA in the absence of dendron 2, and Lanes 4 through 10 represent reactions carried out in the presence of 2.0 mM, 1.0 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.06 mM and 0.03 mM of dendron 2, respectively.
Figure 4: SrtA-mediated assembly of PspA dendrimers using Gly-terminated dendritic scaffold, (A) PspA203 286-LPNTG-His6 (0.5 mM) was incubated with increasing concentrations of divalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37°C for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 1 ;
203 286
PspA " -LPNTG-His6 reacted with SrtA in the absence of dendron 1, Lanes 2 through 6 represent reaction carried out in the presence of 1.0 mM, 0.5 mM, 0.25 mM 0.125 mM and 0.06 mM of dendron 1, respectively. (B) PspA203 286-LPNTG-His6 (0.5 mM) was incubated with varying concentrations of tetravalent Gly-terminated dendron 2 in the presence of 50 M SrtA at 37°C for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%).
203 286
Lane 1 ; PspA " -LPNTG-His6 reacted with SrtA in the absence of dendron 2 and Lanes 2 through 6 represent reactions carried out in the presence of 1.0 mM, 0.5 mM, 0.25 mM, 0.125 mM and 0.06 mM of dendron 2, respectively.
Figure 5: Sortase-Click reaction. His6-tagged protein equipped with a LPXTG-recognition motif is labeled with azide or alkyne by SrtA. The labeled protein is purified by Ni-NTA affinity chromatography and conjugated to appropriate dendron using CuAAC reaction. Figure 6: SrtA-mediated installation of alkyne / azide labels on GFP. (A) 0.5 mM of GFP- LPETG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was loaded on to a Ni-NTA agarose column and the unbound alkyne labeled protein (GFP-LPET-alkyne) was collected and analyzed by SDS-PAGE (12%). Lane 1; standard GFP-LPETG-His6, Lane 2; total reaction mixture, Lane 3; unbound Ni-NTA fraction (GFP-LPET-alkyne), Lane 4; Ni-NTA beads (unreacted protein and SrtA) and Lane 5; Pooled and concentrated stock of GFP-LPET-alkyne. Note that the gels are only qualitative. (B) The reaction of compound 8 and GFP-LPETG-His6 in the presence of SrtA for azide installation was carried out in much the same way as described above for alkyne labeling. Lane 1 ; GFP-LPETG-His6; Lane 2, total reaction mixture, Lane 3; Ni-NTA flow through (GFP-LPET-azide), Lane 4; Ni-NTA beads (unreacted protein and SrtA), and Lane 5; pooled and concentrated GFP-LPET-azide.
Figure 7: ESI-MS characterization of Ni-NTA purified (A) GFP-LPET-alkyne (B) GFP- LPET-azide. The mass difference of 245 units in each case is attributed to loss of Met and Val from the N-terminus of GFP and a possible modification due to oxidation, adding 16 units extra to the mass of the alkyne/azide labeled GFP.
Figure 8: CuAAC mediated assembly of GFP dendrimers (A) GFP-LPET-alkyne ligation to azide-terminated dendrons 3 (divalent) and 4 (tetravalent), respectively. GFP-LPET-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS04 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 1 ; standard GFP-LPETG- His6; Lane 2; GFP-LPET-alkyne, Lane 3; reaction of GFP-LPET-alkyne with 3 and Lane 4; reaction of GFP-LPET-alkyne with 4. The lower bands in Lanes 3 and 4 are presumably respective monovalent derivatives. The bands indicated by an asterisk may represent trivalent species. (B). GFP-LPET-azide ligation to alkyne-terminated dendrons 5 (divalent) and 6 (tetravalent), respectively. GFP-LPET-azide was reacted with 5 or 6 under CuAAC conditions as described above. Lane 1 ; GFP-LPETG-His6, Lane 2; GFP-LPET-azide, Lane 3; reaction of GFP-LPET-azide with 5 and Lane 4; reaction of GFP-LPET-azide with 6.
Figure 9: SrtA-mediated installation of alkyne / azide labels on PspA203 286-LPNTG-His6. (A) Alkyne labeling, 0.5 mM of PspA203 286-LPNTG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was processed as described in Figure 6 and an aliquot was analyzed by SDS-PAGE (12%). Lane 1; standard
203 286
PspA " -LPNTG-His6, Lane 2; total reaction mixture, Lane 3; unbound Ni-NTA fraction (PspA203_286-LPNT-alkyne), Lane 4; Ni-NTA beads (unreacted protein and SrtA) and Lane 5;
203 286
Pooled and concentrated stock of PspA " -LPNT-alkyne. Note that the gels are only
203 286
qualitative. (B) Azide labeling, The reaction of compound 8 and PspA " -LPNTG-His6 in the presence of SrtA for azide installation was carried out in much the same way as described
203 286
above for alkyne labeling. Lane 1 ; PspA " -LPNTG-His6; Lane 2, total reaction mixture, Lane 3; Ni-NTA flow through (PspA203~286-LPNT-azide), Lane 4; Ni-NTA beads (unreacted
203 286
protein and SrtA), and Lane 5; pooled and concentrated PspA " -LPNT-azide.
Figure 10: ESI-MS analysis of (A) PspA203~286-LPNT-alkyne and (B) PspA203~286-LPNT- azide.
Figure 11: CuAAC mediated ligation of PspA203 286-LPNT-alkyne to azide-terminated
203 286
dendrons 3 (divalent) and 4 (tetravalent), respectively. PspA " -LPNT-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS04 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Lane 1 ; standard PspA203 286-LPNTG- His6; Lane 2; PspA203"286-LPNT-alkyne, Lane 3; reaction of PspA203"286-LPNT-alkyne with 3 and Lane 4; reaction of PspA203"286-LPNT-alkyne with 4.
Figure 12: Purification of PspA 203 " 286 dendrimers by size-exclusion chromatography.
203 286
CuAAC reaction was carried out with PspA " -LPNT-alkyne and (A) dendron 3 and (B) dendron 4. Fractions isolated from Superdex200 column from peak 1 and peak 2 were pooled and analyzed by SDS-PAGE (18%).
Figure 13: ESI-MS analysis of purified (A) PspA203"286-divalent and (B) PspA203"286- tetravalent. Figure 14: CuAAC mediated ligation of PspA203 286-LPNT-azide to alkyne-terminated
203 286 dendrons 5 (divalent) and 6 (tetravalent), respectively. Lane 1 ; standard PspA " -LPNTG- His6; Lane 2; PspA203"286-LPNT-azide, Lane 3; reaction of PspA203"286-LPNT-azide with dendron 5 and Lane 4; reaction of PspA 203 " 286 -LPNT-azide with dendron 6. The smearing of high molecular weight bands in lane 4 may be due to dendrimeric nature of tetravalent PspA203"286.
Figure 15: SrtA-mediated installation of alkyne / azide labels on PspA98"286. (A) Alkyne labeling, 0.5 mM of PspA98 286-LPNTG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was processed as described in Figure 6 and an aliquot was analyzed by SDS-PAGE. Lane 1 ; standard PspA98 286-LPNTG- His6, Lane 2; total reaction mixture, Lane 3; unbound Ni-NTA fraction (PspA98 286-LPNT- alkyne), Lane 4; Ni-NTA beads (unreacted protein and SrtA) and Lane 5; Pooled and concentrated stock of PspA98"286-LPNTalkyne. Note that the gels are only qualitative. (B) Azide labeling, The reaction of compound 8 and PspA98 286-LPNTG-His6 in the presence of SrtA for azide installation was carried out in much the same way as described above for alkyne labeling. Lane 1 ; PspA98 286-LPNTG-His6; Lane 2, total reaction mixture, Lane 3; Ni- NTA flow through (PspA98_286-LPNT-azide), Lane 4; Ni-NTA beads (unreacted protein and SrtA), and Lane 5; pooled and concentrated PspA98 286-LPNT-azide.
Figure 16: ESI-MS analysis of Ni-NTA purified (A) PspA98"286-alkyne and (B) PspA98"286- azide. Figure 17: Synthesis of PspA dendrimers. Purification of PspA 98 " 286 -divalent and PspA 98 " 286 - tetravalent dendrimers respectively by size exclusion chromatography. The inset shows SDS- PAGE of the purified material.
Figure 18: ESI-MS analysis of purified and intact (A) PspA98"286-divalent and (B) PspA98"286- tetravalent.
Figure 19: Characterization of PspA 98 " 286 -divalent and PspA 98 " 286 -tetravalent dendrimers by tryptic mapping. A comparison of the tryptic maps of the dendrimers led to the identification of unique peaks labeled A and B in the case of PspA98 286-divalent dendrimer and C and D in the case of PspA98~286-tetravalent dendrimer, respectively.
Figure 20: ESI-MS analysis of peak A, B, C and D obtained from the tryptic digest of PspA- divalent and PspA-tetravalent dendrimer. Figure 21: Analysis of immune response to PspA dendrimers by ELISA. (A) End-point titer for total IgG were estimated for each group using PspA98_286-alkyne as the capture antigen. (B) PspA-specific antibody titers of various IgG subtype induced after immunization with 3 doses of various preparations of PspA dendrimers.
Figure 22: CuAAC mediated ligation of PspA98_286-LPNT-azide to alkyne dendrons 5 (divalent) and 6 (tetravalent), respectively. The CuAAC reaction of PspA98_286-LPNT-azide with 5 and 6 was carried out as described for PspA98_286-LPNT-alkyne. Lane 1 ; standard PspA98 286-LPNTG-His6, Lane 2; PspA98~286-LPNT-azide, Lane 3; reaction of PspA98"286- LPNT-azide with 5 and Lane 4; reaction of Psp A98 286-LPNT-azide with 6.
203 286
Figure 23: CuAAC mediated ligation of PspA " -LPNT-alkyne and per-6-deoxy-6-azido- PCD. A fixed amount of alkyne labelled protein was incubated with increasing concentration of purified azide-derivatized β-CD in the presence of copper sulphate and sodium ascorbate.
Figure 24: Purification of β-CD-PspA 203 " 286 conjugate by size-exclusion chromatography.
203 286
Peak 1 corresponds to multivalent β-CD-PspA " conjugate and peak 2 corresponds to
203 286
unreacted monovalent PspA " -LPNT-alkyne. Table 1: Mass analysis of peptide sequences generated by tryptic digest of PspA98"286 dendrimers.
Table 2: End-point titres of antibody response to multivalent PspA98"286 constructs determined by ELISA using PspA98"286-alkyne as the coating antigen. Anti-PspA antibody titres are presented as the median for groups of eight mice. Titres are defined
as the reciprocal of highest dilution yielding an optical density twice as that of pre-immune mouse sera. I, PspA 9y8o~ 2z86 -alkyne/alum; II, divalent-PspA 9^8^ 286Valum; III, tetravalent-PspA 9"80"
286 /alum; and IV, tetravalent-PspA 98 " 286 /saline (without alum). The control group of mice immunized with dendron 4/alum showed no detectable immune response. A p value < 0.05 was considered statistically significant.
SUMMARY OF THE INVENTION
The present invention relates to synthesis of bio-conjugates and multivalent protein dendrimer assembly by a simple process in which proteins appended with an orthogonal label by facile sortase-mediated ligation are conjugated to a multivalent dendritic scaffold using the versatile copper-catalyzed azide-alkyne cycloaddition reaction. The process mainly involves two steps, wherein protein labeled with alkyne or azide in the first step by the robust action of SrtA is linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction. The unreacted His6 -tagged protein and sortase are then allowed to bind to Ni-NTA beads and the pure labelled protein is collected in the supernatant.
The present invention also provides bio-conjugates generated by the sortase-mediated ligation which comprises of (a) a bio-conjugate comprising LPXTG peptide motif capable of recognition by sortase and (b) an azide or an alkyne terminated multivalent dendritic scaffold. The present invention also provides chemically defined multi-antigenic semisynthetic vaccines. DETAILED DESCRIPTION OF THE INVENTION
The present invention is described with reference to the tables/ figures etc. and specific embodiments; this description is not meant to be construed in a limiting sense. Various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention.
Accordingly, the present invention has various embodiments relating to synthesis of bio- conjugates and well-defined multivalent protein dendrimers by a general and straightforward chemo-enzymatic method.
An embodiment of the present invention relates to a one pot chemo-enzymatic synthetic strategy for synthesis of bio-conjugates and a multivalent protein dendrimer assembly generated by sortase-mediated ligation of LPXTG proteins to a glycine-terminated multivalent dendritic scaffold.
In another embodiment, the process described in the present invention is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and dendrimers from readily accessible His6 -tagged proteins. Interestingly, the inventors have shown a novel two step enzymatic approach, a "Sortase- Click Reaction Suite" according to the present invention, in which proteins appended with an orthogonal label by facile sortase-mediated ligation are conjugated to a multivalent dendritic scaffold using the versatile copper-catalyzed azide-alkyne cycloaddition reaction to obtain a bioconjugates and multivalent dendrimers useful for various therapeutic and diagnostic applications. The process mainly involves two steps, wherein protein labeled with alkyne or azide in the first step by the robust action of SrtA is linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction. The unreacted His6 -tagged protein and sortase are then allowed to bind to Ni-NTA beads and the pure labelled protein is collected in the supernatant.
Figure imgf000013_0001
The present invention for the first time presents a bio -orthogonal Sortase-Click reaction suite as depicted above, involving two extremely specific and reliable reactions that occur under aqueous conditions across a wide pH range of 4 to 9. The method is accessible to all proteins equipped with a LPXTG sortase-recognition sequence and is easily applicable to more common C-terminus His6-tagged proteins. Unlabelled His6-tagged protein and SrtA are easily removed by capture on Ni-NTA beads and pure multivalent proteins, after CuAAC conjugation, are obtained by routine size-exclusion chromatography. Besides, lysine dendritic wedges with orthogonal handles are amenable to easy synthesis by standard solution or solid phase peptide chemistry. SrtA-mediated labelling reaction is known to tolerate a wide range of chemically disparate molecules and therefore can generate a variety of orthogonal handles on proteins for conjugation to compatible dendritic scaffold allowing enormous synthetic flexibility for incorporation of diverse proteins in the scaffold. SrtA-mediated labelling, together with click chemistry described herein is likely to provide synergy in multivalent protein assembly. Besides, copper-free click chemistry can also be adopted using cyclooctyne tagged proteins as demonstrated in linear protein- fusions by Witte et al. Sortase-Click strategy developed here in combination with other orthogonal approaches should facilitate the assembly of a variety of protein dendrimers for unprecedented applications in biology and medicine including construction of chemically defined multi-antigenic semisynthetic immunogens as demonstrated with PspA in this report. Thus overall, the one-pot process for the synthesis of multivalent dendrimeric protein assembly comprises of following steps
(a) ligating hexahistidine - tagged protein having LPXTG peptide motif with orthogonal label selected from alkyne or azide, in the presence of sortase, at a suitable pH such as herein described, for a period in the range of of 10- 12 hours at room temperature to obtain labelled protein;
(b) removing unreacted hexahistidine - tagged proteins and unreacted sortase by binding with Ni-NTA beads and collecting pure labelled protein in supernatant and
(c) conjugating labelled protein of step (a) with multivalent dendritic scaffold by incubation in presence of CUSC , ascorbate at room temperature for 1 -2 hours;
(d) obtaining the protein dendrimer by size exclusion chromatography.
Another important aspect of the present invention relates to multivalent protein dendrimer assembly comprising
(a) a bio-conjugate defined by the following formula
^Protein^> LPXT-G - Orthogonal Label wherein the orthogonal label is selected from the group comprising structures 7, 8, 9 and 10 and
(b) an azide or an alkyne terminated multivalent dendritic scaffold selected from the group comprising structures 1 to 6, 11 and 13
In yet another aspect of the present invention the LPXTG peptide motif is a sortase recognition sequence.
Yet another aspect of the present invention relates to bio-conjugates that are homogeneous protein dendrimer assembly.
In yet another aspect, the present disclosure provides a composition comprising the multivalent dendrimeric protein assembly, useful as therapeutic or diagnostic agent. For administration, the composition may be dispersed in a pharmaceutically acceptable adjuvant or carrier.
Yet another embodiment of the present invention relates to a multivalent vaccine, wherein the vaccine comprises the multivalent dendrimeric protein assembly as claimed in any of the aforesaid claims, wherein the vaccine is effective in preventing or treating various infectious diseases (e.g., viral infections, bacterial infections, fungal infections)..
Exemplary viruses include, but are not limited to, viruses of the Flaviviridae family, such as, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridae family, such as, for example, Hepatitis B Virus; viruses of the Picornaviridae family, such as, for example, Encephalomyocarditis Virus, Human Rhinovirus, and Hepatitis A Virus; viruses of the Retroviridae family, such as, for example, Human Immunodeficiency Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and Rous Sarcoma Virus; viruses of the Coronaviridae family, such as, for example, SARS coronavirus; viruses of the Rhabdoviridae family, such as, for example, Rabies Virus and Vesicular Stomatitis Virus, viruses of the Paramyxoviridae family, such as, for example, Respiratory Syncytial Virus and Parainfluenza Virus, viruses of the Papillomaviridae family, such as, for example, Human Papillomavirus, and viruses of the Herpesviridae family, such as, for example, Herpes Simplex Virus. Diseases caused by gram-positive or gram-negative bacteria, mycobacteria, fungi such as Candida or Aspergillus, helminths, etc., are of interest in certain embodiments. Exemplary bacteria and fungi include those falling within the following groups Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionella, Leptospires Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Menigo cocci), Pasteurellacea (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Treponema, and Staphylococci.
In still another aspect, the present disclosure provides a kit useful for one-pot synthesis of multivalent dendrimeric protein assembly comprising:
(a) hexahistidine - tagged protein having LPXTG peptide motif
(b) orthogonal label is selected from the group comprising structures 7, 8, 9 and 10
(c) Sortase
(d) multivalent dendritic scaffold from the group comprising structures 1 to 6, 11 and 13
(e) CuS04
(f) Na Ascorbate
(g) Tris buffer, pH 7.5
(h) NaCl
The present disclosure with reference to the accompanying examples describes the present invention. A more complete understanding of the invention can be had by reference to the following examples. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way.
Example 1: SrtA-mediated assembly of GFP and PspA dendrimers using Gly- terminated dendritic scaffold
Green fluorescent protein (GFP) was employed to demonstrate the propensity of SrtA to catalyze protein dendrimer assembly and engineered a sortase recognition LPXTG sequence preceding the His6 tag at the C-terminus of GFP. GFP-LPETG-H1S6 (0.5 mM) was incubated with increasing concentrations of divalent/ tetravalent Gly-terminated dendron 1 in the presence of 50 M SrtA at 37oC for 6 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Incubation of GFP- LPETG-H1S6 with glycine-terminated divalent dendron 1 (Figure 2) in the presence of SrtA resulted in the formation of divalent GFP in about 20-30 % yield (Figure 3A). However, reaction of GFP-LPETG-H1S6 with tetravalent dendron 2 (Figure 2) produced low amounts (<5%) of tri- and tetra- valent dendrimers (Figure 3B).
203 286
An identical pattern was observed when PspA -LPNTG-His6 was incubated with dendrons 1 and 2, respectively (Figure 4A and 4B). Taken together, the results indicated that 'native sortase ligation' while useful for one pot synthesis of divalent proteins may not be good enough for the synthesis of higher generation dendrimers.
Example 2: Sortase Click reaction
The Sortase Click reaction of the instant invention is a two step bioorthogonal process wherein a protein labelled with alkyne or azide in the first step by the facile action of SrtA can be linked in the subsequent step to a multivalent azide/alkyne terminated dendritic scaffold using the versatile CuAAC click reaction (Figure 5). 1. SrtA-mediated installation of alkyne / azide labels on GFP
First SrtA-mediated labelling of GFP-LPETG-H1S6 with 7 or 8 was carried out. 0.5 mM of GFP-LPETG-Hise was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37oC for 10-12 h. The reaction mixture was loaded on to a Ni-NTA agarose column and the unbound alkyne labeled protein (GFP-LPET-alkyne) was collected and analyzed by SDS-PAGE (12%). SDS-PAGE analyses suggested that the reaction proceeded well and without any hydrolysis of the protein indicating that 7 and 8 were efficient azide/alkyne labelling substrates (Figure 6 A and 6B). The unreacted His6-tagged protein and sortase were allowed to bind to Ni-NTA beads and the pure labelled protein was collected in the supernatant in about 50%) yield. Mass of the labelled proteins obtained by ESI-MS was in accord with the mass calculated for azide or alkyne labelled GFP constructs (Figure 7 A and 7B).
2. CuAAC mediated assembly of GFP dendrimers
Standard CuAAC reaction conditions were employed for conjugating the azide or alkyne labelled GFP to the respective orthogonally derivatized dendritic scaffold. GFP-LPET-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuSO4/20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). Incubation of 2 equivalents of GFP-alkyne with divalent dendron 3 in the presence of 10 mM CuS04 and 20 mM ascorbate for 1 h at RT produced respective divalent proteins in >80% yield (Figure 8A).
Under identical conditions, reaction of tetravalent scaffold 4 in the presence of four equivalents of alkyne-labelled GFP led to the formation of tetrameric dendrimers in 60-70% yield (Figure 8A). As expected, CuAAC click reaction worked equally well and produced di- and tetra- valent protein dendrimers in comparable yields as above when GFP-azide was coupled to the counterpart alkyne-terminated dendritic scaffolds 5 or 6 (Figure 8B).
Example 3: SrtA-mediated installation of alkyne/azide labels on PspA203 286-LPNTG- HiSfi
To test the generality of the Sortase-Click strategy, a 84 amino acid long fragment corresponding to residues 203-286 of pneumococcal surface protein A (PspA) from Streptococcus pneumoniae was chosen. The sortase-recognition motif and hexahistidine tag
203 286
at the C-terminus were engineered and the recombinant (PspA " -LPNTG-His6) protein
203 286
was obtained. PspA -LPNTG-His6 was labelled with 7 and 8 in the presence of SrtA (Figure 9A and 9B).
(A) Alkyne labeling, 0.5 mM of PspA203 286-LPNTG-His6 was incubated with 5 mM of 7 in the presence of 20 M SrtA at 37°C for 10-12 h. The reaction mixture was processed as described in Figure 6 and an aliquot was analyzed by SDS-PAGE (12%>).
203 286
(B) Azide labeling, The reaction of compound 8 and PspA " -LPNTG-His6 in the presence of SrtA for azide installation was carried out in much the same way as described above for alkyne labeling. The identity of the associated product (PspA203~286-LPNT-alkyne or PspA203~286-LPNT-azide) was confirmed by ESI-MS measurements (Figure 10A and 10B). Example 4:
(A) CuAAC mediated ligation of PspA203 286-LPNT-alkyne to azide-terminated dendrons 3 (divalent) and 4 (tetravalent)
PspA203~286-LPNT-alkyne (0.25 mM) was incubated with 3 (0.125 mM) or 4 (0.0625 mM) in presence of 10 mM CuS04 / 20 mM Na Ascorbate in 50 mM Tris, pH 7.5 and 150 mM NaCl at RT for 1 h. An aliquot of the reaction mixture was analyzed by SDS-PAGE (12%). SrtA- mediated ligations produced about 50-60%) yields suggesting that the labelling reaction was as efficient as that seen with GFP. The CuAAC conjugation of PspA203_286-LPNT-alkyne to the orthogonal di- or tetravalent dendritic scaffold (3 and 4) yielded > 95 % dendrimeric product as judged by SDS-PAGE (Figure 11 and 12). Mass of the purified protein dendrimers (20789 Da and 41618 Da) were in accord with the expected mass of the divalent (20789 Da) or tetravalent (41619) constructs (Figure 13A and 13B).
(B) CuAAC mediated ligation of PspA203 286-LPNT-azide to alkyne-terminated dendrons 5 (divalent) and 6 (tetravalent)
203 286
The reaction of PspA " -LPNT-azide with the alkyne terminated dendritic scaffolds 5 or 6 occurred in a similar fashion and produced >90%> yields corroborating the robustness of the process (Figure 14).
Example 5: Application of protein dendrimers obtained by Sortase-Click reaction: For this, we chose a longer sequence (188 residues) encompassing the surface exposed immunogenic domain of PspA (PspA98 286) from Streptococcus pneumoniae. The choice of PspA was inspired by the fact that it is a lead candidate for a protein-based pneumococcal vaccine. PspA thus can be a good model antigen to construct a protein dendrimer for evaluating the effect of multivalency on immunogenicity. PspA98"286 was engineered in the
203 286
same way as PspA " to obtain a construct equipped with LPNTG followed by the hexa- His tag (PspA98 286-LPNTG-His6). The Sortase-Click reaction suite (labelling and conjugation) was followed strictly as followed in the case o f GFP or PspA 2Z0U3J 2 Z806U and the desired divalent and tetravalent protein dendrimers were obtained in >80% yield (Figure 15 and 16). The high yields facilitated easy isolation of the PspA98 286-LPNT-alkyne dendrimers by size exclusion chromatography (Figure 17A and 17B).
The experimental mass (Figure 18A and 18B) of divalent (45096 Da) and tetravalent (90237 Da) proteins were in agreement with their calculated mass (45097 Da and 90234 Da, respectively). Furthermore, tryptic digest of the divalent and tetravalent protein constructs generated expected peptides including the unique fragment carrying two or four copies of the C-terminus tryptic peptide linked to divalent or tetravalent scaffold (Figure 19 and 20, Table
1) . Collectively, these results establish that the proteins were linked to the MAP scaffold by usual triazole formation and no untoward modification of the protein side chains occurred during CuAAC reaction.
Example 6: Demonstration of the high potential of Sortase-Click method in the construction of homogeneous multivalent vaccines; Effect of multivalency on immunogenicity:
Three groups of eight mice (female, BALB/c) were immunized subcutaneously thrice at 2-
98 286 98 286
week intervals with 25 g of PspAyo~z -LPNT-alkyne monovalent, PspA^0 -divalent and
PspA 98 " 286 -tetravalent in alum. A fourth group was administered PspA 98 " 286 -tetravalent dendrimer (without alum) to gauge the intrinsic effect of multivalency on immunogenicity. The serum anti-PspA end point titre was determined for individual mice following three immunizations with various PspA dendrimer preparations using ELISA with PspA98"286- LPNT-alkyne as the capture antigen. The median PspA98"286-specific total IgG titre revealed a hierarchy of immunogenicity as tetravalent > divalent > monovalent (Figure 21A and Table
2) . The subtying of antibody response (Figure 2 IB and Table 2) exhibited the same trend with preponderance of IgGl indicating a bias towards Th2 response as observed previously. Interestingly, immunization with PspA-tetravalent dendrimer without alum also elicited an immune response comparable to that observed when the same dendrimer was administered in the presence of alum. Thus, multivalent presentation of pneumococcal surface protein-A elicited enhanced immune response, both in the presence and absence of alum. The results demonstrate the high potential of Sortase-Click method in the construction of homogeneous multivalent vaccines facilitating structure-activity studies as well as easy adherence to regulatory and quality control norms.
Synthetic peptide scaffolds are complemented by various naturally occurring sugar based polymeric scaffolds for macromolecular assemblage of multivalent bioconjugates. Cyclodextrin (CD), a cyclic polymer of D-glucose, is one such example (Fig. 1). 6- aminosugars are effective nucleophilic substrates in sortase-catalyzed ligation reaction. 6- aminohexoses or appendages thereof, can be efficiently ligated to peptides and proteins encoded with a LPXTG sortase recognition sequence. The robustness, simplicity and elegance of this unprecedented sortase-catalyzed transamidation reaction have been applied to the one-pot synthesis of a variety of amide-linked neoglycopeptides and proteins. This invention describes the propensity of SrtA to ligate LPXTG containing peptides and proteins to a CD scaffold for multivalent display of ligands. Accordingly, appropriately derivatized β-CD has been prepared and used to obtain bioconjugates by direct SrtA-mediated ligation or through Sortase-Click reaction.
Bioorthogonal ligation was carried out with an alkyne labelled protein and per-6-deoxy-6-
203 286 azido-P-CD, an intermediate in the synthesis of amine functionalized β-CD. PspA " -
203 286
LPNT-alkyne was prepared by sortase mediated ligation of PspA " -LPNTG-His6 with peptide 9 using the procedure described above. Under similar CuAAC conditions, incubation of a fixed amount of PspA 203 " 286 -LPNT-alkyne with varying concentration of azide- derivatized β-CD (compound 13, figure 2) produced a high molecular weight band appearing at ~ 250 KDa indicating the formation of multivalent product (Figure 23). About 70-80 % of monovalent protein was converted into multivalent PspA 203 " 286 -β-CD conjugate. Importantly, no other protein band was observed between monovalent and the high molecular weight specie indicating the homogeneity of the multivalent protein conjugate.
SDS-PAGE depicted an abnormal mobility of multivalent PspA 203 " 286 conjugated to β-CD. To confirm the molecular identity of the conjugate, a bulk reaction reaction was set up and multivalent β-CD-PspA 203 " 286 conjugate was isolated by size-exclusion chromatography
(Figure 24). Large size difference between monovalent and multivalent PspA 203 " 286 allowed complete resolution of the two species on Superdex200 column. Purified protein showed a single band on SDS-PAGE (15%) indicating that the β-CD-PspA203"286 conjugate was 98-99% pure (Figure 24, inset)

Claims

The Claims
A multivalent dendrimeric protein assembly, comprising
(a) a bio-conjugate defined by the following formula
Protein -LPXT-Gr Orthogonal Label wherein the orthogonal label is selected from the group comprising structures 7, 8, 9 and 10 and (b) an azide or an alkyne terminated multivalent dendritic scaffold selected from the group comprising structures 1 to 6, 11 and 13.
2. A one-pot process for the synthesis of multivalent dendrimeric protein assembly as claimed in claim 1, comprising the following steps: (a) ligating hexahistidine - tagged protein having LPXTG peptide motif with orthogonal label selected from alkyne or azide, in the presence of sortase, at a suitable pH such as herein described, for a period in the range of 10-12 hours at room temperature to obtain labelled protein;
(b) removing unreacted hexahistidine - tagged proteins and unreacted sortase by binding with Ni-NTA beads and collecting pure labelled protein in supernatant and
(c) conjugating labelled protein of step (a) with multivalent dendritic scaffold by incubation in presence of CUSC , ascorbate at room temperature for 1-2 hours;
(d) obtaining the protein dendrimer by size exclusion chromatography.
3. The process as claimed in claim 1, wherein the X in sortase recognition LPXTG peptide motif is any natural or unnatural amino acid.
4. A multivalent vaccine, wherein the vaccine comprises the multivalent dendrimeric protein assembly as claimed in any of the aforesaid claims, wherein the vaccine is effective in preventing or treating viral and bacterial diseases.
5. A composition useful as therapeutic or diagnostic agent comprising the multivalent dendrimeric protein assembly as claimed in any of the aforesaid claims along with pharmaceutically acceptable adjuvant and carrier.
6. Multivalent dendrimeric protein assembly for use in the manufacture of a medicament for the treatment or prophylaxis of viral and bacterial diseases.
7. A kit useful for one-pot synthesis of multivalent dendrimeric protein assembly as claimed in claim 1 comprising:
(a) Hexahistidine - tagged protein having LPXTG peptide motif
(b) orthogonal label is selected from the group comprising structures 7, 8, 9 and 10
(c) Sortase
(d) Multivalent dendritic scaffold from the group comprising structures 1 to 6, 11 and 13.
(e) CuS04
(f) Na Ascorbate
(g) Tris buffer, pH 7.5
(h) NaCl
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