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WO2013065009A1 - Suite de réaction de sortase-click pour la synthèse d'un assemblage de protéines de dendrimères multivalents - Google Patents

Suite de réaction de sortase-click pour la synthèse d'un assemblage de protéines de dendrimères multivalents 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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Kanchan Gupta
Shikha SINGH
Naeem KHAN
Devinder SEHGAL
Rajendra Prasad Roy
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National Institute of Immunology
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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

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Abstract

La présente invention concerne la synthèse de bioconjugués et d'un assemblage de dendrimères de protéine multivalents bien défini, par un procédé chimio-enzymatique général et direct. Le procédé comprend un procédé simple en deux étapes, dans lequel des protéines pourvues d'un marqueur orthogonal par ligation facile médiée par la sortase sont conjuguées à un échafaudage dendritique multivalent, au moyen de la réaction polyvalente de cycloaddition azide-alcyne catalysée par du cuivre. La stratégie de «sortase-Click» permet d'obtenir un rendement satisfaisant de dendrimères, dans des conditions modérées, à partir de protéines marquées par His6 facilement disponibles, et constitue une stratégie générale efficace pour l'assemblage covalent de bioconjugués et de dendrimères de protéine.
PCT/IB2012/056083 2011-11-01 2012-11-01 Suite de réaction de sortase-click pour la synthèse d'un assemblage de protéines de dendrimères multivalents Ceased WO2013065009A1 (fr)

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US11155584B2 (en) 2016-09-23 2021-10-26 Duke University Unstructured non-repetitive polypeptides having LCST behavior
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US11649275B2 (en) 2018-08-02 2023-05-16 Duke University Dual agonist fusion proteins
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US12257308B2 (en) 2018-04-30 2025-03-25 Duke University Stimuli-responsive PEG-like polymer-based drug delivery platform
US12296018B2 (en) 2018-01-26 2025-05-13 Duke University Albumin binding peptide-drug (AlBiPeD) conjugates and methods of making and using same

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US10392611B2 (en) 2013-05-30 2019-08-27 Duke University Polymer conjugates having reduced antigenicity and methods of using the same
US9592303B2 (en) 2013-05-30 2017-03-14 Duke University Enzyme-catalyzed synthesis of site-specific and stoichiometric biomolecule-polymer conjugates
US10364451B2 (en) 2013-05-30 2019-07-30 Duke University Polymer conjugates having reduced antigenicity and methods of using the same
WO2015095227A3 (fr) * 2013-12-16 2015-12-10 Genentech, Inc. Composés peptidomimétiques et conjugués anticorps-médicament de ceux-ci
US10632210B2 (en) 2013-12-16 2020-04-28 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
US10124069B2 (en) 2013-12-16 2018-11-13 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015130846A3 (fr) * 2014-02-25 2015-11-05 Duke University Compositions et procédés utilisables en vue de la modification dirigée de polypeptides
CN106795268A (zh) * 2014-09-30 2017-05-31 汉高股份有限及两合公司 不含nco的化合物及其在可固化组合物中的用途
US10577463B2 (en) 2014-09-30 2020-03-03 Henkel Ag & Co. Kgaa NCO-free compounds and usage thereof in a curable composition
US10385115B2 (en) 2015-03-26 2019-08-20 Duke University Fibronectin type III domain-based fusion proteins
US10017577B2 (en) 2015-06-15 2018-07-10 Genentech, Inc. Antibodies and immunoconjugates
US11458205B2 (en) 2015-08-04 2022-10-04 Duke University Genetically encoded intrinsically disordered stealth polymers for delivery and methods of using same
US11752213B2 (en) 2015-12-21 2023-09-12 Duke University Surfaces having reduced non-specific binding and antigenicity
US11467156B2 (en) 2016-06-01 2022-10-11 Duke University Nonfouling biosensors
US11135301B2 (en) 2016-09-14 2021-10-05 Duke University Triblock polypeptide-based nanoparticles for the delivery of hydrophilic drugs
US11155584B2 (en) 2016-09-23 2021-10-26 Duke University Unstructured non-repetitive polypeptides having LCST behavior
US12084480B2 (en) 2016-09-23 2024-09-10 Duke University Unstructured non-repetitive polypeptides having lcst behavior
US11648200B2 (en) 2017-01-12 2023-05-16 Duke University Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature triggered hierarchical self-assembly
US11554097B2 (en) 2017-05-15 2023-01-17 Duke University Recombinant production of hybrid lipid-biopolymer materials that self-assemble and encapsulate agents
US11680083B2 (en) 2017-06-30 2023-06-20 Duke University Order and disorder as a design principle for stimuli-responsive biopolymer networks
US12296018B2 (en) 2018-01-26 2025-05-13 Duke University Albumin binding peptide-drug (AlBiPeD) conjugates and methods of making and using same
US12257308B2 (en) 2018-04-30 2025-03-25 Duke University Stimuli-responsive PEG-like polymer-based drug delivery platform
US11649275B2 (en) 2018-08-02 2023-05-16 Duke University Dual agonist fusion proteins
US11512314B2 (en) 2019-07-12 2022-11-29 Duke University Amphiphilic polynucleotides
US11965164B2 (en) 2019-07-12 2024-04-23 Duke University Amphiphilic polynucleotides

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