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EP-3986465-B1 - SITE-SPECIFIC, KINETICALLY INERT CONJUGATION OF LABELS AND/OR CARRIERS TO TARGET MOLECULES SUCH AS HIS-TAGGED PROTEINS VIA METAL COMPLEX REAGENTS

EP3986465B1EP 3986465 B1EP3986465 B1EP 3986465B1EP-3986465-B1

Inventors

  • BENK, Amelie S.
  • SCHENK, Franziska
  • WEGNER, Seraphine
  • COMBA, PETER
  • SPATZ, JOACHIM P.

Dates

Publication Date
20260513
Application Date
20200618

Claims (15)

  1. A complex comprising: a) a metal cation; b) a metal cation ligand being CO 3 2- or HCO 3 - ; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier.
  2. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain is a polydentate ligand that comprises one or more carboxylic acid groups and/or one or more amine groups and/or one or more aromatic amines and/or phosphates.
  3. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptides such as peptides with the consensus sequence (GHHPH) n G; with n= 1 to 3; see SEQ ID NOs: 1 to 3) or cadystin, triazacyclononane (TACN), diethylenetriamine-pentaacetate (DTPA), phytochelatin, carboxymethylaspartate (CMA), phosphonates, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionicdiacetic acid (NPDA), nitriloisopropionicdiacetic acid (NIPDA), N-(hydroxylethyl)ethylenediaminetriacetic acid (HEDTA), 1,4,7,10-tetraazacyclodo-decane-N,N',N",N‴-tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclonane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4.7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetra-decane-N,N',N",N‴-tetraacetic acid (TETA), ethylenedicysteine, ethylenediaminetetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononane phosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyridimidine and derivatives thereof, preferably NTA, IDA and derivatives thereof.
  4. The complex of any one of claims 1 to 3, wherein the metal cation is a metal cation having a water ligand exchange rate of 10 -1 s -1 or lower, preferably 10 -2 s -1 or lower.
  5. The complex of any one of claims 1 to 3, wherein the metal cation is selected from the group consisting of: Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Pt 2+ , Pt 4+ , Ru 2+ , Ru 3+ , La 3+ , Eu 3+ , Os 2+ , Pd 4+ , Mo 3+ , Fe 3+ , Ru 3+ , Gd 3+ ,Tc 3+ , Re 3+ , Sm 3+ , Tb 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Pm 3+ , Dy 3+ , Ho 3+ , Er 3+ , Tm 3+ , Yb 3+ + , V 2+ , Mn 4+ , Fe 2+ and Lu 3+ , preferably Co 3+ .
  6. The complex of any one of claims 1 to 5, wherein the complex comprises a [Co(III)(NTA)CO 3 ] 2- complex, a [Co(III)(NTA)HCO 3 ] - complex, a [Co(III)(IDA)CO 3 ] - complex, a [Co(III)(IDA)HCO 3 ] complex or a hydrate thereof, wherein the label and/or carrier is attached to NTA or IDA.
  7. The complex of any one of claims 1 to 6, wherein the label comprises a fluorophore, a diagnostic, a targeting moiety, a therapeutic agent, a PEG molecule, a lipid, biotin and/or its derivatives, proteins, peptides, a toxin and/or a reactive group selected from a thiol, azide, alkyne, nitrone, tetrazine and tetrazole and/or wherein the carrier is a polymer, a hydrogel, a microparticle, a nanoparticle, a sphere (including nano- and microsphere), a bead, a quantum dot, a prosthetic or a solid surface.
  8. A composition comprising a complex as defined in any one of claims 1 to 7.
  9. A kit comprising: a) a metal cation, preferably a metal cation as defined claim 4 or 5; b) a metal cation ligand being CO 3 2- or HCO 3 - ; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier, preferably a metal cation chelating domain as defined in any one of claims 2, 3 and 7.
  10. A method for producing a complex according to any one of claims 1 to 7 comprising incubating in a solution: (i) a metal cation; (ii) a metal cation ligand as defined in claim 1 b); and (iii) a metal cation chelating domain as defined in claim 1 c), preferably a metal cation chelating domain as defined in any one of claims 2, 3 and 7 and/or a metal cation as defined in of claim 4 or 5, wherein the method optionally comprises collecting and/or purifying the complex.
  11. The method of claim 10, wherein the metal cation is Co 3+ and wherein Co 3+ and CO 3 2- or HCO 3 - are provided in form of a neutral complex with counter ions, such as in form of a salt, or in form of a charged complex comprising the Co 3+ and CO 3 2- or HCO 3 - , preferably wherein the neutral complex is sodium tris-carbonatocobalte(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ]*3H 2 O) or potassium tris-carbonatocobalte(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ]*3H 2 O).
  12. A method for attaching a label and/or a carrier to a target molecule comprising the step of incubating the complex of any one of claims 1 to 7 or the composition of claim 8 with a target molecule, wherein the target molecule comprises a protein, peptide or nucleic acid, preferably a protein or DNA, preferably a protein or DNA that can exchange the metal cation ligand in the complex and even more preferably wherein said target molecule comprises at least 4 histidine residues or histidine-like residues in a sequence [H n S m ] k , wherein H is a histidine residue or a histidine-like residue, wherein S is a spacer amino acid residue, wherein n is in each case independently 1 to 4, wherein m is in each case independently 0 to 6, and wherein k is 2 to 6, wherein the method optionally comprises the step of recovering and/or purifying the target molecule with the label and/or carrier linked thereto.
  13. The method of claim 12, wherein the method further comprises washing the complex of the invention in a solution comprising HCO 3 - or CO 3 2- , preferably at a concentration of at least 1 mM, preferably 10 mM and most preferably 1 M, before the incubation and/or wherein the incubation is performed in a solution comprising HCO 3 - or CO 3 2- , preferably in a concentration of at least 1 mM, preferably 10 mM and most preferably 1M.
  14. The method of claim 12 or 13, wherein the incubation is performed in an aqueous solution containing one or more buffer substances selected from the group consisting of: ACES, AMPSO, BES, BisTris, BisTris propane, borate, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, carbonate/bicarbonate buffers, phosphate buffers (e.g. PBS) and Tris, preferably Bis-Tris, MES, HEPES and PIPES.
  15. A labeled or carrier complex-attached target molecule obtainable by the method as defined in any one of claims 12 to 14, wherein the label or carrier complex comprises a) a metal cation; b) a metal cation ligand being CO 3 2- or HCO 3 - ; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier.

Description

The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) a metal cation ligand being CO32- or HCO3- and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and/or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention. In recent years, chemically modified proteins became a very important tool for many biological applications. Various biochemical and cellular techniques in life science research such as fluorescence-based assays, western blot and protein purification rely on labeled or immobilized proteins. But also for the development of biopharmaceuticals (e.g. antibody-drug-conjugates, PEGylation and lipidation), medical diagnostics including biosensors, bioimaging and even medical engineering, protein conjugation is a crucial production step. Thereby in all application fields a simple, stable, site-specific modification method, which does not impede protein function, is desirable. In addition, also other biological molecules such as nucleic acids are often labeled or attached to carriers. In classical labeling/immobilization procedures, small molecules are attached covalently to reactive groups (such as primary amines in lysines or thiols in cysteines) in the unmodified protein of interest via e.g. N-hydroxy-succinimide (NHS) derived reagents or maleimides in a fast and efficient way (Chen and Wu, 2016). However, all these approaches possess intrinsic disadvantages due to the ubiquitous availability of these reactive sites in a protein. Hence, the control of these labeling reactions is limited, resulting in huge batch to batch variability with a lack of site-specificity, inhomogeneous immobilization/labeling stoichiometry and even destabilization and loss of protein functionality (Lindhoud et al., 2012). To address this problem, in recent years several new techniques for site-specific protein conjugation were developed. The most prominent is thereby the Avi-tag system, in which a biotin moiety is attached site-specifically to a short peptide tag by the enzymatic catalysis with biotin ligase BirA (Tirat et al., 2006). But also in other methods, enzymes such as sortase (Popp et al., 2007), transglutaminase (Lin and Ting, 2006), lipoic acid ligase (Fernandez-Suarez, M. et al., 2007) and phospho-pantheinyl-transferases (Yin et al., 2005) are used to catalyze the oriented conjugation of proteins via short recognition peptide tags. Another approach is the expansion of the genetic code through the ribosomal incorporation of bio-orthogonal functional groups such as tetratines, alkynes, azides or norbornenes via non-natural amino acids (Ou et al., 2011; Deiters et al., 2003; Lang et al. 2012). Using specific linking chemistry, the target protein can be modified at the integrated group in a very precise way. Alternatively, to unnatural amino acids, conventional amino acids with a unique reactivity and low abundance on the protein surface can be used for site-specific labeling. Most methods in this context use engineered cysteine substitutions that provide accessible thiol groups for selective targeting (Junutula et al., 2008; Cal et al. 2014). Not only surface exposed, but also N-terminal amino acids can be a useful reaction point for protein conjugation. For example, N-terminal cysteines can be selectively targeted by native chemical ligation with thioester derivates (Dawson et al., 1994) or if incorporated in a short N-terminal tag, the protein can be labeled by perfluoroaromatic reagents (Zhang et al., 2016). Alternatively, proteins can be conjugated site-specifically by the oxidation of N-terminal serine leading to a targetable unique aldehyde group (Gaertner and Offord, 1996) or by the acylation of an N-terminal glycin-histidine tag (Martos-Maldonado et al., 2018). The above described methods have in common that they require specialized molecular tags, non-natural amino acids and/or additional amino acids being incorporated that are not frequently used for other applications. Accordingly, often laborious genetic engineering and re-e