NL2033410B1 - N-terminal protein modification - Google Patents
N-terminal protein modification Download PDFInfo
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- NL2033410B1 NL2033410B1 NL2033410A NL2033410A NL2033410B1 NL 2033410 B1 NL2033410 B1 NL 2033410B1 NL 2033410 A NL2033410 A NL 2033410A NL 2033410 A NL2033410 A NL 2033410A NL 2033410 B1 NL2033410 B1 NL 2033410B1
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- 101800001452 P1 proteinase Proteins 0.000 title description 3
- 230000009145 protein modification Effects 0.000 title description 3
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 250
- 230000021615 conjugation Effects 0.000 claims abstract description 161
- 238000000034 method Methods 0.000 claims abstract description 91
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- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 29
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
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- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 2
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- 108091023037 Aptamer Proteins 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for providing a cargo-conjugated protein (130), wherein the method comprises: a first conjugation stage (510) comprising exposing a protein (100) to a linker (20) to provide a linker-conjugated protein (120), wherein the linker (20) has a structure according to formula (I): ? N / \ R wherein R comprises a first click chemistry group (21) selected from the group comprising a 10 2-azatricyclo[10.4.0.04’9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl moiety, a bicyclo[6.l.0]non-4-yne moiety, an (E)—1-(cyclooct-4-en-1-yloxy) moiety, an azide moiety, a terminal alkyne moiety, a 4-(6-methyl-1,2,4,5-tetrazin-3 -yl)phenyl moiety, a 2- (cyanobenzo[d]thiazol-6-yl)amino moiety, and a 1,2-aminothiol moiety, a second conjugation stage (520) comprising eXposing the linker-conjugated protein (120) to a cargo (30) to provide 15 the cargo-conjugated protein (130), wherein the cargo (30) comprises a second click chemistry group (31), wherein the second click chemistry group (31) is configured to conjugate to the first click chemistry group (21).
Description
N-terminal protein modification
The invention relates to a method for providing a cargo-conjugated protein. The invention further relates to a linker. The invention further relates to a linker-conjugated protein.
The invention additionally relates to a cargo-conjugated protein.
Methods to provide cargo-conjugated proteins are known in the art. For instance,
US10370407B2 relates to methods for preparing a protein conjugate having a defined number of conjugate groups. The method includes: forming a mixture containing a macrocyclic matrix material and a plurality of proteins; eluting the proteins to obtain a first separated protein fraction and a second separated protein fraction, wherein substantially all of the proteins in the first separated protein fraction have the same number of handle moieties; contacting the handle moieties with a conversion reagent under conditions sufficient to convert the handle moieties in the first separated protein fraction to reactive moieties, and contacting the reactive moieties with a conjugation reagent under conditions sufficient to form a plurality of protein conjugates, wherein substantially all of the protein conjugates in the plurality have the same number of conjugate groups. Methods also include recovering enzymes and other proteins from mixtures for isolation and/or reuse of the enzymes and proteins.
Proteins are biochemical workhorses in all living cells. The many thousands of different proteins sustain the functions of the cell, from copying DNA and catalyzing basic metabolism to producing cellular motion and more. For the understanding of biological processes and their (dys)regulation, including diseases, it may be critical to analyze proteins and the protein composition of cells.
A critical step for protein analysis and other biotechnology applications may be protein terminus modification. Proteins may comprise an amino acid sequence with two terminal endings, an amino-terminal amino acid at the N-terminal end and a carboxyl-terminal amino acid at the C-terminal end. For the analysis, immobilization, and purification of proteins, it may be necessary to attach a cargo, such as a DNA oligomer, to the N-terminal end. The attachment of the cargo may require high efficiency for downstream applications.
The prior art may describe N-terminal cargo conjugation methods, but they may typically suffer from low efficiency and/or low specificity. Especially, prior art methods may result in substantial off-target conjugation, i.e., they (also) modify the amine side-chain of lysine amino acids.
The prior art may for instance describe approaches using a 2- pyridinecarboxaldehyde (2PCA) moiety, which may facilitate targeting the N-terminal end of a protein. However, such prior art approaches may yet be relatively slow and/or inefficient in terms of overall conversion efficiency.
Further, prior art methods may be limited in the type of cargo that may be attached to the N-terminal end of the protein, especially in regard to the size and functionality of the type of cargo.
Hence, it is an aspect of the invention to provide an alternative method for providing a cargo-conjugated protein, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
In a first aspect, the invention provides a method for providing a cargo- conjugated protein. In embodiments, the method may comprise a first conjugation stage and a second conjugation stage. The first conjugation stage may comprise exposing a protein to a linker, such as a bifunctional linker, to provide a linker-conjugated protein. Especially, the linker may be configured to conjugate to an amino-terminal (or: “N-terminal”) end of the protein. Further, the linker may have a structure according to formula (I): î
N
UO 0 2 Se N
R
In embodiments, R may comprise a first click chemistry group selected from the group comprising a 2-azatricyclo[10.4.0.0**Jhexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl (also: “dibenzoazacyclooctyne” or “DCBO”) moiety, a bicyclo[6.1.0]non-4-yne (also: “bicyclononyne” or “BCN”) moiety, an (E)-l-(cyclooct-4-en-1-yloxy) (also: “trans- cyclooctene” or “TCO”) moiety, an azide moiety, a terminal alkyne moiety, a 4-(6-methyl- 1,2,4,5-tetrazin-3-yl)phenyl (also: “(methyl)tetrazine” or ‘“tetrazine”) moiety, a 2- (cyanobenzo[d]thiazol-6-yl)amino moiety (also: “2-cyanobenzothiazole” or “CBT”), and a 1,2- aminothiol moiety. The second conjugation stage may comprise exposing the linker-conjugated protein to a cargo to provide the cargo-conjugated protein. In specific embodiments, the cargo may comprise a functionalized cargo and/or a bulky moiety, especially a polymer. The cargo may comprise a second click chemistry group. Further, the second click chemistry group may be configured to conjugate to the first click chemistry group.
Therefore, in embodiments the invention provides a method for providing a cargo-conjugated protein, wherein the method comprises: a first conjugation stage comprising exposing a protein to a linker to provide a linker-conjugated protein, wherein the linker is configured to conjugate to an N-terminal end of the protein, wherein the linker has a structure according to formula (I), wherein R comprises a first click chemistry group selected from the group comprising a DCBO moiety, a BCN moiety, a TCO moiety, an azide moiety, a terminal alkyne moiety, a tetrazine moiety, a CBT moiety, and a 1,2-aminothiol moiety; and a second conjugation stage comprising exposing the linker-conjugated protein to a functionalized cargo to provide the cargo-conjugated protein, wherein the cargo comprises a second click chemistry group, wherein the second click chemistry group is configured to conjugate to the first click chemistry group.
With the present invention, the method for providing a cargo-conjugated protein utilizing a linker with the 2-pyridinecarboxaldehyde (2PCA) moiety structure according to formula (I) may provide a two-step process with a particularly high efficiency and/or specificity for the N-terminal conjugation of proteins. Specifically, the term “2PCA moiety structure according to formula (T)” may herein refer to a structure according to formula (I) without the
R-group. Effectively, the term “2PCA moiety structure according to formula (I)” may thus refer to a moiety based on 6-(1-piperazinylmethyl)-2-pyridinecarboxaldehyde (or “2PPCA"). 2PCA moieties may selectively bind to the N-terminal end of the amino acid sequence of a protein, and may do so with higher efficiency compared to other approaches for N-terminal conjugation described in the prior art. Further, the specific 2PCA moiety structure according to formula (I) comprised by the linker in the present invention may provide the linker with particularly high efficiency, reaction speed, and/or specificity compared to other 2PCA moieties. Additionally, the specific 2PCA moiety structure according to formula (I) in the present invention may facilitate N-terminal conjugation with cargo of relatively small to relatively large molecular weight, and may be particularly suitable for the conjugation of cargo with large molecular weight such as e.g. a bulky moiety, especially a polymer. Hence, the present invention may provide a particularly efficient, fast, and/or specific process for the N-terminal protein modification that may be used in protein analysis and biotechnology applications such as e.g. the structural characterization or sequencing of a protein.
As indicated, the invention provides a method for providing a cargo-conjugated protein. The terms “protein” and “cargo” will be defined first in the following section.
The term “protein” may herein refer to a chain of amino acids of any length, such as a chain of at least 2 amino acids, especially a chain of at least 10 amino acids, more especially a chain of 30 or more amino acids. The term protein may herein thus also refer to a peptide, an oligopeptide, and a polypeptide. In embodiments, the protein may comprise a natural protein, 1.e., a native protein (or peptide) produced by a wildtype organism. Further, the protein may comprise a section of a natural protein, such as obtained after proteolysis. Further yet, the protein may comprise a modified natural protein, such as a protein conjugated with synthetic molecules. Alternatively, the protein may comprise a recombinant protein, i.e, a protein produced by genetically modified organisms. The protein may especially comprise a number of amino acids selected from the range of 2 - 4000, such as 3-2500, especially 5-1000.
In further embodiments, the protein may comprise at most 1000 amino acids, such as at most 500 amino acids, especially at most 100 amino acids, such as at most 50 amino acids. In further embodiments, the protein may comprise at least 2 amino acids, such as at least 3 amino acids, especially at least 5 amino acids, more especially at least 10 amino acids. In further embodiments, the protein may especially comprise a peptide, such as an oligopeptide or polypeptide, and the number of amino acids may be selected from the range of 2 — 60, especially from the range of 2 — 40, such as from the range of 3 — 40.
Proteins may comprise a linear sequence of covalently bound amino acids, beginning on one end of the amino acid sequence at the N-terminal amino acid and ending on the other end of the amino acid sequence at the carboxyl-terminal (or: “C-terminal”) amino acid. Hence, the amino acid sequence begins at an N-terminal end and ends at a C-terminal end.
As essentially all proteins comprise an N-terminal end and a C-terminal end, these may provide suitable general attachment points for biotechnological applications. Herein, the invention provides a selective and effective method for attachment of a cargo to the N-terminal end of a protein.
The term “cargo” may herein refer to any compound that may be relevant to attach to a protein. For instance, the cargo may comprise a bulky moiety, a small-molecule moiety, a polymer, a biopolymer, an oligonucleotide, a peptide, an aptamer, an antibody, a drug, a transcription factor, a purification tag, a nanoparticle, a chromophore, and/or an anchoring tag.
In embodiments, the cargo may comprise a bulky moiety. The bulky moiety may comprise an organic compound with a spatial arrangement of atoms resulting in steric hindrance. Hence, the bulky moiety may comprise an A-value of at least 1.70, such as an A- value of at least 1.80, further an A-value of at least 1.90, especially an A-value of at least 2.00, moreover an A-value of at least 3.00, further an A-value of at least 4.00. The steric hindrance of the bulky moiety may facilitate spatial arrangement of other compounds relative to the bulky 5 moiety. Hence, the protein conjugated to the bulky moiety may be spatially arranged relative to the bulky moiety suitable for use in biotechnological applications.
In further embodiments, the cargo may have a molecular weight of > 200, especially > 500 Da, moreover > 1,000 Da. In further embodiments, the cargo may have a molecular weight of < 50,000 Da, especially < 10,000 Da, moreover < 5,000 Da.
In further embodiments, the cargo, especially the bulky moiety, may comprise a polymer. The polymer may comprise a plurality of repeating subunit compounds (also: “subunits” or “monomers”). A monomer may comprise at least two different locations at which the monomer may be chemically bonded to another monomer. This may facilitate linking the plurality of monomers together such that they form a monomer sequence (or “chain”). Thereby, in a monomer sequence, a first monomer may be chemically bonded on a primary location of the first monomer to a secondary location of a second monomer. In turn, the secondary monomer may be chemically bonded on a primary location of the second monomer to a secondary location of a third monomer. The third monomer may then be chemically bonded on a primary location of the third monomer to a secondary location of a fourth monomer, and so forth.
In embodiments, the polymer may (essentially) comprise the entire monomer sequence.
In embodiments, the monomer sequence may comprise a plurality of chemically identical monomers. The monomer sequence may also comprise a plurality of chemically distinct monomers, i.e., each monomer (comprised by the monomer sequence) may comprise at least one substructure independently selected from a group of different substructures, wherein at least two of the monomers comprise a different substructure.
The monomer sequence may comprise at least two repeating monomers, further at least five monomers, especially at least ten monomers, moreover at least thirty monomers.
In further embodiments, the polymer may especially comprise a biopolymer, i.e., a native organic polymer produced by a wildtype organism. The biopolymer may comprise a polynucleotide, a polypeptide, a polyethylene, or a polysaccharide. More especially, the biopolymer may comprise a DNA polynucleotide, with a DNA oligonucleotide as the monomer. In other embodiments, the biopolymer may comprise an RNA polynucleotide, with an RNA oligonucleotide as the monomer. Hence, in specific embodiments the cargo may comprise a biopolymer, wherein the biopolymer comprises DNA oligonucleotide subunits.
In embodiments, the cargo may be functionalized. The cargo may comprise a tag suitable for selectively arranging a (resulting) cargo-conjugated protein on a target location.
The target location may be comprised by e.g. a surface structure (such as a membrane layer) or a nanoparticulate structure (such as a micelle). In further embodiments, the tag may comprise an antibody. In other embodiments, the cargo may comprise the structure comprising the target location, such as e.g. the surface structure or the nanoparticulate structure. Especially, in such embodiments the cargo may comprise a bulky moiety, such as especially a polymer, providing the structure Comprising the target location. Hence, the cargo may facilitate the arrangement of the cargo-conjugated protein to a target location at the N-terminal end, facilitating structural analysis and/or other (biotechnological) applications.
In embodiments, the functionalized cargo may comprise a tag suitable for detection of a signal. The tag may in certain embodiments be stimulated to send out the signal to be detected. For example, the tag may comprise a chromophore, such as a fluorophore.
Especially, the tag may comprise a fluorescent dye. Hence, the cargo may send out a signal for analysis in biotechnological applications such as structural analysis of a protein.
Various specific examples of (functionalized) cargos are described herein. It will be clear to the person skilled in the art, however, that the invention is not limited to such examples, but may further cover a large variety of other cargo compounds with a variety of other functions.
Herein, the term “cargo-conjugated protein” may refer to a compound comprising at least part of a protein and at least part of a cargo. The cargo-conjugated protein may in embodiments comprise (essentially) the (whole) protein. Further, the cargo-conjugated protein may comprise (essentially) the (whole) cargo. It will be clear to the person skilled in the art that the protein and/or the cargo may be (locally) modified during the conjugation process that provides the cargo-conjugated protein. For example, the N-terminal end of the protein may react with the linker to form a protein-linker conjugate, and thereby one or more atoms of the protein may be removed from or re-arranged in the protein-linker conjugate relative to the protein. Similarly, one or more atoms of the cargo in the resultant cargo- conjugated protein may be removed and/or re-arranged relative to the cargo. Hence, the cargo- conjugated protein may comprise a protein part and a cargo part.
In embodiments, the protein part may comprise at least 90 wt% of the protein, such as at least 95 wt%, especially at least 98 wt%.
In further embodiments, the cargo part may comprise at least 80 wt% of the cargo, such as at least 90 wt%, especially at least 95 wt%.
Such a cargo-conjugated protein may provide a protein part with additional functionalities facilitated by the cargo, e.g. when the cargo part is a chromophore sending out a signal to be detected in a biotechnological application. Such a cargo-conjugated protein may further provide a protein part in a target location, e.g. when the cargo is a structure comprising the target location that presents the protein part for structural analysis or protein activity in a biotechnological application e.g. in embodiments the protein comprises a bi-specific antibody (i.e, an antibody comprising two distinct epitope targets) and the cargo comprises a structure comprising the target location, facilitating efficient exposure of two distinct epitope targets to the bi-specific antibody. In other embodiments the protein comprises an enzyme and the cargo comprises a structure comprising the target location, facilitating exposure of multiple catalytic sites to enhance enzyme activity. Conjugation of a protein to cargo comprising a structure comprising the target location may additionally lead to increased protein stability and/or protein solubility. In all the aforementioned examples, the cargo may alternatively comprise a tag suitable for selectively arranging the cargo-conjugated protein to a target location (comprised by a structure).
Hence, the current invention facilitates the process of providing a cargo- conjugated protein via another compound, a “linker”, that may be able to conjugate to both the protein and the cargo in a predictable and efficient manner. The resultant cargo-conjugated protein may hence comprise (1) a protein part, (ii) a cargo part, and (iii) a linker part, formed as a result of the linker conjugating to both the protein and the cargo. In embodiments, the linker part may be conjugated to both the protein part and the linker part. Especially, the protein part and the cargo part may not be directly conjugated to each other.
Herein, the term “linker” may refer to a compound that facilitates conjugation of a protein to a cargo. The linker may conjugate to a protein or to a cargo. Especially, the linker may be conjugated to a protein and a cargo, and may hence comprise a bifunctional linker. As such, the linker may remain comprised by a (resultant) cargo-conjugated protein in the form of a linker part. In embodiments, the linker may be configured to conjugate to a target location of a protein or a cargo. The linker may especially be configured to conjugate to an N- terminal end of a protein. Further, the linker may be configured to conjugate to a specific spatial arrangement of a cargo, especially when the cargo comprises a bulky moiety such as a biopolymer. Hence, the linker may facilitate conjugation to a target location of a protein and to a target location of a cargo, thereby facilitating a predetermined spatial arrangement of the protein relative to the cargo.
In general, a method to provide a cargo-conjugated protein via use of a linker may comprise at least two steps: (i) the conjugation of a linker (part) with a protein, and (ii) the conjugation of a linker (part) with a cargo. Depending on the method, these two steps may be performed in any order. In embodiments of the present invention, the first step may comprise the conjugation of a linker with a protein to provide a linker-conjugated protein. Herein, this may happen during the first conjugation stage (described further below). The second step may comprise the conjugation of a linker part comprised by a resultant linker-conjugated protein with a cargo to provide a cargo-conjugated protein. Herein, this may happen during the second conjugation stage (described further below). Hence, in embodiments of the present invention, a linker-conjugated protein may be an intermediary form obtained in the process of providing a cargo-conjugated protein, especially after a first conjugation stage. Such a linker-conjugated protein may comprise (i) a protein part and (11) a linker part. These two parts may be formed as a result of the linker conjugating to the protein.
In embodiments of the present invention, the linker may comprise a 2- pyridinecarboxaldehyde (2PCA) moiety. 2PCA moieties have been reported in the prior art, such as in MacDonald JI ef af., “One-step site-specific modification of native proteins with 2- pyridinecarboxyaldehydes”, Nat Chem Biol. 2015 May;11(5):326-31 and Li DZ et al, “N- terminal o-amino group modification of antibodies using a site-selective click chemistry method”, MAbs. 2018 Jul; 10(5):712-719, which are hereby herein incorporated by reference. 2PCA moieties may facilitate conjugation to the N-terminal end of a protein with a relatively high specificity.
Specifically, the linker may comprise a 6-(1-piperazinylmethyl)-2- pyridinecarboxaldehyde (2PPCA) moiety, such as in a structure according to formula (I).
Such linker may provide a particularly high efficiency and/or specificity.
Further, 2PPCA may be a particularly flexible linker suitable for a wider variety of click chemistry approaches for bio-orthogonal cargo attachment compared to other 2PCA moieties.
Q
YN
LN
R
Herein, the R in the structure according to formula (I) may comprise a first click chemistry group. “Click chemistry” comprises a category of chemical reactions between mutually compatible classes of organic moieties, referred to herein as “click chemistry groups”.
Click chemistry groups may be defined by some characteristics; (1) click chemistry groups may be readily conjugated to a wide variety of (organic) compounds on a first position, and (ii) may conjugate in a predictable, selective, and specific manner to a mutually compatible click chemistry group on a second position. Thereby, click chemistry may facilitate the conjugation of at least two (organic) compounds by conjugating at least two (organic) compounds to two mutually compatible click chemistry groups on two respective first positions, that may conjugate to each other in a click chemistry reaction on a second position. Especially, click chemistry reactions may have several characteristics: modularity, insensitivity to solvent parameters, high chemical yields, insensitivity towards oxygen and water, regiospecificity, stereospecificity, bio-orthogonality, non-toxicity, and a large thermodynamic driving force.
Further, click chemistry reactions may in general make use of simple reaction conditions using readily available materials and reagents, and produce little to no byproducts. Hence, click chemistry allows for the predictable and specific conjugation of a wide variety of (organic) compounds to one another via the highly selective conjugation of a first click chemistry group with a compatible second click chemistry group. In the present invention, click chemistry may be utilized for the conjugation of a linker part (in a linker-conjugated protein) to a cargo.
In embodiments, R may comprise a first click chemistry group selected from the group comprising a DCBO moiety, a BCN moiety, a TCO moiety, an azide moiety, a terminal alkyne moiety, a tetrazine moiety, a CBT moiety, and a 1,2-aminothiol moiety. Further, the cargo may comprise a second click chemistry group. The second click chemistry group may especially be configured to conjugate to the first click chemistry group. The second click chemistry group may especially comprise a second click chemistry group selected from the group comprising a DCBO moiety, a BCN moiety, a TCO moiety, an azide moiety, a terminal alkyne moiety, a tetrazine moiety, a CBT moiety, and a 1,2-aminothiol moiety. Hence, the linker (part) may comprise a first click chemistry group and the cargo may comprise a second click chemistry group that may be configured to be compatible in a click chemistry reaction (during the second conjugation stage). The linker-conjugated protein (obtained after the first conjugation stage) may comprise a linker part comprising the first click chemistry group, and is thus configured to conjugate with the cargo that may comprise the second click chemistry group (such as during the second conjugation stage). After the click chemistry reaction between a first click chemistry group (of a linker part comprised by a linker-conjugated protein) and a second click chemistry group (conjugated to a cargo part), the (resultant) cargo-conjugated protein may comprise a first click chemistry group part and a second click chemistry group part. Hence, the (resultant) cargo-conjugated protein may comprise the following parts: (i) a protein part, (ii) a cargo part, (iii) a linker part, (iv) a first click chemistry group part, and (v) a second click chemistry group part.
Compatible combinations of click chemistry groups may be selected from the combinations shown in Table 1. Especially, the first click chemistry group may be selected from either the primary click chemistry group or the secondary click chemistry group. The second click chemistry group may then be selected from a respective compatible other click chemistry group (i.e. a secondary click chemistry group if the first click chemistry group comprises a primary click chemistry group, and vice versa).
Table 1. Compatible combinations of click chemistry groups
Specifically, in embodiments, click chemistry groups may be selected from the list of the compounds in Table 2. For example, the first click chemistry group may comprise a terminal alkyne moiety and the second click chemistry group may comprise an azide moiety.
Further, the first click chemistry group may comprise an azide moiety and the second click chemistry may comprise a DCBO moiety. Additionally, the first click chemistry group may comprise an azide moiety and the second click chemistry group may comprise a BCN moiety.
Further, the first click chemistry group may comprise a tetrazine moiety and the second click chemistry group may comprise a TCO moiety. Additionally, the first click chemistry group may comprise CBT moiety and the second click chemistry group may comprise a 1,2- aminothiol moiety. Any of these click chemistry groups may be possible options for either the first click chemistry group or the second click chemistry group, given that the other click chemistry group is compatible (see Table 1). Hence, for example, the first click chemistry group may comprise an azide moiety and the second click chemistry group may comprise a terminal alkyne moiety. Further, the first click chemistry group may comprise a DCBO moiety and the second click chemistry may comprise an azide moiety. Additionally, the first click chemistry group may comprise a BCN moiety and the second click chemistry group may comprise an azide moiety. Further, the first click chemistry group may comprise a TCO moiety and the second click chemistry group may comprise a tetrazine moiety. Additionally, the first click chemistry group may comprise 1,2-aminothiol moiety and the second click chemistry group may comprise a CBT moiety. While the aforementioned compatible combinations of click chemistry groups may be especially relevant for the present invention, it will be clear to the skilled person that the method of the invention is not limited to the compatible combinations of click chemistry groups mentioned in table 2.
Table 2. Click chemistry groups, click chemistry group types, and number of ethylene glycol residues.
Ethylene
Type Click chemistry group glycol residues
DCBO 4-(2-azatricyclo[10.4.0.0%"Thexadeca-1(16),4,6,8,12,14- anime"
DCBO 6-(2-azatricyclo[10.4.0.0*°Jhexadeca-1(16),4,6,8,12,14-
Ee
DCBO 3-(2-(2-(4-(2-azatricyclo[10.4.0.0**Jhexadeca- moiety 1(16),4,6,8,12,14-hexaen-10-yn-2-y1)-4- 2 oxobutanamido)ethoxy ethoxy )propanoyl
DCBO 3-(2-(2-(6-(2-azatricyclo[10.4.0.0*'Thexadeca- moiety 1(16),4,6,8, 12,14-hexaen-10-yn-2-yl})-6- 2 oxohexanamido)ethoxy)ethoxy)propanoyl
DCBO 20-(2-azatricyclo[10.4.0.0**Jhexadeca-1(16),4,6,8,12,14- moiety hexaen-10-yn-2-yl)-17,20-dioxo-4,7,10,13-tetraoxa-16- 4 azaicosanoyl
DCBO 22-(2-azatricyclo[10.4.0.0*°Jhexadeca-1(16),4,6,8,12,14- moiety hexaen-10-yn-2-yl)-19,22-dioxo-4,7,10,13-tetraoxa-16- 4 azadocosanoyl me ensen 0 / 2-(4-(6-methyl-1,2 4, 5-tetrazin-3-yl)phenyl)acetyl moiety
Tetrazine | 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12,15-
Ethylene
Type Click chemistry group glycol residues
Tetrazine | 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-oxo- ; moiety 6,9,12,15,18-pentaoxa-3-azahenicosan-2 1-oyl )
Tetrazine | 1-(4-(6-methyl-1,2 4, 5-tetrazin-3-yl)phenyl)-2-oxo- moiety 6,9,12,15,18,21,24,27-octaoxa-3-azatriacontan-30-oyl (E)-2-(cyclooct-4-en-1-yloxy)carbonyl 0
TCO (E)-1-(eyclooct-4-en-1-yloxy)-1-0x0-5,8, 11, 14-tetraoxa-2- 4 azaheptadecan-17-oyl
Azide 2-azidoacetyl moiety
Azide 3-azidopropanoyl moiety
Azide 4-azidobutyroyl moiety
Azide / 3-(2-azidoethoxy)propanoyl 1 moiety
Azide / 3-(2-(2-azidoethoxy)ethoxy)propanoyl 2 moiety
Azide / 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoyl 3 moiety
Azide1-azido-3,6,9,12-tetraoxapentadecan-15-oyl 4 moiety
Terminal alkyne 4-pentynoyl moiety
Terminal alkyne 5-hexynoyl moiety
Terminal alkyne 4,7,10, 13-tetraoxahexadec-15-ynoyl 3 moiety
Ethylene
Type Click chemistry group glycol residues
Terminal alkyne 4,7,10,13,16-pentaoxanonadec-18-ynoyl 4 moiety
Terminal alkyne 4,7,10,13,16, 19-hexaoxadocos-21-ynoyl 5 moiety ie [omnis 2-(cyanobenzo[d]thiazol-6-yl)amino moiety 1,2- aminothiol | (S)-2-amino-3-mercaptopropanol moiety
Ee OO / (bicyclo[6. 1.0]non-4-yn-9-ylmethoxy)carbonyl moiety
Hence, in embodiments, the first click chemistry group may comprise a DBCO moiety, especially a DBCO moiety selected from the group comprising 4-(2- azatricyclo[10.4.0.0* Thexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl, 6-(2- azatricyclo[10.4.0.0*°]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-6-oxocaproyl, 3-(2-(2- (4-(2-azatricyclo[10.4.0.0*"Thexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4- oxobutanamido)ethoxy)ethoxy)propanoyl, 3-(2-(2-(6-(2-azatricyclo[10.4.0.0%[hexadeca- 1(16),4,6,8, 12, 14-hexaen-10-yn-2-yl)-6-oxohexanamido)ethoxy)ethoxy)propanoyl, 20-(2-aza- tricyclo[10.4.0.0%?]hexadeca-1(16),4,6,8, 12, 14-hexaen-10-yn-2-yl1)-17,20-diox0-4,7,10,13- tetraoxa-16-azaicosanoyl, and 22-(2-azatricyclo[10.4.0.0%’Jhexadeca-1(16),4,6,8,12,14- hexaen-10-yn-2-y1)-19,22-diox0-4,7,10,13-tetraoxa-16-azadocosanoyl.
Similarly, in embodiments, the second click chemistry group may comprise a
DBCO moiety, especially 4-(2-azatricyclo[10.4.0.0+*Jhexadeca-1(16),4,6,8, 12, 14-hexaen-10- yn-2-yl)-4-oxobutanoyl, 6-(2-azatricyclo[10.4.0.0*9]hexadeca-1(16),4,6,8, 12,14-hexaen-10- yn-2-yl)-6-oxocaproyl, 3-(2-(2-(4-(2-azatricyclo[10.4.0.0*"Thexadeca-1(16),4,6,8,12,14- hexaen-10-yn-2-yl)-4-oxobutanamido)ethoxy)ethoxy)propanoyl, 3-(2-(2-(6-(2-azatricyclo- [10.4.0.0**Jhexadeca-1(16),4,6,8, 12, 14-hexaen-10-yn-2-yl)-6- oxohexanamido)ethoxy)ethoxy)propanoyl, 20-(2-azatrieyclo[10.4.0.0*°Jhexadeca-
1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-17, 20-dioxo0-4,7,10, 13 -tetraoxa-16-azaicosanoyl, and 22-(2-azatricyclo[10.4.0.0*?Jhexadeca-1(16),4,6,8, 12, 14-hexaen-10-yn-2-yl)-19,22-dioxo- 4,7,10,13-tetraoxa-16-azadocosanoyl.
Hence, in embodiments, the first click chemistry group may comprise a TCO moiety, especially a TCO moiety selected from the group comprising (E)-2-(cyclooct-4-en-1- yloxy)carbonyl and (E)-1-(cyclooct-4-en-1-yloxy)-1-0x0-5,8, 11, 14-tetraoxa-2-azaheptadecan- 17-0yl.
Similarly, in embodiments, the second click chemistry group may comprise a
TCO moiety, especially (E)-2-(cyclooct-4-en-1-yloxy)carbonyl and (E)-1-(cyclooct-4-en-1- yloxy)-1-ox0-5,8,11,14-tetraoxa-2-azaheptadecan-17-oyl.
Hence, in embodiments, the first click chemistry group may comprise an azide moiety, especially an azide moiety selected from the group comprising 2-azidoacetyl, 3- azidopropanoyl, 4-azidobutyroyl, 3-(2-azidoethoxy)propanoyl, 3-(2-(2-azidoethoxy)ethoxy)- propanoyl, 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoyl and 1-azido-3,6,9,12-tetra- oxapentadecan-15-oyl.
Similarly, in embodiments, the second click chemistry group may comprise an azide moiety, especially 2-azidoacetyl, 3-azidopropanoyl, 4-azidobutyroyl, 3-(2- azidoethoxy)propanoyl, 3-(2-(2-azidoethoxy)ethoxy)propanoyl, 3-(2-(2-(2-azidoethoxy)- ethoxy)ethoxy)propanoyl and 1-azido-3,6,9,12-tetraoxapentadecan-15-oyl.
Hence, in embodiments, the first click chemistry group may comprise a terminal alkyne moiety, especially a terminal alkyne moiety selected from the group comprising 4- pentynoyl, S-hexynoyl, 4,7,10,13-tetraoxahexadec-15-ynoyl, 4,7,10,13,16-pentaoxanonadec- 18-ynoyl and 4,7,10,13,16,19-hexaoxadocos-21-ynoyl.
Similarly, in embodiments, the second click chemistry group may comprise a terminal alkyne moiety, especially 4-pentynoyl, 5-hexynoyl, 4,7,10,13-tetraoxahexadec-15- ynoyl, 4,7,10,13,16-pentaoxanonadec-18-ynoyl and 4,7,10,13,16,19-hexaoxadocos-21-ynoyl.
Hence, in embodiments, the first click chemistry group may comprise a tetrazine moiety, especially a tetrazine moiety selected from the group comprising 2-(4-(6-methyl- 1,2,4,5-tetrazin-3-yl)phenyl)acetyl, 1-(4-(6-methyl-1,2 4,5-tetrazin-3-yl)phenyl)-2-oxo- 6,9,12,15-tetraoxa-3-azaoctadecan-18-oyl, 1-(4-(6-methyl-1,2 4, 5-tetrazin-3-yl)phenyl)-2- 0x0-6,9,12,15, 18-pentaoxa-3-azahenicosan-21-oyl, and 1-(4-(6-methyl-1,2 4 5-tetrazin-3- yl)phenyl)-2-0x0-6,9,12,15,18,21,24,27-octaoxa-3-azatriacontan-30-oyl.
Similarly, in embodiments, the second click chemistry group may comprise a tetrazine moiety, especially 2-(4-(6-methyl-1,2 4 5-tetrazin-3-yl)phenyl)acetyl, 1-(4-(6-
methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12, 1 5-tetraoxa-3-azaoctadecan-18-oyl, 1-(4- (6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12,15,18-pentaoxa-3-azahenicosan-2 1 -oyl, and 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12,15,18,21,24,27-octaoxa-3-aza- triacontan-30-oyl.
Hence, in embodiments, the first click chemistry group may comprise a CBT moiety, especially 2-(cyanobenzo[d]thiazol-6-yl)amino. Similarly, in embodiments, the second click chemistry group may comprise a CBT moiety, especially 2-(cyanobenzo[d]thiazol-6- yl)amino.
Hence, in embodiments, the first click chemistry group may comprise a 1,2- aminothiol moiety, especially (S)-2-amino-3-mercaptopropanol. Similarly, in embodiments, the second click chemistry group may comprise a 1,2-aminothiol moiety, especially (S)-2- amino-3-mercaptopropanol.
Hence, in embodiments, the first click chemistry group may comprise a BCN moiety, especially (bicyclo[6. 1.0]non-4-yn-9-ylmethoxy)carbonyl. Similarly, in embodiments, the second click chemistry group may comprise a BCN moiety, especially (bicyclo[6. 1.0]non- 4-yn-9-ylmethoxy)carbonyl. Click chemistry compounds (see Table 2) may comprise ethylene glycol residues, especially oligoethylene glycol residues [-O[CH2CH20]x-]. A click chemistry compound may comprise n ethylene glycol residues. In embodiments, n may be selected from the range of 0-10, such as from the range of 1-8. Especially, n > 1, such as , n > 2 especially, n > 4. Oligoethylene glycol residues comprised by click chemistry groups may serve to enhance water solubility of the linker and/or cargo, and the (resultant) cargo-conjugated protein. The higher the water solubility, the smaller proportion of an organic solvent may be needed for the click chemistry reaction. This may be particularly beneficial as high proportions of organic solvent may lead to protein precipitation or denaturation. Most especially, n = 4 which may provide an optimal balance in linker length and water solubility.
Especially, in embodiments, the first click chemistry group may comprise a
DCBO moiety and the second click chemistry group may comprise an azide moiety.
Alternatively, the second click chemistry group may comprise a DCBO moiety and the first click chemistry group may comprise an azide moiety. In other embodiments of the present invention, the first click chemistry group may comprise a TCO moiety and the second click chemistry group may comprise a tetrazine moiety. Alternatively, the second click chemistry group may comprise a TCO moiety and the first click chemistry group may comprise a tetrazine moiety.
In embodiments, the method may comprise a first conjugation stage and a second conjugation stage. The first conjugation stage may primarily comprise conjugating the linker to the protein to provide a linker-conjugated protein. The second conjugation stage may primarily comprise conjugating the cargo to the linker-conjugated protein to provide the cargo- conjugated protein. The term “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method. The first conjugation stage and the second conjugation stage may especially be temporally separated, wherein the second conjugation stage is temporally arranged after the first conjugation stage. Hence, the method may provide a method for the conjugation of a cargo to a protein using a linker to provide a cargo-conjugated protein.
Certain embodiments may comprise further stages beyond the two conjugation stages as described above, such as a linker preparation stage, a quenching stage, a purification stage, and a buffer exchange stage. It may be clear to the skilled person that any of such additional stages may be combined within an embodiment of the method as described herein.
In embodiments, the method may comprise a linker preparation stage. In particular, the linker may be provided for the first conjugation stage during the linker preparation stage. Especially, the linker preparation stage may precede the first conjugation stage. The linker preparation stage may comprise reacting a first reactant and a second reactant to provide the linker. In such embodiments where the linker comprises a structure according to formula (I), the first reactant may especially comprise 6-(piperazin-1-ylmethyl)2- pyridinecarboxaldehyde. Further, the second reactant may especially comprise NHS-R, where
R may be selected from the group of first click chemistry groups described above. Hence, the linker preparation stage provides a linker comprising the 2PCA moiety structure according to formula (I) and a first click chemistry group.
In embodiments comprising the linker preparation stage, the linker preparation stage may be performed under predetermined linker preparation conditions, i.e. reactant concentration, reaction time and reaction temperature (or “linker preparation temperature”).
The linker preparation stage may comprise providing, especially combining, the first reactant and the second reactant at a mole ratio selected from the range of 5:1 - 1:125, such as selected from the range of 2:1 — 1:50, especially selected from the range of 1:1 - 1:25, moreover selected from the range of 1:2 — 1:15, such as at (about) 1:5. Such a suitable mole ratio of first reactant to second reactant may provide a high efficiency and high yield of the linker preparation stage, especially in embodiments with an excess of second reactant over first reactant. Thereby, after the linker preparation stage there may be an excess of unconjugated second reactant (i.e. NHS-
R).
Further, the linker preparation stage may have a linker preparation duration selected from the range of 2 — 96 hours, such as selected from the range of 6 — 72 hours, especially selected from the range of 12 — 48 hours, moreover selected from the range of 18 — 36 hours, such as at (about) 24 hours. Such a linker preparation duration may provide a suitable time-period for the first reactant and the second reactant to react and provide the linker at a high yield.
Further, the linker preparation stage may comprise exposing the first reactant and the second reactant to a reaction temperature selected from the range of 15 — 60 °C, such as selected from the range of 22 — 50 °C, especially selected from the range of 30 — 40 °C, moreover selected from the range of 35 — 40 °C, such as at (about) 37 °C. Such a reaction temperature may provide a suitable temperature at which the reaction between the first reactant and the second reactant may occur with high efficiency and with limited, especially without, degradation of the reactants or the (resultant) linker.
Hence, the predetermined linker preparation conditions during the linker preparation stage may be optimized for providing the linker at high efficiency and yield. For example, the linker preparation stage may in an embodiment comprise providing, especially combining, the first reactant and the second reactant at a mole ratio of 1:5, with a linker preparation duration of 24 hours, and at a reaction temperature of 37 °C.
In further embodiments, the linker preparation stage may be performed in a linker preparation solution. Along with a first reactant and a second reactant, a linker preparation solution may comprise further compounds facilitating the reaction between the first reactant and the second reactant, i.e. an organic solvent, an organic base, and water. The linker preparation stage may hence comprise providing the first reactant and the second reactant in an organic solvent. The organic solvent may be selected from the group comprising dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dichloromethane, chloroform, tetrahydrofuran (THF), and 1,4-dioxane. The organic solvent may especially comprise DMSO. The linker preparation stage may further comprise providing the first reactant and the second reactant in the presence of an organic base. The organic base may especially comprise triethylamine.
Especially, the linker preparation stage may comprise providing the first reactant and the second reactant in an organic solvent (such as DMSO) in the presence of an organic base (such as triethylamine).
Further, the second reactant and the organic base may be provided in a mole ratio selected from the range of 10:1 — 1:40, such as selected from the range of 7:1 — 1:28, especially selected from the range of 4:1 — 1:16, moreover selected from the range of 2:1 — 1:8,
such as at (about) 1:2. Thereby, the linker preparation solution may comprise a base solution that facilitates the reaction between the first reactant and the second reactant. Hence, the organic solvent and the organic base provide further linker preparation conditions (e.g., a mole ratio of second reactant to organic base) facilitating the linker preparation stage to provide the linker in a linker preparation solution.
In specific embodiments, the organic solvent may be provided during the linker preparation stage at a range of 0 — 100 vol% of the linker preparation solution. In specific embodiments, (essentially) no organic solvent may be provided. In general, in embodiments, the organic solvent may be provided at a range of 0.1 — 100 vol%, like 10 — 90 vol%, especially 15-85 vol%, moreover 25 — 75 vol%. In embodiments, the organic solvent may comprise >10 vol% of the linker preparation solution, such as >15 vol%, especially >20 vol%, moreover >25 vol%. In embodiments, the organic solvent may be provided at a range of 10 mM -100 mM in the linker preparation solution, such as a range of 20 mM — 90 mM, especially a range of 25 mM — 70 mM, moreover a range of 30 mM — 60 mM.
Hence, in embodiments, the linker preparation stage may comprise providing a mixture of the first reactant, the second reactant, the organic base, the water, and optionally the solvent. In further embodiments, the mixture may comprise at least 10 vol% water, such as at least 15 vol% water, especially at least 25 vol%, such as at least 50 vol% water. In further embodiments, the mixture may comprise at least 60 vol% water, such as at least 70 vol%, especially at least 80 vol%. In further embodiments, the mixture may comprise at most 99 vol% water, such as at most 95 vol%, especially at most 90 vol%.
Especially, the second reactant may comprise n ethylene glycol residues selected from the range of n = 1 — n = 8, especially from the range of n = 1 — n= 5. In further embodiments, especially n > 1, such as n > 2, moreover n > 4. More especially, the second reactant may comprise the DCBO or the TCO moiety. Such embodiments with the provided click chemistry groups and linker preparation conditions may be particularly suitable for providing a linker in a linker preparation solution suitable for the present invention, as the linker may be provided at high yield with (relatively) low amounts of organic solvent. Such embodiments may be beneficial as the organic solvent may be detrimental to the (later stages of the) method for providing the cargo-conjugated protein. The organic solvent may be diluted to <25 vol% after the linker preparation duration, such as <20 vol%, especially <15 vol®%, moreover <10 vol%.
In embodiments, the method may further comprise a quenching stage. The quenching stage may especially follow the linker preparation stage. The quenching stage may precede the first conjugation stage. The quenching stage may comprise exposing the linker to a quenching base. The quenching base may hydrolyze the excess unconjugated NHS-R esters remaining in the linker preparation solution after the linker preparation stage. Thereby, the excess unconjugated NHS-R esters may no longer be available for reacting with other amine groups, such as on a protein or a cargo. Especially, the quenching stage may comprise exposing the linker in a linker preparation solution to a quenching base, i.e., the quenching stage may comprise adding a quenching base to the linker preparation solution. The quenching base may be selected from the group comprising dimethylamine, diethylamine, sodium hydroxide, and potassium hydroxide, especially diethylamine. The quenching base may in other embodiments comprise at least two or more from the group comprising dimethylamine, diethylamine, sodium hydroxide, and potassium hydroxide, especially sodium hydroxide and potassium hydroxide.
The quenching stage may be performed under quenching conditions, i.e, a quenching base molarity and a quenching duration. The quenching stage may comprise providing the quenching base at a molarity selected from the range of 0.005 — 2 M, such as from the range of 0.01 — 1 M, especially from the range of 0.02 — 0.5 M, moreover from the range of 0.05 — 0.2 M, such as at about 0.1 M. Thereby the quenching base may be provided in sufficient amount to quench (essentially) all remaining unconjugated NHS-R esters from the linker preparation solution.
The quenching stage may have a quenching duration selected from the range of 1-24 hours, such as from the range of 2 — 18 hours, especially from the range of 3 — 12 hours, moreover from the range of 4 — 9 hours, such as (about) 6 hours. Such a quenching duration may provide sufficient time for the quenching base to quench (essentially) all remaining unconjugated NHS-R esters from the linker preparation solution.
Hence, the quenching base may hydrolyze and thereby quench (essentially) all (remaining) unconjugated second reactant, especially from a linker preparation solution, such that they will no longer be available for reacting with other amine groups, such as on a protein or a cargo. Thereby the linker may be provided in a quenched solution suitable for use in later stages of the present method that may comprise exposure to a protein or a cargo.
In embodiments, the method may further comprise a purification stage. The purification stage may especially follow the linker preparation stage. In certain embodiments, the purification stage may follow the quenching stage (following the linker preparation stage).
The purification stage may precede the first conjugation stage. The purification stage may comprise subjecting the linker (in a linker preparation solution or quenched solution) to a chromatography method.
Especially, the chromatography method may comprise liquid chromatography.
Liquid chromatography is an established method known to the skilled person for separating the various compounds in a liquid mixture (such as the linker preparation solution or quenched solution) employing the principle of adsorption. Different molecules in the liquid mixture may flow at different speeds across a solid medium with specific characteristics for the adsorption of the desired compound. Thus liquid chromatography may be used to separate different molecules based on their flow rate. More especially, the chromatography method may comprise high-performance liquid chromatography (HPLC). In HPLC, the liquid mixture may be pressurized for improved efficiency and yield.
The chromatography method may purify the linker to provide a purified linker in a purified solution. Thereby the linker may be available for the first conjugation stage in a purified solution, with little to no contamination by other compounds from the linker preparation stage or quenching stage. After the purification stage, the first conjugation stage may comprise exposing the protein to the purified linker.
Hence, in embodiments, one or more stages may precede the first conjugation stage to provide the linker comprising the desired first click chemistry group in a solution suitable for conjugating with a protein. The linker may thereby be provided in a linker preparation solution, quenched solution, or a purified solution. Specific embodiments may comprise yet further stages for modification of the linker or any of the solutions comprising the linker. Thereby the linker is provided for use in the first conjugation stage.
In embodiments, the first conjugation stage may comprise exposing a protein to the linker (in a linker preparation solution, a quenched solution, or a purified solution) to provide a linker-conjugated protein (in a first conjugation solution). The first conjugation stage may especially be performed under first conjugation conditions, such as ,i.e., a mole ratio of a protein to a linker, a first conjugation duration, and a first conjugation temperature. The protein and the linker may be provided in a buffered solution, such as a buffered saline solution, especially a phosphate buffered saline solution (PBS). A buffered solution may prevent the pH of the solution from (substantially) changing over the course of the first conjugation stage.
The protein and the linker may be provided at a mole ratio selected from the range of 1:10 — 1:5000, such as from the range of 1:50 — 1:2000, especially from the range of 1:100 - 1:1000, moreover from the range of 1:200 — 1:700, such as at (about) 1:400. Such a mole ratio may provide an excess of linker to protein. Such a mole ratio may result in a high efficiency and high yield of the first conjugation stage. Further, a linker may (in general) be more cost-efficient to produce or procure compared to a protein.
The first conjugation stage may further comprise a first conjugation duration selected from the range of 2 — 96 hours, such as selected from the range of 6 — 72 hours, especially selected from the range of 12 — 48 hours, moreover, selected from the range of 18 — 36 hours, such as at (about) 24 hours. Such a conjugation duration may provide a suitable time- period for the linker and the protein to react and provide the linker-conjugated protein at a high yield. The first conjugation stage may in specific embodiments be performed under mixing conditions for at least part of the first conjugation duration, especially for the entire first conjugation duration. Mixing conditions may comprise e.g. shaking, stirring, blending, or agitating of the first conjugation solution. Mixing conditions may comprise (essentially) continuous mixing of the first conjugation solution. Mixing conditions may be selected (via e.g. rotations per minute of a magnetic stirring bead or a platform shaker) such that the protein and the linker are efficiently exposed to each other in the first conjugation solution. Mixing conditions may further improve the efficiency and yield of the first conjugation stage.
The first conjugation stage may further comprise exposing the linker and the protein to a first conjugation temperature selected from the range of 15 — 60 °C, such as selected from the range of 22 — 50 °C, especially selected from the range of 30 — 40 °C, moreover selected from the range of 35 — 40 °C, such as at (about) 37 °C. Such a first conjugation temperature may be suitable for conjugating the linker and the protein at high efficiency without degradation of the linker, the protein, or the (resultant) linker-conjugated protein.
Hence, the predetermined first conjugation conditions during the first conjugation stage may be selected for providing the linker-conjugated protein at high efficiency and yield. For example, the first conjugation stage may in an embodiment comprise providing a protein and a linker in PBS at a mole ratio of 1:400, for a first conjugation duration of 24 hours under shaking conditions at a first conjugation temperature of 37 °C. Hence, the first conjugation stage may provide the linker-conjugated protein, especially in a first conjugation solution.
Following the first conjugation stage, the (resultant) linker-conjugated protein may have a structure according to formula (II):
R2 may herein comprise a protein. R2 may especially comprise a protein part (that has been conjugated at the N-terminal end). R3 may herein comprise a first click chemistry group (as described above). R3 may especially comprise a first click chemistry group part. The main depicted chemical structure according to formula (II) may comprise the linker part that has been conjugated to both the protein (part) and the first click chemistry group.
In further embodiments, the method may further comprise a buffer exchange stage following the first conjugation stage. The buffer exchange stage may in certain embodiments precede the second conjugation stage. The buffer exchange stage may comprise removing the excess (remaining) unconjugated linker (from the first conjugation solution). The buffer exchange stage may comprise a buffer exchange method to provide the linker-conjugated protein. Especially, the buffer exchange stage may comprise subjecting linker-conjugated protein in a first conjugation solution to a buffer exchange method.
The buffer exchange method may be selected from the group consisting of desalting column chromatography and dialysis. Desalting column chromatography is a type of chromatography based on size exclusion chromatography, thereby filtering compounds in a liquid mixture (i.e., the first conjugation solution) based on molecular size. Dialysis filters compounds in a liquid mixture (i.e., the first conjugation solution) based on their diffusion rate through a semipermeable membrane separating compartments with different ionic contents.
Hence, the buffer exchange stage may comprise providing the linker-conjugated protein in a filtered first conjugation solution. In such a filtered first conjugation solution, (essentially) all excess unconjugated linker remaining in the first conjugation solution may be removed during the buffer exchange stage. Such excess unconjugated linker in the first conjugation solution may otherwise be conjugated to a cargo during the second conjugation stage. Hence, in embodiments comprising a buffer exchange stage, the linker-conjugated protein may be provided for the second conjugation stage in a filtered first conjugation solution.
In embodiments, the first conjugation stage (and optionally a buffer exchange stage) may be followed by a second conjugation stage. The second conjugation stage may comprise exposing the linker-conjugated protein (in either a first conjugation solution or a filtered first conjugation solution) to a cargo to provide the cargo-conjugated protein (in a second conjugation solution). The second conjugation stage may especially be performed under second conjugation conditions, such as ‚i.e, a mole ratio of a cargo to a linker-conjugated protein, a second conjugation duration, and a second conjugation temperature.
The cargo and the linker-conjugated protein may be provided at a mole ratio selected from the range of 1:2 — 40:1, especially from the range of 1:1 — 20:1, moreover from the range of 2:1 — 10:1, such as at (about) 5:1 of cargo to linker-conjugated protein. In particular, the cargo and the linker-conjugated protein may be provided at a mole ratio of > 5:1, especially > 10:1, moreover > 15:1. Such a mole ratio may result in a high efficiency and high yield of the second conjugation stage, especially in embodiments with an excess of cargo over linker-conjugated protein. Further, the cargo may be more cost-efficient and easy to produce or procure compared to the linker-conjugated protein.
The second conjugation stage may comprise a second conjugation duration selected from the range of 2 — 96 hours, such as selected from the range of 6 — 72 hours, especially selected from the range of 12 — 48 hours, moreover selected from the range of 18 — 36 hours, such as at (about) 24 hours. Such a second conjugation duration may provide a suitable time-period for the cargo and the linker-conjugated protein to conjugate and provide the cargo-conjugated protein at a high yield.
The second conjugation stage may comprise a second conjugation temperature selected from the range of 2 — 50 °C, such as selected from the range of 5 — 40 °C, especially selected from the range of 10 — 30 °C, moreover selected from the range of 15 — 25 °C, such as at (about) 20 °C. Such a second conjugation temperature may be suitable for the conjugation between the cargo and the linker-conjugated protein at high efficiency without degradation of the cargo, the linker-conjugated protein, or the (resultant) cargo-conjugated protein.
Hence, the predetermined second conjugation conditions during the second conjugation stage may be optimized for providing the cargo-conjugated protein at high efficiency and yield. For example, in embodiments the second conjugation stage may provide the cargo and the linker-conjugated protein at a mole ratio of 5:1 for a second conjugation duration of 24 hours at a second conjugation temperature of 20 °C. Hence, the second conjugation stage may provide the cargo-conjugated protein, especially in a second conjugation solution.
The cargo-conjugated protein obtained during the second conjugation stage may comprise a structure according to formula (III):
SR ow Bong
Herein R2 may comprise a protein. R2 may especially comprise a protein part, conjugated to the structure according to formula (II) at the N-terminal end of the protein. Herein
R4 may comprise (1) a first click chemistry group part, (i1) a second click chemistry group part, and (iii) a cargo, especially a cargo part conjugated to the second click chemistry group part.
Hence, all components of the cargo-conjugated protein may be represented herein (as described above): (1) a protein part, (11) a cargo part, (ii1) a linker part, (iv) a first click chemistry group part, and (v) a second click chemistry group part. The main depicted chemical structure according to formula (III) may comprise the linker part that has been conjugated to both the protein (part) as R2 and the first click chemistry group as part of R4. Especially, the linker part comprised by the structure according to formula (IIT) may be directly conjugated to the first click chemistry group part comprised by R4 in the structure according to formula (III). The first click chemistry group part may be conjugated to the second click chemistry group part, especially on a different site from where the first click chemistry group part is conjugated to the structure according to formula (III). The second click chemistry group part may be conjugated to the cargo part on a different site from where the second click chemistry group part is conjugated to the first click chemistry group part.
In a further aspect, the invention provides a linker having a structure according to formula (I), especially where R is selected from the list of moieties of Table 2: 7 rm
EN
‘R
Hence, in embodiments, R may comprise a DBCO moiety, especially a DBCO moiety selected from the group comprising 4-(2-azatricyclo[10.4.0.0%?]hexadeca- 1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl, 6-(2-azatricyclo[10.4.0.0*Thexadeca- 1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-6-oxocaproyl, 3-(2-(2-(4-(2-azatricyclo- [10.4.0.0*Thexadeca-1(16),4.6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanamido)ethoxy)- ethoxy)propanoyl, 3-(2-(2-(6-(2-azatricyclo[10.4.0.0* Thexadeca-1(16),4.6,8,12,14-hexaen- 10-yn-2-yl)-6-oxohexanamido)ethoxy ethoxy )propanoyl, 20-(2-azatricyclo[10.4.0.0%?]- hexadeca-1(16),4,6,8,12, 14-hexaen-10-yn-2-yl1)-17,20-diox0-4,7, 10, 13-tetraoxa-16- azaicosanoyl, and 22-(2-azatricyclo[ 10.4.0.0*°Jhexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2- y1)-19,22-dioxo-4,7, 10, 1 3-tetraoxa-16-azadocosanoyl,
Further, in embodiments, R may comprise a TCO moiety, especially a TCO moiety selected from the group comprising (E)-2-(cyclooct-4-en-1-yloxy)carbonyl and (E)-1- (cyclooct-4-en-1-yloxy)-1-ox0-5,8,11, 14-tetraoxa-2-azaheptadecan-17-oyl.
Moreover, in embodiments, R may comprise an azide moiety, especially 2- azidoacetyl, 3-azidopropanoyl, 4-azidobutyroyl, 3-(2-azidoethoxy)propanoyl, 3-(2-(2- azidoethoxy)ethoxy)propanoyl, 3-(2-(2-(2-azidoethoxy)ethoxy ethoxy )propanoyl and 1-azido- 3,6,9, 12-tetraoxapentadecan-15-oyl.
Additionally, in embodiments, R may comprise a terminal alkyne moiety, especially a terminal alkyne moiety selected from the group comprising 4-pentynoyl, 5- hexynoyl, 4,7,10,13-tetraoxahexadec-15-ynoyl, 4,7,10,13,16-pentaoxanonadec-18-ynoyl and 4,7,10,13,16,19-hexaoxadocos-2 1 -ynoyl.
Hence, in embodiments, R may comprise a tetrazine moiety, especially a tetrazine moiety selected from the group comprising 2-(4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenylacetyl, 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12,15-tetraoxa-3- azaoctadecan-18-oyl, 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-0x0-6,9,12,15,18- pentaoxa-3-azahenicosan-21-oyl, and 1-(4-(6-methyl-1,2 4, 5-tetrazin-3-yl)phenyl)-2-oxo- 6,9,12,15,18,21,24,27-octaoxa-3-azatriacontan-30-oyl.
Hence, in embodiments, R may comprise a CBT moiety, especially 2- (cyanobenzo[d]thiazol-6-yl)amino.
Hence, in embodiments, R may comprise a 1,2-aminothiol moiety, especially (S)-2-amino-3-mercaptopropanol.
Hence, in embodiments, R may comprise a BCN moiety, especially (bicyclo[6. 1.0]non-4-yn-9-ylmethoxy)carbonyl.
Such a linker may be used for an N-terminal conjugation with a protein, such as in the present invention. Such a linker may additionally be utilized for a click chemistry reaction with another click chemistry group. Especially, the other click chemistry group may be compatible to the click chemistry group comprised by R, with viable combinations listed in
Table 1.
For example, R may comprise a terminal alkyne moiety and the other click chemistry group may comprise an azide moiety. Further, R may comprise an azide moiety and the other click chemistry may comprise a DCBO moiety. Additionally, R may comprise an azide moiety and the other click chemistry group may comprise a BCN moiety. Further, R may comprise a tetrazine moiety and the other click chemistry group may comprise a TCO moiety.
Additionally, R may comprise a CBT moiety and the other click chemistry group may comprise a 1,2-aminothiol moiety.
Any of these click chemistry groups may be possible options for either R or the other click chemistry group, given that the two click chemistry groups are compatible (see
Table 1). Hence, for example, R may comprise an azide moiety and the other click chemistry group may comprise a terminal alkyne moiety. Further, R may comprise a DCBO moiety and the other click chemistry may comprise an azide moiety. Additionally, R may comprise a BCN moiety and the other click chemistry group may comprise an azide moiety. Further, R may comprise a TCO moiety and the second click chemistry group may comprise a tetrazine moiety.
Additionally, R may comprise 1,2-aminothiol moiety and the other click chemistry group may comprise a CBT moiety.
In another aspect, the invention provides a linker-conjugated protein obtainable as described herein. Especially, the linker-conjugated protein may comprise a structure according to formula (IT) (as described above):
As such, R2 may comprise a protein part. R3 may comprise a first click chemistry group part. Hence, the components of the linker-conjugated protein may be represented herein (as described above): (1) a protein part, (ii) a linker part, and (iii) a first click chemistry group part. Especially, the linker part comprised by the structure according to formula (IT) may be directly conjugated to the first click chemistry group part comprised by R3 in the structure according to formula (II). The first click chemistry group part of the linker- conjugated protein may be available to be conjugated to a compatible second click chemistry group (part) as described above, especially on a different site from where the first click chemistry group part is conjugated to the structure according to formula (IT).
In an additional aspect, the invention provides a cargo-conjugated protein obtainable as described herein. Especially, the cargo-conjugated protein may comprise a structure according to formula (IT) (as described above):
UQ. a sa
As such, R2 may comprise a protein part. R4 may comprise (i) a first click chemistry group part, (11) a second click chemistry group part, and (iii) a cargo part. Hence, all components of the cargo-conjugated protein may be represented herein (as described above): (1) a protein part, (ii) a cargo part, (iii) a linker part, (iv) a first click chemistry group part, and (v) a second click chemistry group part. Especially, the linker part comprised by the structure according to formula (III) may be directly conjugated to the first click chemistry group part comprised by R4 in the structure according to formula (IIT). The first click chemistry group part may be conjugated to the second click chemistry group part, especially on a different site from where the first click chemistry group part is conjugated to the structure according to formula (III). The second click chemistry group part may be conjugated to the cargo part on a different site from where the second click chemistry group part is conjugated to the first click chemistry group part.
In embodiments, the cargo-conjugated protein may especially be used in biotechnological applications. For example, the cargo-conjugated protein may be used for protein sequencing, i.e., the amino acid sequence of the protein part comprised by the cargo- conjugated protein may be determined. Further, the cargo-conjugated protein may be used for protein structural characterization, i.e., the protein structure of the protein part comprised by the cargo-conjugated protein may be determined. Moreover, the cargo-conjugated protein may be used for protein activity within a biotechnological process, 1.e., the functionality of the protein may be employed to facilitate a biotechnological process. Especially, in such applications, the cargo part may comprise a functionalized cargo (as described above).
In a further aspect, the invention may provide a protein array comprising the cargo-conjugated protein according to the invention. For example, the cargo may comprise a polymer comprised by a structure comprising the target location. A protein may be conjugated to such structure comprising the target location. Hence, such a structure may provide a protein array comprising a protein. Such structure comprising a protein array may be used for a variety of biotechnological applications. In embodiments, a protein comprised by the protein array may be sequenced. Further, a protein comprised by the protein array may be structurally investigated. Moreover, a protein comprised by the protein array may be utilized for (specific) protein activity within a biotechnological process.
The embodiments described herein are not limited to a single aspect of the invention. For example, an embodiment describing the method may further relate to the (resulting) cargo-conjugated protein. Similarly, an embodiment of the cargo-conjugated protein may further relate to embodiments of the method. In particular, an embodiment of the method describing structural formulae with specific groups (e.g., a specific selection of possible R groups) may indicate that the cargo-conjugated protein may, in embodiments, have such groups (such as the specific selection of possible R groups).
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1 schematically depicts embodiments of (i) a method for providing a cargo-conjugated protein, (ii) a linker, and (iii) a cargo-
conjugated protein. Fig. 2 schematically depicts further embodiments of a method for providing a cargo-conjugated protein. The schematic drawings are not necessarily on scale.
Fig. 1 schematically depicts embodiments of a method for providing a cargo-conjugated protein 130. The method may comprise a first conjugation stage 510 and a second conjugation stage 520. Depicted on top of Fig. 1 1s the first conjugation stage 510. The first conjugation stage 510 may comprise exposing a protein 100 to a linker 20 to provide a linker-conjugated protein 120. In specific embodiments, the linker 20 may be configured to conjugate to an N-terminal end 101 of the protein 100. Further, the linker 20 may have a structure according to formula (I): 7 rm
EN
‘R
In embodiments, R may comprise a first click chemistry group 21 selected from the group comprising a 2-azatricyclo[10.4.0.0* Thexadeca-1(16),4,6,8,12, 14-hexaen-10-yn-2- yl (also: “dibenzoazacyclooctyne” or “DCBO”) moiety, a bicyclo[6.1.0]non-4-yne (also: “bicyclononyne” or “BCN”) moiety, an (E)-1-(cyclooct-4-en-1-yloxy) (also: “trans- cyclooctene” or “TCO”) moiety, an azide moiety, a terminal alkyne moiety, a 4-(6-methyl- 1,2,4,5-tetrazin-3-yl)phenyl (also: “(methyl)tetrazine” or ‘“tetrazine”) moiety, a 2- (cyanobenzo[d]thiazol-6-yl)amino moiety (also: “2-cyanobenzothiazole” or “CBT”), and a 1,2- aminothiol moiety. The resultant linker-conjugated protein 120 1s depicted in the middle of Fig. 1. As a result of the first conjugation stage, the resultant linker-conjugated protein 120 may have a structure according to formula (IT):
OL an
R2 comprises a protein part 105 and R3 comprises a first click chemistry group 21. The main chemical structure depicted in formula (II) may comprise the linker part 25. Depicted on the bottom of Fig. 1 is the second conjugation stage 520. The second conjugation stage 520 may comprising exposing the linker-conjugated protein 120 to a cargo 30 to provide the cargo- conjugated protein 130. The cargo 30 may comprise a second click chemistry group 31. In Fig. 1, the first and second click chemistry groups are schematically depicted as a jack and a plug,
respectively. The second click chemistry group 31 may be configured to conjugate to the first click chemistry group 21. As a result of the second conjugation stage, the resultant cargo- conjugated protein 130 may have a structure according to formula (III): = gg
R2 comprises a protein part 105 and R4 comprises (1) a first click chemistry group part 22, (ii) a second click chemistry group part 32, and (iii) a cargo part 35. The main chemical structure depicted in formula (III) may comprise the linker part 25.
In embodiments, the first conjugation stage S10 may comprise providing the protein 100 and the linker 20 at a ratio selected from the range of 1:100 — 1:1000. The first conjugation stage 510 may further comprise a first conjugation duration selected from the range of 12 — 48 hours. The first conjugation stage 510 may be performed under shaking conditions for at least part of the first conjugation duration. The first conjugation stage 510 may also comprise a first conjugation temperature selected from the range of 15° — 50° C.
In specific embodiments, the second conjugation stage 520 may comprise providing the cargo 30 and the linker-conjugated protein 120 at a mole ratio selected from the range of 1:1 — 20:1. The second conjugation stage 520 may further comprise a second conjugation duration selected from the range of 12 — 48 hours. The second conjugation stage 520 may also comprise a second conjugation temperature selected from the range of 5° — 40°
C.
In further embodiments, the cargo 30 may comprise a biopolymer. The biopolymer may especially comprise a DNA oligonucleotide. The protein 100 may comprise an amino acid sequence of at least 30 amino acids.
The second click chemistry group 31 may be selected from the group comprising a DCBO moiety, a TCO moiety, an azide moiety, a terminal alkyne moiety, a tetrazine moiety, a 2-cyanobenzothiazole moiety, and a 1,2-aminothiol moiety.
Further depicted in Fig. 1 is an embodiment of a linker 20 having a structure according to formula (I):
O
U Noo
J N° TD) ~ LN, A
R is herein selected from the group consisting of 4-(2-azatricyclo[10.4.0.0*"]hexadeca- 1(16),4,6,8,12, 14-hexaen-10-yn-2-yl)-4-oxobutanoyl, 6-(2-azatricyclo[10.4.0.0*°]hexadeca- 1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-6-oxocaproyl, 3-(2-(2-(4-(2-azatricyclo- [10.4.0.0*9]hexadeca-1(16),4,6,8, 12,14-hexaen-10-yn-2-y1)-4-oxobutanamido)ethoxy)- ethoxy)propanoyl, 3-2-{2-6-(2-azatricyclo[10.4.0.0*’Jhexadeca-1(16),4,6,8, 12,14-hexaen- 10-yn-2-yl)-6-oxohexanamido)ethoxy)ethoxy)propanoyl, 20-(2-azatricyclo- [10.4.0.0*+*Jhexadeca-1(16),4,6,8, 12, 14-hexaen-10-yn-2-yl)-17,20-dioxo-4,7,10,13-tetraoxa- l6-azaicosanoyl, 22-(2-azatricyclo[10.4.0.0*"]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2- y1)-19,22-diox0-4,7, 10, 13-tetraoxa-16-azadocosanoyl, 2-(4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)acetyl, 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl phenyl )-2-0x0-6,9,12, 1 5-tetraoxa-3- azaoctadecan-18-oyl, 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl )-2-0x0-6,9,12, 15, 18- pentaoxa-3-azahenicosan-2 1-oyl 1-(4-(6-methyl-1,2,4, 5-tetrazin-3-yl)phenyl)-2-oxo- 6,9,12,15,18,21,24,27-octaoxa-3-azatriacontan-30-oyl, (E)-2-(cyclooct-4-en-1- yloxy)carbonyl, (E)-1-(cyclooct-4-en-1-yloxy)-1-ox0-5,8,11,14-tetraoxa-2-azaheptadecan-17- oyl, 2-azidoacetyl, 3-azidopropanoyl, 4-azidobutyroyl, 3-(2-azidoethoxy)-propanoyl, 3-(2-(2- azidoethoxy)ethoxy)propanoyl, 3-(2-(2-(2-azidoethoxy)ethoxy)-ethoxy)propanoyl, 1-azido- 3,6,9,12-tetraoxapentadecan-15-oyl, 4-pentynoyl, 5-hexynoyl, 4,7,10,13-tetraoxahexadec-15- ynoyl, 4,7,10,13,16-pentaoxanonadec-18-ynoyl, and 4,7,10,13,16, 19-hexaoxadocos-21-ynoyl, 2-(cyanobenzo[d]thiazol-6-yl)amino, (S)-2-amino-3-mercaptopropanol, and (bicyclo[6. 1.0]non-4-yn-9-ylmethoxy)carbonyl,
In a further aspect, Fig. 1 depicts a linker-conjugated protein 120 obtainable with the method described herein. The linker-conjugated protein 120 has a structure according to formula (IT): egen a
R2 herein comprises a protein part 105. R3 comprises a first click chemistry group part 22. The main chemical structure depicted in formula (II) may comprise the linker part 25.
In another aspect, Fig. 1 depicts a cargo-conjugated protein 130 obtainable with the method described herein. The cargo-conjugated protein 130 has a structure according to formula (IIT):
- ag
R2 herein comprises a protein part 105. R4 comprises (1) a first click chemistry group part 22, (ii) a second click chemistry group part 32, and (iii) a cargo part 35. The main chemical structure depicted in formula (IIT) may comprise the linker part 25.
Fig. 2 schematically depicts a further embodiment of the method for providing a cargo-conjugated protein 130. On top of Fig. 2, the depicted embodiment comprises a linker preparation stage 501 comprising reacting a first reactant 11 and a second reactant 12 to provide the linker 20. Especially, the first reactant 11 may comprise 6-(piperazin-1-ylmethyl)2- pyridinecarboxaldehyde and the second reactant 12 may comprise NHS-R. The linker preparation stage 501 may comprise providing the first reactant 11 and the second reactant 12 at a mole ratio selected from the range of 1:1 — 1:25. The linker preparation stage 501 may further comprise a reaction duration selected from the range of 12 — 48 hours. The linker preparation stage 501 may also comprise reacting the first reactant 11 and the second reactant 12 at a reaction temperature selected from the range of 22 — 50 °C. The linker preparation stage 501 may comprise providing the first reactant 11 and the second reactant 12 in the presence of an organic solvent 15, an organic base 16, and water. The organic solvent 15 may be selected from the group comprising dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dichloromethane, chloroform, tetrahydrofuran (THF), and 1,4-dioxane. The organic base 16 may comprise triethylamine. The second reactant 12 and the organic base 16 may be provided in a mole ratio selected from the range of 10:1 — 1:40.
In specific embodiments, the first click chemistry group 21 may be selected from the group comprising a DCBO moiety and the second click chemistry group 31 may be selected from the group comprising an azide moiety. In alternative specific embodiments, the first click chemistry group 21 may be selected from the group comprising a TCO moiety and the second click chemistry group 31 may be selected from the group comprising a tetrazine moiety. In such embodiments, the linker preparation stage 501 may comprise providing the organic solvent 15 at >20 vol%. Herein, R may comprise n oligoethylene glycol residues, wherein n may be selected from the range of n =1 —n = 8.
Below the linker preparation stage 501 in the depicted embodiment, Fig. 2 depicts a quenching stage 502 following the linker preparation stage 501. The quenching stage 502 may comprise exposing the linker 20 to a quenching base 17. The quenching base 17 may be selected from the group comprising dimethylamine, diethylamine, sodium hydroxide, and potassium hydroxide, most especially diethylamine. The quenching stage 502 may have a quenching duration selected from the range of 3 — 12 hours.
Below the quenching stage 502 in the depicted embodiment, Fig. 2 depicts a purification stage S03 following the linker preparation stage 501. Especially here, the purification stage 503 is depicted following the quenching stage 502. The purification stage 503 may comprise subjecting the linker 20 to a chromatography method to purify the linker 20.
The chromatography method may especially comprise high-performance liquid chromatography.
Depicted below the purification stage 503, Fig. 2 depicts a first conjugation stage 510. The first conjugation stage 510 may comprise providing the protein 100 and the linker 20 in a buffer solution 25 to provide the linker-conjugated protein 120.
Depicted in Fig. 2 below the first conjugation stage 510, the method may further comprise a buffer exchange stage 511 following the first conjugation stage 510. The buffer exchange stage 511 may comprise removing excess linker 20 via a buffer exchange method to provide the linker-conjugated protein 120 in a filtered solution 26. The bufter exchange method may especially be selected from the group consisting of desalting column chromatography and dialysis.
Depicted below the buffer exchange stage 511, Fig. 2 depicts a second conjugation stage 520. The second conjugation stage 520 may comprise providing the linker- conjugated protein 120 and the cargo 30 to provide the cargo-conjugated protein 130.
The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably.
The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.
Moreover, the terms “about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.
The term “comprise” also includes embodiments wherein the term “comprises” means “consists of”.
The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of" but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.
The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.
It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.
In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.
Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.
The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.
The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.
Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.
The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.
Claims (16)
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| NL2033410A NL2033410B1 (en) | 2022-10-28 | 2022-10-28 | N-terminal protein modification |
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|---|---|---|---|---|
| US10370407B2 (en) | 2015-01-21 | 2019-08-06 | The Regents Of The University Of California | Affinity-assisted protein modification and recycling |
| US20200199175A1 (en) * | 2018-12-20 | 2020-06-25 | Indian Institute Of Science Education And Research | Method for synthesis of protein amphiphiles |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10370407B2 (en) | 2015-01-21 | 2019-08-06 | The Regents Of The University Of California | Affinity-assisted protein modification and recycling |
| US20200199175A1 (en) * | 2018-12-20 | 2020-06-25 | Indian Institute Of Science Education And Research | Method for synthesis of protein amphiphiles |
Non-Patent Citations (4)
| Title |
|---|
| LI DE-ZHI ET AL: "N-terminal [alpha]-amino group modification of antibodies using a site-selective click chemistry method", MABS, vol. 10, no. 5, 13 April 2018 (2018-04-13), US, pages 712 - 719, XP055889300, ISSN: 1942-0862, DOI: 10.1080/19420862.2018.1463122 * |
| LI DZ ET AL.: "N-terminal a-amino group modification of antibodies using a site-selective click chemistry method", MABS, vol. 10, no. 5, July 2018 (2018-07-01), pages 712 - 719, XP055889300, DOI: 10.1080/19420862.2018.1463122 |
| MACDONALD JI ET AL.: "One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes", NAT CHEM BIOL, vol. 11, no. 5, May 2015 (2015-05-01), pages 326 - 31, XP055940089, DOI: 10.1038/nchembio.1792 |
| SCHOONEN LISE ET AL: "Modular, Bioorthogonal Strategy for the Controlled Loading of Cargo into a Protein Nanocage", BIOCONJUGATE CHEMISTRY, vol. 29, no. 4, 6 February 2018 (2018-02-06), US, pages 1186 - 1193, XP093055747, ISSN: 1043-1802, Retrieved from the Internet <URL:http://pubs.acs.org/doi/pdf/10.1021/acs.bioconjchem.7b00815> DOI: 10.1021/acs.bioconjchem.7b00815 * |
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