Site-specific conjugation of single domain antibody fragments (VHH) using equilibrium transfer alkylation reagent (ETAC)

Abstract

To provide a conjugation method that is site-specific, stable under physiological conditions, and not prone to protein aggregation as seen with disulfide bonds.SOLUTION: The invention is directed to a method for providing a polypeptide with a detectable label, wherein the polypeptide comprises at least 5 amino acids of which a first amino acid is Cysteine and at least one second amino acid is selected from the group consisting of Serine, Threonine, Tyrosine, Lysine, Histidine and Arginine, characterized by the steps a) initiating Michael addition between the first and second amino acids by providing a specific reagent and b) adding a nucleophilic agent to remove the Michael addition products between two second amino acids by retro-Michael addition.SELECTED DRAWING: Figure 1

Description

Background Art

[0001] Antibodies and their antibody fragments, such as VHHs, are an important class of biomolecules for immunotherapy. VHHs are increasing in importance for new therapeutic approaches due to their unique molecular properties, such as small size, simple folding, and improved tissue penetration. One potential new use of VHHs is their use as adapter molecules in combination with AdCAR™ T cells.

[0002] Therefore, VHHs need to be modified or tagged for such purposes. One approach is to chemically conjugate chemical tags, such as biotin, thiamine, peptides, DBCO, oligonucleotides, peptide nucleic acids (PNAs), etc., to be recognized by AdCAR™ T cells in order to bridge tumor cells and CAR T cells to kill the tumor cells. Using the adapter molecule together with AdCAR™ T cells allows for control of dosing and as a result ensures safety, since this can be easily removed if the need arises and minimizes the risk to the patient. For it to be suitable as a pharmaceutical, the chemical conjugation of the adapter molecule must be site-specific and result in a homogeneous, well-defined product that is stable under physiological conditions after injection.

[0003] One situation in which site-specific conjugation may be possible is the incorporation of engineered cysteines. However, free cysteines can form disulfide bonds that lead to protein aggregation during production or purification. Another strategy currently in use is the conjugation of free cysteines based on maleimide. The limitation of this approach is that maleimide-thiol adducts can generate chemically labile conjugations under physiological reducing conditions. The adapter molecules that would result from the retro-Michael addition that occurs here are non-functional and may block the epitopes of the conjugated adapter molecules [1,2].

[0004] Methods for site-specific conjugation of single-domain antibody fragments are well known, such as the addition of maleimide to engineered cysteines, sortase reactions, etc. [3].

[0005] For example, US Patent No. 11,021,544 (B2) discloses the use of an ETAC system (Thiobridging) for dimerizing VHHs. Here, VHHs characterized by C-terminal extensions containing cysteine moieties are chemically linked via the ETAC system (Thiobridging) to generate nanobody dimers into which diagnostic agents, therapeutic agents, or labeling agents are introduced.

[0006] Furthermore, publication [5] discloses that conjugation by bisalkylation using reagents can be equilibrium-controlled by addition-elimination reactions that depend on Michael reactions. Covalent conjugation occurs between two spatially close amino acids (e.g., two cysteine thiols derived from a disulfide or two histidines in a C-terminal or N-terminal his-tag).

[0007] Publication [6] describes the possibility of the addition of multiple reagents by modifying the His-tag with ETAC reagent. The modification of the His-tag is not stable against strong Michael donors such as DTT. To stabilize the conjugate, the reduction of the ketone in the introduced ETAC linker is achieved using triacetoxyborohydride, which may not be compatible with protein stability.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0010] Therefore, there is a need for a conjugation method that is site-specific, stable under physiological conditions, and less likely to cause protein aggregation as seen in disulfide bonds. [Means for Solving the Problems]

[0011] The inventors have herein demonstrated that an ETAC-biotin linker can specifically monolabel a single VHH unit by linking one terminal cysteine to a histidine residue located immediately adjacent to said terminal cysteine, thus targeting the cysteine-histidine motif. By providing an excess of the ETAC-biotin linker, the ETAC linker reacted with multiple Michael acceptors within the VHH. The inventors were able to remove the ETAC-biotin label that crosslinked only the histidine residue by adding a competing nucleophile (e.g., TCEP). This led to the enrichment of the single-site monospecific conjugate VHH. No further treatment was required to remove the non-conjugated products (as confirmed by LC-MS experiments). The dual Michael addition reaction that targets one cysteine and one adjacent histidine in the same sequence, followed by the removal of the multiple label targets using a nucleophile such as TCEP to selectively enrich the site-specific single-label VHH, is, to the best of the inventors' knowledge, a unique approach and has not been reported yet.

[0012] The subject matter of the present invention is the method according to claim 1 for providing a polypeptide having a detectable label, wherein said polypeptide comprises at least 5 amino acids, of which the first amino acid is C (cysteine) and at least one second amino acid is selected from the group consisting of S (serine), T (threonine), Y (tyrosine), K (lysine), H (histidine) and R (arginine), a) Formula (I)

Chemical formula

[0013] The term "polypeptide" refers to any molecule composed of a chain of at least 5 amino acids. This includes small peptides that are polypeptides with a molecular weight of 300 Da to 7 kDa, single domain antibodies (VHH) with a molecular weight of 8 to 15 kDa, antibody fragments typically in the range of 20 to 50 kDa, and full-size antibodies such as IgG species with a molecular weight of about 140 to 160 kDa. It includes various molecules up to larger forms.

[0014] Specific binding to each tumor antigen was observed in ETAC-biotin conjugated VHH. This indicates correct folding and function. Furthermore, the ETAC-biotin conjugate VHH induced tumor cell lysis and cytokine secretion of the biotin-specific adapter CAR.

[0015] Crosslinking modification of VHH will improve the stability against deconjugation compared to a single attachment site with maleimide or the like, and increase the rigidity of the introduced labeling agent to enhance the binding to secondary reagents (e.g., CAR T cells).

Brief Description of the Drawings

[0016]

Figure 1

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Modes for Carrying Out the Invention

[0017] A polypeptide having at least one reduced cysteine (C) and at least one nucleophilic amino acid (X) is treated with an ETAC reagent. The ETAC compound undergoes a Michael addition reaction with one C and amino acid X, or amino acid X and another amino acid X. By treating this reaction product with a Michael donor, such as a reducing agent, the ETAC conjugated to amino acid X and another amino acid X is removed.

[0018] VHH contains a sequence of terminal cysteine and histidine. The terminal cysteine is reduced while leaving the intramolecular disulfide bridge intact to enable conjugation of VHH with the ETAC reagent. Next, the ETAC compound undergoes a Michael addition reaction with one of the terminal reduced cysteine and histidine. In addition, the ETAC compound is conjugated only to histidine. By treating this reaction product with a Michael donor, such as a reducing agent, the ETAC compound cross-linked only with histidine is removed from VHH. The ETAC compound linked to cysteine and histidine is stable even in the presence of a Michael donor and remains cross-linked.

[0019] In certain embodiments of the present invention, the at least one second amino acid is provided at the C-terminus of the polypeptide.

[0020] Nucleophiles include dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine (PPh 3 ), hydrazine (N 2 H 4 ), tetrakis(dimethylamino)ethylene (TDAE), mercaptoethanol, formic acid, trichlorosilane (HSiCl 3 ), phosphine (PH 3 ), dimethylsulfide borane (Me 2 S·BH 3 ), thioacetic acid, tris(trimethylsilyl)phosphine, tri-n-hexylphosphine, ammonia (NH 3) It may be selected from the group consisting of cysteine, selenocysteine, thiophenol, glutathione (GSH), iodide, methylamine, and hydrogen sulfide.

[0021] Preferably, the reagent has the formula (II)

Chem.

Chem.

[0022] Most preferably, the reagent has one of the formulas (IV) to (VII).

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[0023]

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[0024]

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[0025]

Chem.

[0026] Usage The method of the present invention is particularly useful for providing a detectable label to a polypeptide in a CAR - adapter molecule, in a cell staining reagent, in a cell separation / labeling / enrichment / depletion process, in protein purification, and for cell activation. Literature 1. Baldwin, A.D.; Kiick, K.L. 「Tunable Degradation of Maleimide - Thiol Adducts in Reducing Environments」 Bioconjug. Chem. 2011, 22, 1946 - 1953, doi:10.1021 / BC200148V / SUPPL_FILE / BC200148V_SI_001.PDF. 2. Baldwin, A.D.; Kiick, K.L. 「Reversible Maleimide - Thiol Adducts Yield Glutathione - Sensitive Poly(Ethylene Glycol) - Heparin Hydrogels」 Polym. Chem. 2012, 4, 133 - 143, doi:10.1039 / C2PY20576A. 3. Schumacher, D.; Helma, J.; Schneider, A. F. L.; Leonhardt, H.; Hackenberger, C. P. R. "Nanobodies: Chemical Functionalization Strategies and Intracellular Applications" Angew. Chem. Int. Ed. Engl. 2018, 57, 2314, doi:10.1002 / ANIE.201708459. 4. "Nanobody Dimers Linked via C-Terminally Engineered Cysteins" U.S. Patent No. 11021544 (B2) 2021 5. Peciak, K.; Laurine, E.; Tommasi, R.; Choi, J. W.; Brocchini, S. "Site-Selective Protein Conjugation at Histidine" Chem. Sci. 2019, 10, 427 - 439, doi:10.1039 / C8SC03355B. 6. Cong, Y.; Pawlisz, E.; Bryant, P.; Balan, S.; Laurine, E.; Tommasi, R.; Singh, R.; Dubey, S.; Peciak, K.; Bird, M.; et al. "Site-Specific PEGylation at Histidine Tags" Bioconjug. Chem. 2012, 23, 248 - 263, doi:10.1021 / BC200530X / SUPPL_FILE / BC200530X_SI_001.PDF.

Example

[0027] Example 1 Site-Specific Conjugation of EGFR-Specific VHH with ETAC-Biotin Linker 1.1 Conjugation of EGFR-Specific VHH with Multiple ETAC-Biotin Linkers The inventors used an anti-EGFR specific VHH as a model VHH, which has a C-terminal histidine tag, followed by a flexible glycine-serine linker and a glycine-capped cysteine residue as a Michael donor.

[0028] The cysteine introduced at the end of the EGFR VHH was reduced with 3 molar equivalents of TCEP at 21 °C for 1 hour. This reaction was carried out at pH 7.8 in PBS buffer (PE buffer) containing 5 mM EDTA. Next, TCEP was removed by a desalting column containing Sephadex G-25 resin and the PE buffer. Thereafter, an ETAC-biotinylated molecule was added to the reduced and purified EGFR VHH. The ETAC-biotinylated molecule was pre-dissolved in DMSO and added at 4 molar equivalents to the EGFR VHH. A sufficient amount of DMSO was added to the reaction mixture so that the final concentration in the final reaction mixture was 10% DMSO. The conjugation reaction was maintained at 21 °C for 2 hours. Next, the reaction mixture was applied to a molecular weight cut-off column (Merck Millipore) pre-equilibrated with PBS buffer and washed with 4 column volumes of 1× PBS. The protein concentration of the final product was analyzed by absorption at 280 nanometers. The product of the coupling reaction was analyzed by liquid chromatography coupled to mass spectrometry (Figure 3a). The analysis results showed that each VHH molecule was bound to 1 to 5 ETAC-biotin linker units, and the main composition consisted of 2 ETAC-biotin linkers per VHH. 1.2 Concentration of Single ETAC-Biotin Linker-Labeled EGFR-Specific VHH by Retro-Michael Addition The ETAC-biotin label that did not react with cysteine was selectively removed by a retro-Michael reaction by adding a nucleophilic reducing agent. Therefore, 250 mM TCEP was added to 13 μL of VHH at 37 °C for 10 minutes. This was confirmed by mass spectrometry to result in a VHH polypeptide with a molecular weight of 15905 Da containing only 1 ETAC-biotin label (Figure 3b). This results in the desired final product after the TCEP step.

[0029] The site of conjugation of ETAC-biotin to the EGFR-specific VHH was analyzed by peptide mass fingerprinting (PMF) (Figure 4). PMF indicated successful conjugation of the ETAC-biotin label to cysteine and histidine residues at the carboxy terminus of the VHH. This demonstrated linkage of one terminal cysteine with a histidine residue proximal to that terminal cysteine. This represents a unique site-specific labeling site that can be addressed by ETAC-based crosslinkers and serves as an alternative motif to cysteine-cysteine.

[0030] 1.3. Use of ETAC-biotin linker-labeled EGFR-specific VHH in flow cytometry To demonstrate the application of ETAC biotin-labeled EGFR-specific VHH to flow cytometry, tumor cells known to express EGFR were detected using EGFR-specific VHH molecules. For this purpose, 10 μg / mL of ETAC-biotin-labeled EGFR-specific VHH was incubated with EGFR-positive tumor cells at 4 °C for 1 hour. The cell suspension was washed with phosphate buffer containing 0.5% BSA (PBS buffer), and secondary staining was performed with a PE-labeled biotin-specific secondary antibody (REA746). Next, the cell suspension was washed with PBS. Binding of ETAC-biotin-labeled EGFR-specific VHH to EGFR-positive tumor cells and the presence of the ETAC-biotin linker were detected by secondary staining with a PE-labeled biotin-specific secondary antibody. The signal intensity of unstained tumor cells (upper histogram in Figure 5) or cells stained with the biotin-specific secondary antibody alone (middle histogram in Figure 5) was substantially lower compared to EGFR-positive tumor cells incubated with ETAC-biotin-labeled anti-EGFR VHH and the biotin-specific secondary PE-labeled antibody (lower histogram in Figure 5). This confirmed conjugation of the ETAC-biotin linker to the EGFR-specific VHH. Additionally, this data confirmed that the conjugation process did not interfere with the functional property of the EGFR-specific VHH to bind to its epitope. 1.4. Use of an ETAC-biotin linker-labeled EGFR-specific VHH as an adapter molecule for adapter CAR T cells CAR T cells are immune cells genetically engineered to express a chimeric antigen receptor (CAR) that is not naturally present in T cells. This receptor is specific for a particular antigen expressed on the surface of tumor cells. CAR T cells induce tumor cell lysis after association with tumor cells. In contrast, adapter CAR T cells express a CAR specific for a tagged polypeptide rather than an antigen expressed by tumor cells. This polypeptide, such as VHH, mediates the binding between adapter CAR T cells and tumor cells. In the absence of this adapter, tumor cell lysis is not induced. By using different adapter molecules, one CAR T cell can target two or more tumor antigens. This mode of action enhances safety and therapeutic efficacy. 1.4.1 ETAC-biotin linker-specific adapter CAR T cells ETAC-biotin CAR T cells contained a biotin-specific scFv as the binding moiety. This scFv was linked to the human CD8 transmembrane domain via the hIgG4 hinge domain. The signaling domain was composed of 4-1BB and CD3ζ.

[0031] 1.4.2 Generation and titration of LV particles Lentiviral vector particles were produced by transient transfection of HEK-293T cells. Lentiviral vector particles were pseudotyped with VSV-G. For transfection, HEK-293T cells were seeded in DMEM supplemented with 2 mM L-glutamine and 10% FCS in a T175 culture flask 3 days before transfection. On the day of transfection, the culture medium was removed and replaced with DMEM supplemented with 2 mM L-glutamine. The cells were transfected with a three-plasmid system encoding VSV-G, gag / pol / rev, and a psi-positive transfer vector.

[0032] After 48 hours, the supernatant was collected and centrifuged at 1000 rpm for 10 minutes to remove cell debris. Furthermore, the supernatant was filtered through a 0.45 μm filter. The pellet was resuspended in ice-cold PBS and stored at -80°C.

[0033] 1.4.3 Transduction, culture and analysis of ETAC-biotin linker-specific adapter CAR T cells Anti-biotin adapter CAR T cells were produced using primary T cells from healthy donors. T cells were isolated from PBMC using a pan-T cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Prior to transduction, 2 × 10^6 T cells were seeded in 24-well plates using 2 mL of TexMACS medium (Miltenyi) supplemented with IL-7 (Miltenyi Biotec), IL-15 (Miltenyi Biotec) and TransAct (Miltenyi Biotec).

[0034] After 24 hours, the T cells were transduced at an MOI of 5 by adding the corresponding volume of lentiviral vector particles. On the third day after activation, the culture medium was removed and replaced with TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec).

[0035] On the sixth day after transduction, the frequency of anti-biotin adapter CAR-positive T cells was analyzed by flow cytometry using biotinylated PE conjugate (Miltenyi Biotec). On the tenth day after transduction, anti-biotin adapter CAR T cells were used for functional assays.

[0036] Example 5 Functionality of EGFR-specific VHH as an adapter molecule for ETAC-biotin linker-specific adapter CAR T cells To test the application of ETAC biotin-conjugated EGFR-specific VHH as an adapter molecule for biotin-specific anti-biotin adapter CAR T cells, antigen-positive tumor cells and ETAC biotin-labeled EGFR-specific VHH were cultured either in the presence or absence of biotin-specific adapter CAR T cells. Thus, 20,000 biotin-specific adapter CAR T cells and 5,000 tumor cells were seeded in RPMI containing 2 mM L-glutamine and 10% FCS in a 96-well flat-bottom plate.

[0037] Next, 50 μL of ETAC-biotin-labeled EGFR-specific VHH was added to a final concentration of 5 nM. A functional tumor-specific adapter molecule was used as a positive control. The co-cultures were incubated at 37 °C and 5% CO 2 2. Tumor cell lysis was analyzed by live cell fluorescence microscopy of tumor cells using the IncuCyte® S3 system. The data presented were obtained from unrelated donors (n = 3) and plotted as mean and ±1 standard deviation (Figure 6). Tumor cell lysis was induced only in the presence of biotin-specific adapter CAR T cells. The titer of ETAC-biotin-linked VHH was comparable to that of the control adapter. These results indicate proper bridging of tumor cells and adapter CAR T cells via ETAC-biotin-labeled VHH. In summary, this indicates that ETAC-biotin linker-labeled EGFR-specific VHH is a functional adapter molecule for biotin-specific adapter CAR T cells.

[0038] Example 2 Site-specific conjugation of CD56-specific VHH with ETAC-biotin linker We used an anti-CD56-specific VHH as a model VHH, which has a C-terminal histidine tag followed by a flexible glycine-serine linker and a glycine-capped cysteine residue as a Michael donor.

[0039] The cysteine introduced at the C-terminus of CD56 VHH was reduced with 3 molar equivalents of TCEP at 21 °C for 1 hour. This reaction was carried out at pH 7.8 in PBS buffer (PE buffer) containing 5 mM EDTA. Next, TCEP was removed using a desalting column containing Sephadex G-25 resin and PE buffer. Thereafter, the ETAC-biotinylated molecule was added to the reduced and purified CD56 VHH. The ETAC-biotinylated molecule was pre-dissolved in DMSO and added at 4 molar equivalents to CD56 VHH. A sufficient amount of DMSO was added to the reaction mixture so that the final concentration in the final reaction mixture was 10% DMSO. The conjugation reaction was maintained at 21 °C for 2 hours. Next, the reaction mixture was applied to a molecular weight cut-off column (Merck Millipore) pre-equilibrated with PBS buffer and washed with 4 column volumes of 1× PBS. The protein concentration of the final product was analyzed by absorption at 280 nanometers. The product of the coupling reaction was analyzed by liquid chromatography coupled to mass spectrometry (Figure 7a). The analysis results showed that each VHH molecule was bound to 1 to 4 ETAC-biotin linker units, and the main composition consisted of 2 ETAC-biotin linkers per VHH. 2.2 Concentration of CD56-specific VHH labeled with a single ETAC-biotin linker by retro-Michael addition ETAC-biotin labels that did not react with cysteine were selectively removed by a retro-Michael reaction by adding a nucleophilic reducing agent. Therefore, 250 mM TCEP was added to 13 μL of VHH at 37 °C for 10 minutes. This was confirmed by mass spectrometry to result in a VHH polypeptide with a molecular weight of 16,240 Da containing only 1 ETAC-biotin label (Figure 7b). This results in the desired final product after the TCEP step.

[0040] 2.3 Use of CD56-specific VHH labeled with an ETAC-biotin linker in flow cytometry To demonstrate the application of ETAC-biotinylated CD56-specific VHH to flow cytometry, tumor cells known to express CD56 were detected using CD56-specific VHH molecules. To show the specific binding of ETAC-biotinylated CD56-specific VHH, 5 μg / mL of conjugated VHH was incubated with CD56-positive tumor cells and CD56-negative cells at 4 °C for 1 hour. The cell suspension was washed with PEB. The binding of ETAC-biotinylated CD56-specific VHH was detected by secondary staining using a PE-labeled biotin-specific secondary antibody (REA746). CD56-specific IgG1 (REA196-PE) was used as a positive control. Next, the cell suspension was washed with PEB. The signal intensity of PE-positive tumor cells was analyzed by flow cytometry in antigen-positive and antigen-negative cells. The frequency of CD56-positive cells was plotted for antigen-positive and antigen-negative cells after incubation with either ETAC-biotinylated CD56-specific VHH or CD56-specific IgG1 (REA196-PE) as a positive control. Specific binding of ETAC-biotinylated VHH was detected (Figure 8a). The CD56 ETAC-biotinylated VHH staining pattern was equivalent to that of the positive control. Binding of ETAC-biotinylated CD56 VHH was not detected by flow cytometry in antigen-negative cells (Figure 8b).

Claims

1. 1. A method for providing a polypeptide bearing a detectable label, said polypeptide comprising at least five amino acids, wherein a first amino acid is C (cysteine) and at least one second amino acid is selected from the group consisting of S (serine), T (threonine), Y (tyrosine), K (lysine), H (histidine) and R (arginine); a) a compound represented by formula (I) 【Chemistry 1】 (I) [In the formula, R １ =-biotin, thiamine, peptides of 2-30 amino acids, oligonucleotides of 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophores, -fluorescent proteins R ２ = Substituted or unsubstituted aromatic residue R ３ = H, F, NO ２ , alkyl having 1 to 5 carbon atoms R ４ = direct bond, substituted or unsubstituted alkyl having 1 to 20 carbon atoms, amine or amide residue] initiating a Michael addition between the first amino acid and the second amino acid by providing a reagent of b) adding a nucleophile to remove the Michael addition product between the two second amino acids by retro-Michael addition. A method comprising:

2. 2. The method of claim 1, wherein at least one second amino acid is provided at the C-terminus of the polypeptide.

3. The nucleophile is dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine (PPh ３ ), hydrazine (N ２ H ４ ), tetrakis(dimethylamino)ethylene (TDAE), mercaptoethanol, formic acid, trichlorosilane (HSiCl ３ ), phosphine (PH ３ ), dimethylsulfide borane (Me ２ S.B.H. ３ ), thioacetic acid, tris(trimethylsilyl)phosphine, tri-n-hexylphosphine, ammonia (NH ３ 3. The method according to claim 1 or 2, characterized in that the cysteine, selenocysteine, thiophenol, glutathione (GSH), iodide, methylamine, hydrogen sulfide is selected from the group consisting of:

4. The reagent is represented by the formula (II) 【Chemistry 2】 (II) [In the formula, R １ =-biotin, thiamine, peptides of 2-30 amino acids, oligonucleotides of 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophore R ２ = Substituted or unsubstituted aromatic residue R ３ = H, F, NO ２ , alkyl having 1 to 5 carbon atoms R ４ = direct bond, substituted or unsubstituted alkyl having 1 to 20 carbon atoms, amine or amide residue R ５ = direct bond, substituted or unsubstituted alkyl or amine residue having 1 to 20 carbon atoms R ６ = direct bond, substituted or unsubstituted alkyl or amine residue having 1 to 20 carbon atoms R ７ = a substituted or unsubstituted alkyl, carboxy, amine or hydroxyl residue having 1 to 20 carbon atoms] The method according to any one of claims 1 to 3, characterized in that it comprises:

5. The reagent has the formula (III) 【Chemistry 3】 (III) [In the formula, R １ =-biotin, thiamine, peptides of 2-30 amino acids, oligonucleotides of 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophore R ２ = Substituted or unsubstituted aromatic residue R ３ = H, F, NO ２ , alkyl having 1 to 5 carbon atoms R ４ = direct bond, substituted or unsubstituted alkyl having 1 to 20 carbon atoms, amine or amide residue R ６ = direct bond, substituted or unsubstituted alkyl or amine residue having 1 to 20 carbon atoms R ７ = a substituted or unsubstituted alkyl, carboxy, amine or hydroxyl residue having 1 to 20 carbon atoms] The method according to any one of claims 1 to 4, characterized in that it comprises:

6. The method according to any one of claims 1 to 5, characterized in that the C-terminus of the polypeptide is provided with 2 to 10 histidine units (H).

7. The method according to any one of claims 1 to 6, characterized in that said polypeptide is a single domain antibody (VHH), an antibody fragment or a full-sized antibody.

8. Use of the method according to any one of claims 1 to 7 for providing a detectable label to a polypeptide in a CAR-adapter molecule, in a cell staining reagent, in a cell separation / labelling / enrichment / depletion process, in protein purification and for cell activation.

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