Site-specific coupling of single domain antibody fragments with equilibrium transfer alkylating reagent ETAC

Abstract

The present invention relates to a method for providing a polypeptide having a detectable tag, the polypeptide comprising at least 5 amino acids wherein the first amino acid is C and at least one second amino acid is selected from S, T, Y, K, H and R, comprising the steps of: a) initiating a Michael addition reaction between the first amino acid and the second amino acid by providing an agent according to formula (I), wherein: R1 independently represents-biotin, thiamine, a peptide having from 2 to 30 amino acids, an oligonucleotide having from 6 to 100 nucleotides,-DBCO, DBCO,-N3, N3, a fluorophore or a fluorescent protein, R2 independently represents a substituted or unsubstituted aromatic residue, R3 independently represents H, F, NO2 or an alkyl group having from 1 to 5 carbon atoms, r4 independently represents a direct bond, a substituted or unsubstituted alkyl group, an amine or amide residue having 1 to 20 carbon atoms; b) adding a nucleophilic reagent to remove the Michael addition product between the two second amino acids by a reverse Michael addition reaction. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to site-specific conjugation of single domain antibody fragments (Single Domain Antibody Fragments, SdAb, such as VHH (Variable Domain of Heavy Chain of Heavy-Chain Antibody)) using an equilibrium transfer alkylation reagent (ETAC). Background Art

[0002] Antibodies and their antibody fragments, such as VHH, are an important class of biomolecules in immunotherapy. Due to their unique molecular properties, such as small size, simple folding, and improved tissue permeability, VHHs have become more important for new therapeutic approaches. A potential new application of VHHs is their use as linker molecules to bind to AdCAR TM T cells.

[0003] Therefore, for such purposes, it is necessary to modify or label VHHs. One approach is chemical conjugation to chemical tags (such as biotin, thiamine, peptides, DBCO, oligonucleotides, peptide nucleic acids (PNAs), etc.) to be recognized by AdCAR TM T cells, thereby crosslinking tumor cells and CAR T cells to kill tumor cells. The use of linker molecules conjugated to AdCAR TM T cells lies in its ability to control the dose and the resulting safety, as it can be easily removed if needed, characterized by minimal risk to the patient. For applicability to drugs, the chemical conjugation of the linker molecule must be site-specific and produce a homogeneous, stable, and well-defined product under physiological conditions after injection.

[0004] An opportunity to achieve site-specific conjugation is the incorporation of engineered cysteines. However, free cysteines can form disulfide bonds, leading 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 method is that the maleimide-thiol adduct produces an unstable chemical conjugation under physiological reducing conditions. The occurring retro-Michael addition will result in non-functional linker molecules and potentially blocking epitopes for the conjugated linker molecules [1,2].

[0005] There are many known methods for site-specific conjugation of single domain antibody fragments, such as maleimide addition to engineered cysteines or Sortase reactions [3].

[0006] For example, US11021544B2 discloses the use of an ETAC system (sulfur bridging) in the dimerization of VHHs, wherein the VHHs are linked via the ETAC system (sulfur bridging) with a C-terminal extension containing a cysteine moiety to produce a chemically linked nanobody dimer incorporating a diagnostic, therapeutic, or labeling agent.

[0007] Furthermore, publication [5] discloses that the coupling for the double alkylation reaction using a reagent can be balanced and controlled by an addition-elimination reaction dependent on the Michael Reaction. Covalent coupling occurs between two amino acids in spatial proximity (e.g., two cysteine thiols from a disulfide, or two histidines in a C-terminal or N-terminal histidine tag).

[0008] In publication [6], the possibility of multiple additions of a reagent through the modification of a histidine tag by an ETAC reagent has been documented. The modification of the histidine tag is unstable to strong Michael donors such as DTT. To stabilize the conjugate, triacetoxyborohydride, which may be incompatible with the stability of the protein, was used to achieve the reduction of the ketone introduced into the ETAC linker.

[0009] Therefore, there is a need for a site-specific coupling method that is stable under physiological conditions and does not readily undergo visible protein aggregation such as disulfide bonds. SUMMARY OF THE INVENTION

[0010] It can be demonstrated herein that by linking a terminal cysteine to a histidine residue directly adjacent to the terminal cysteine, an ETAC-biotin linker can specifically monoconjugate a single VHH unit, thereby targeting the cysteine-histidine motif. By providing an excess of the ETAC-biotin linker, the ETAC linker reacts with multiple Michael acceptors within the VHH. In the presence of a competitive nucleophile (e.g., TCEP), we were able to remove the ETAC-biotin tag crosslinked to a single histidine residue, resulting in the enrichment of site-specific, mono-specifically conjugated VHHs. The uncoupled products can be removed without further treatment (confirmed by LC-MS experiments). To the best of our knowledge, targeting a cysteine and an adjacent histidine in the same sequence for a double Michael addition reaction, followed by the elimination of multi-tag targets using a nucleophile such as TCEP to selectively enrich site-specific mono-tagged VHHs is a unique method and has not been reported.

[0011] The object of the present invention is a method for providing a polypeptide with a detectable label according to claim 1, wherein the polypeptide comprises at least 5 amino acids, wherein the first amino acid is C (cysteine), and at least one second amino acid is selected from S (serine), T (threonine), Y (tyrosine), K (lysine), H (histidine) and R (arginine), characterized by the following steps: a) Initiating a Michael addition reaction between the first amino acid and the second amino acid by providing a reagent according to formula (I), wherein: R1 independently represents - biotin, thiamine, a peptide having 2 - 30 amino acids, an oligonucleotide having 6 - 100 nucleotides, - DBCO, DBCO, - N3, N3 and - a fluorophore (such as FITC, Cy dye, Vio dye), - a fluorescent protein (such as GFP, RFP), R2 independently represents a substituted or unsubstituted aromatic residue (such as toluene or phenyl), R3 independently represents H, F, NO2, an alkyl group having 1 - 5 carbon atoms, R4 independently represents a direct bond, a substituted or unsubstituted alkyl group, an amine or an amide residue (such as (-CH2-CH2-CH2-CH2-CH2-CH2-)(LC) or polyethylene glycol (PEG)), having 1 - 20 carbon atoms; b) Adding a nucleophile to remove the Michael addition product between the two second amino acids by a retro-Michael addition reaction.

[0012] The term "polypeptide" refers to any molecule composed of a chain of at least 5 amino acids. This includes anything from small peptides (which are polypeptides with a molecular weight in the range of 300 Da - 7 kDa) to larger forms of molecules such as single-domain antibodies (VHH) with a molecular weight between 8 - 15 kDa, antibody fragments typically in the range of 20 - 50 kDa, and full-size antibodies such as IgG species with a molecular weight of approximately 140 - 160 kDa.

[0013] ETAC-biotin-linked VHH shows specific binding to its respective tumor antigen, indicating correct folding and function. In addition, ETAC-biotin conjugated VHH induces tumor cell lysis and cytokine secretion of a biotin-specific adaptor CAR.

[0014] Compared to a single attachment site via, for example, maleimide, the Bridged Modification of VHH can provide better stability against decoupling and higher rigidity of the introduced labeling agent, thus potentially enhancing the binding to a second reagent (such as CAR T cells). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shows the general concept of site-specific conjugation of a polypeptide using an ETAC reagent.

[0016] Figure 2 Shows the general concept of site-specific conjugation of a single-domain antibody using an ETAC reagent.

[0017] Figure 3 Shows the ESI-MS spectra of ETAC-biotin-labeled EGFR-specific VHH before and after the inverse Michael addition reaction according to Example 1.1.

[0018] Figure 4 Shows the peptide mass fingerprint (PMF) of ETAC-biotin-labeled EGFR-specific VHH according to Example 1.2, indicating the conjugation site.

[0019] Figure 5 Shows the binding of ETAC-biotin-labeled EGFR-specific VHH to antigen-positive cells according to Example 1.3.

[0020] Figure 6 Shows the specific killing of EGFR-positive tumor cells by biotin-specific adaptor CAR T cells in the presence of an ETAC-biotin-labeled EGFR-specific VHH adaptor molecule according to Example 1.4.

[0021] Figure 7 Shows the ESI-MS spectra of ETAC-biotin-labeled CD56-specific VHH before and after the inverse Michael addition reaction according to Examples 2.1 and 2.2.

[0022] Figure 8 Shows the binding of ETAC-biotin-labeled CD56-specific VHH to antigen-positive or antigen-negative cells according to Example 2.3. DETAILED DESCRIPTION

[0023] 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 reacts with C and amino acid X, or with amino acid X and a second amino acid X, in a Michael addition reaction. The ETAC coupled to amino acid X and the second amino acid X is removed by treating the reaction product with a Michael donor, such as a reducing agent.

[0024] The VHH contains a terminal cysteine and histidine sequence. To enable the VHH to couple with the ETAC reagent, the terminal cysteine is reduced while the intramolecular disulfide bond bridge remains intact. Next, in the Michael addition, the ETAC compound reacts with the terminal reduced cysteine and one of the histidines. Additionally, the ETAC compound couples only with histidine. The ETAC compound cross-linked only to histidine is removed from the VHH by treating the reaction product with a Michael donor, such as a reducing agent. The ETAC compound linked to cysteine and histidine is stable in the presence of a Michael donor and remains cross-linked.

[0025] In an embodiment of the present invention, at least one second amino acid is provided at the C-terminus of the polypeptide.

[0026] The nucleophile can be selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine (PPh3), hydrazine (N2H4), tetrakis(dimethylamino)ethylene (TDAE), mercaptoethanol, formic acid, trichlorosilane (HSiCl3), phosphine (PH3), dimethylsulfide borane (Me2S·BH3), thioacetic acid, tris(trimethylsilyl)phosphine, tri-n-hexylphosphine, ammonia (NH3), cysteine, selenocysteine, benzenethiol, glutathione (GSH), iodide, methylamine, hydrogen sulfide.

[0027] Preferably, the reagent has the formula (II) or the formula (III) wherein, R1 independently represents - biotin, thiamine, a peptide having 2 - 30 amino acids, an oligonucleotide having 6 - 100 nucleotides, - DBCO, DBCO, - N3, N3, - fluorophore, R2 independently represents a substituted or unsubstituted aromatic residue, R3 independently represents H, F, NO2, an alkyl group having 1 - 5 carbon atoms, R4 independently represents a direct bond, a substituted or unsubstituted alkyl, amine or amide residue having 1 - 20 carbon atoms, R5 independently represents a direct bond, a substituted or unsubstituted alkyl or amine residue having 1 to 20 carbon atoms, R6 independently represents a direct bond, a substituted or unsubstituted alkyl or amine residue having 1 to 20 carbon atoms, R7 independently represents a substituted or unsubstituted alkyl, carboxyl, amine or hydroxyl residue having 1 to 20 carbon atoms.

[0028] Most preferably, the reagent has one of the formulas (IV)-(VII)

[0029] Use of the method

[0030] The method of the present invention can be particularly used to provide polypeptides with detectable tags in CAR-linker molecules, cell staining reagents, cell separation / labeling / enrichment / depletion processes, protein purification, and for cell activation.

[0031] 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 US11021544B2 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.

[0032] Examples

[0033] Example 1. Site-specific coupling of EGFR-specific VHH with ETAC-biotin-linker

[0034] 1.1 Coupling of EGFR-specific VHH with multiple ETAC-biotin-linkers

[0035] As a model VHH, we used an anti-EGFR-specific VHH with a C-terminal histidine tag, followed by a flexible glycine-serine linker and a glycine-capped cysteine residue as a Michael donor.

[0036] The cysteine introduced at the end of EGFR VHH was reduced with 3 molar equivalents of TCEP at 21 °C for 1 h. This reaction was carried out in PBS buffer containing 5 mM EDTA (PE buffer) at pH 7.8. Next, TCEP was removed by passing through a desalting column containing Sephadex G-25 resin and PE buffer. Subsequently, the ETAC-biotin labeled molecule was added to the reduced and purified EGFR VHH. The ETAC-biotin labeled molecule was pre-dissolved in DMSO and added to EGFR VHH at 4 molar equivalents. Sufficient DMSO was added to the reaction mixture to bring the final concentration of DMSO in the final reaction mixture to 10%. The coupling reaction was maintained at 21 °C for 2 h. Next, the reaction mixture was applied to a molecular weight cut-off column (Merck Millipore) previously equilibrated with PBS buffer and washed with four column volumes of 1X PBS. The protein concentration of the final product was analyzed by absorption at 280 nm.

[0037] The product of the coupling reaction was analyzed by liquid chromatography coupled with mass spectrometry ( Figure 3 a). The analysis results showed that each VHH molecule was bound to 1-5 ETAC-biotin-linker units, and its main configuration consisted of two ETAC-biotin-linkers per VHH.

[0038] 1.2 Enrichment of single ETAC-biotin-linker-labeled EGFR-specific VHH by inverse Michael addition reaction

[0039] The ETAC-biotin tag that did not react with one cysteine was selectively removed via a retro-Michael reaction by adding a nucleophilic reducing agent. Thus, at 37 °C, 250 mM TCEP was added to every 13 μL of VHH for 10 min. This yielded a VHH polypeptide containing a single ETAC-biotin tag, which was confirmed by mass spectrometry to have an MW of 15905 Da ( Figure 3 b). After the TCEP step, the desired product was finally obtained.

[0040] The coupling site of ETAC-biotin with EGFR-specific VHH was analyzed by peptide mass fingerprint (PMF) ( Figure 4 ). PMF indicated that the ETAC-biotin tag was successfully coupled to the cysteine and histidine residues at the carboxyl terminus of VHH. This demonstrated that one terminal cysteine was linked to a histidine residue close to this terminal cysteine. This describes a unique site-specific labeling site that can be addressed by an ETAC-based crosslinker, providing an alternative motif for cysteine-cysteine.

[0041] 1.3 Use of ETAC-biotin-linker-labeled EGFR-specific VHH in flow cytometry

[0042] To demonstrate the application of ETAC-biotinylated EGFR-specific VHH in flow cytometry, EGFR-specific VHH molecules were used to detect tumor cells known to express EGFR. Thus, 10 μg / mL of ETAC-biotinylated EGFR-specific VHH was incubated with EGFR-positive tumor cells at 4 °C for 1 h. The cell suspension was washed with PE buffer (PEB buffer) containing 0.5% BSA and secondary stained with a PE-labeled biotin-specific secondary antibody (REA746). Next, the cell suspension was washed with PEB. By using the PE-labeled biotin-specific secondary antibody for secondary staining, the binding of ETAC-biotinylated EGFR-specific VHH to EGFR-positive tumor cells and the presence of ETAC-biotin-linker were detected. Compared with EGFR-positive tumor cells incubated with ETAC-biotinylated anti-EGFR VHH and biotin-specific PE-labeled secondary antibody ( Figure 5 bottom row histogram), the signal intensity of unstained tumor cells ( Figure 5 top row histogram) or cells stained only with biotin-specific secondary antibody ( Figure 5 middle row histogram) was significantly reduced. This verified the conjugation of ETAC-biotin-linker with EGFR-specific VHH. In addition, this data verified that the conjugation process did not disrupt the functional properties of EGFR-specific VHH that binds its epitope.

[0043] 1.4 Use of ETAC-biotin-linker-labeled EGFR-specific VHH as an adaptor molecule for CART cells (Adapter CART cells)

[0044] CAR T cells are immune cells that have been genetically engineered to express a chimeric antigen receptor (CAR) that does not naturally occur in T cells. The receptor is specific for a certain antigen expressed on the surface of tumor cells. After engaging with tumor cells, CAR T cells will induce tumor cell lysis. In contrast, adapter CAR T cells express a CAR that is specific for a tagged polypeptide and not specific for the antigen expressed by tumor cells. This polypeptide, such as VHH, will mediate the binding between adapter CAR T cells and tumor cells. In the absence of such an adapter, tumor cell lysis will not be induced. By using different adapter molecules, one type of CAR T cell can target more than one tumor antigen. This mode of action improves safety and therapeutic efficacy.

[0045] 1.4.1 Generation of ETAC-biotin-linker-specific adaptor CART cells

[0046] ETAC-biotin CAR T cells contain a biotin-specific scFv as the binding moiety. The scFv is linked to the human CD8 transmembrane domain via an hIgG4 hinge domain. The signal transduction domain consists of 4-1BB and CD3ζ.

[0047] 1.4.2 Generation and titration of LV particles

[0048] Lentiviral vector particles are produced by transient transfection of HEK-293T cells. The lentiviral vector particles are pseudotyped with VSV-G. For transfection, HEK-293T cells are seeded in DMEM supplemented with 2 mM L-glutamine and 10% FCS in a T175 culture flask 3 days prior to transfection. On the day of transfection, the medium is removed and replaced with DMEM supplemented with 2 mM L-glutamine. The cells are transfected with a three-plasmid system encoding VSV-G, gag / pol / rev, and a psi-positive transfer vector.

[0049] After 48 h, the supernatant is collected and centrifuged at 1000 rpm for 10 min to remove cell debris. In addition, the supernatant is filtered through a 0.45-μm filter. The pellet is resuspended in ice-cold PBS and stored at -80 °C.

[0050] 1.4.3 Transduction, culture and analysis of ETAC-biotin-linker-specific adaptor CART cells

[0051] Anti-biotin linker CAR T cells are produced using primary T cells from healthy donors. T cells are isolated from PBMCs using the PAN T cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Prior to transduction, 2E6 T cells are seeded in a 24-well plate containing 2 mL of TexMACS medium (Miltenyi) supplemented with IL-7 (Miltenyi Biotec), IL-15 (Miltenyi Biotec), and TransAct (Miltenyi Biotec).

[0052] After 24 h, the T cells are transduced at an MOI of 5 by adding the appropriate volume of lentiviral vector particles. On the third day after activation, the medium is removed and replaced with TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec).

[0053] On the sixth day after transduction, the frequency of anti-biotin linker CAR-positive T cells is analyzed by flow cytometry using a biotinylated PE conjugate (Miltenyi Biotec). On the tenth day after transduction, the anti-biotin linker CAR T cells are used for functional assays.

[0054] 1.5 Functionality of EGFR-specific VHH as an adaptor molecule for ETAC-biotin-linker-specific adaptor CART cells Figure 6

[0055] To test the application of ETAC-biotinylated EGFR-specific VHH as a bridging molecule for biotin-specific anti-biotin adaptor CAR T cells, antigen-positive tumor cells and ETAC-biotin-labeled EGFR-specific VHH were cultured in the presence or absence of biotin-specific adaptor CAR T cells. Thus, 20,000 biotin-specific adaptor 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.

[0056] Next, 50 μL of ETAC-biotin-labeled EGFR-specific VHH was added to a final concentration of 5 nM. As a positive control, a functionalized tumor-specific bridging molecule was used. The co-culture was incubated at 37 °C and 5% CO 2 2. The S3 system was used to analyze tumor cell lysis by live cell fluorescence microscopy of tumor cells. The data shown were obtained from independent donors (n = 3) and plotted as mean and ±1 standard deviation ( Example 2. Site-specific coupling of CD56-specific VHH with ETAC-biotin-linker ).

[0057] Tumor cell lysis was induced only in the presence of biotin-specific adaptor CAR T cells. The potency of the ETAC-biotin-linked VHH was comparable to that of the control adaptor. These results indicate that tumor cells and adaptor CAR T cells are properly cross-linked by ETAC-biotinylated VHH.

[0058] In summary, this indicates that ETAC-biotin-linker-labeled EGFR-specific VHH is a functionalized bridging molecule for biotin-specific adaptor CAR T cells.

[0059] 2.1 Coupling of CD56-specific VHH with multiple ETAC-biotin-linkers

[0060] Figure 7

[0061] As a model VHH, we used an anti-CD56-specific VHH with a C-terminal histidine tag, followed by a flexible glycine-serine linker, and a glycine-capped cysteine residue as a Michael donor.

[0062] The cysteine introduced at the C-terminus of CD56 VHH was reduced with 3 molar equivalents of TCEP at 21 °C for 1 h. This reaction was carried out in PBS buffer containing 5 mM EDTA (PE buffer) at pH 7.8. Next, TCEP was removed by passing through a desalting column containing Sephadex G-25 resin and PE buffer. Subsequently, the ETAC-biotinylated molecule was added to the reduced and purified CD56 VHH. The ETAC-biotinylated molecule was pre-dissolved in DMSO and added to CD56 VHH at 4 molar equivalents. Sufficient DMSO was added to the reaction mixture to bring the final concentration of DMSO in the final reaction mixture to 10%. The coupling reaction was maintained at 21 °C for 2 h. Next, the reaction mixture was applied to a molecular weight cut-off column (Merck Millipore) pre-equilibrated with PBS buffer and washed with four column volumes of 1X PBS. The protein concentration of the final product was analyzed by absorption at 280 nm.

[0063] The product of the coupling reaction was analyzed by liquid chromatography coupled with mass spectrometry ( 2.2 Enrichment of single ETAC-biotin-linker-labeled CD56-specific VHH by inverse Michael addition reaction a). The analysis results showed that each VHH molecule was bound to 1 - 4 ETAC-biotin-linker units, and the major configuration consisted of two ETAC-biotin-linkers per VHH.

[0064] Figure 7

[0065] The ETAC-biotin tag that did not react with one cysteine was selectively removed via a retro-Michael reaction by adding a nucleophilic reducing agent. Thus, 250 mM TCEP was added to every 13 μL of VHH at 37 °C for 10 min. This yielded a VHH polypeptide containing a single ETAC-biotin tag, which was confirmed by mass spectrometry to have an MW of 16240 Da ( 2.3 Use of ETAC-biotin-linker-labeled CD56-specific VHH in flow cytometry b). After the TCEP step, the desired product was finally obtained.

[0066] Figure 8

[0067] To demonstrate the application of ETAC-biotinylated CD56 VHH in flow cytometry, CD56-specific VHH molecules were used to detect tumor cells known to express CD56. To demonstrate the specific binding of ETAC-biotinylated CD56-specific VHH, 5 μg / mL conjugated VHH was incubated with CD56-positive tumor cells and CD56-negative cells at 4 °C for 1 h. 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. By flow cytometry, the signal intensity of PE-positive tumor cells on antigen-positive and antigen-negative cells was analyzed. After incubation with ETAC-biotinylated CD56-specific VHH or CD56-specific IgG1 (REA196-PE) as a positive control, the frequency of CD56-positive cells on antigen-positive and antigen-negative cells was plotted. Specific binding of ETAC-biotinylated VHH was detected ( Figure 8 a). The CD56 ETAC-biotinylated VHH staining pattern was comparable to the positive control. Binding of ETAC-biotinylated CD56 VHH was not detected on antigen-negative cells by flow cytometry ( ​ b).

Claims

1. A method for providing a polypeptide having a detectable tag, wherein the polypeptide comprises at least 5 amino acids, wherein the first amino acid is cysteine ​​C, and at least one second amino acid is selected from serine S, threonine T, tyrosine Y, lysine K, histidine H and arginine R, characterized in that Follow these steps: a) initiating a Michael addition reaction between a first amino acid and a second amino acid by providing a reagent according to formula (I) in: R1 independently represents -biotin, thiamine, a peptide having 2-30 amino acids, an oligonucleotide having 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophore, or -fluorescent protein, R2 independently represents a substituted or unsubstituted aromatic residue, R3 independently represents H, F, NO2, an alkyl group having 1 to 5 carbon atoms, R4 independently represents a direct bond, a substituted or unsubstituted alkyl, amine or amide residue having 1 to 20 carbon atoms; b) adding a nucleophile to remove the Michael addition product between the two second amino acids by a reverse Michael addition reaction.

2. The method according to claim 1, characterized in that At least one second amino acid is provided at the C-terminus of the polypeptide.

3. The method according to claim 1 or 2, characterized in that: The nucleophilic reagent is selected from dithiothreitol (DTT), tri(2-carboxyethyl)phosphine (TCEP), triphenylphosphine (PPh3), hydrazine (N2H4), tetrakis(dimethylamino)ethylene (TDAE), mercaptoethanol, formic acid, trichlorosilane (HSiCl3), phosphine (PH3), dimethylborane sulfide (Me2S·BH3), thioacetic acid, tri(trimethylsilyl)phosphine, tri-n-hexylphosphine, ammonia (NH3), cysteine, selenocysteine, thiophenol, glutathione (GSH), iodide, methylamine, and hydrogen sulfide.

4. The method according to any one of claims 1 to 3, characterized in that The reagent has the formula (II), in: R1 is independently represented by -biotin, thiamine, a peptide having 2-30 amino acids, an oligonucleotide having 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophore, R2 independently represents a substituted or unsubstituted aromatic residue, R3 is independently represented by H, F, NO2, an alkyl group having 1 to 5 carbon atoms, R4 independently represents a direct bond, a substituted or unsubstituted alkyl, an amine or an amide residue having 1 to 20 carbon atoms, R5 independently represents a direct bond, a substituted or unsubstituted alkyl group or an amine residue having 1 to 20 carbon atoms, R6 independently represents a direct bond, a substituted or unsubstituted alkyl group or an amine residue having 1 to 20 carbon atoms, R7 independently represents a substituted or unsubstituted alkyl, carboxyl, amine or hydroxyl residue having 1 to 20 carbon atoms.

5. The method according to any one of claims 1 to 4, characterized in that The reagent has the formula (III), in: R1 independently represents -biotin, thiamine, a peptide having 2-30 amino acids, an oligonucleotide having 6-100 nucleotides, -DBCO, DBCO, -N3, N3, -fluorophore, R2 independently represents a substituted or unsubstituted aromatic residue, R3 independently represents H, F, NO2, an alkyl group having 1 to 5 carbon atoms, R4 independently represents a direct bond, a substituted or unsubstituted alkyl, amine or amide residue having 1 to 20 carbon atoms, R6 independently represents a direct bond, a substituted or unsubstituted alkyl group or an amine residue having 1 to 20 carbon atoms, R7 independently represents a substituted or unsubstituted alkyl, carboxyl, amine or hydroxyl residue having 1 to 20 carbon atoms.

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

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

8. Use of the method according to any one of claims 1 to 7 in CAR-engagement molecules, cell staining reagents, cell separation / labeling / enrichment / depletion processes, protein purification and providing polypeptides with detectable tags for cell activation.

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