Site-specific tyrosine modification of immunoglobulins

Abstract

The present disclosure provides a method of modifying an antibody by attaching a molecule with a functional handle to the antibody.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 416,373, filed on October 14, 2022, the entire disclosure of which is hereby incorporated by reference into this application for all purposes. Background Art

[0002] Functionalized antibodies can be used to form conjugates with small organic compounds (such as small molecule drugs) and biologics (such as proteins and peptides) as well as cells and tissues. In the biotechnology and pharmaceutical fields, technologies that can selectively functionalize antibodies are highly desired. Summary of the invention

[0003] In one aspect, the present disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprising: (a) incubating a molecule containing at least one tyrosine amino acid with a small molecule containing a terminal olefin and a functional handle and tyrosinase in a solution; (b) irradiating the solution of step (a) with visible wavelength light; (c) Purification of the modified molecules.

[0004] In some embodiments, the visible wavelength light ranges from 380 nm to 700 nm. In particular, in some embodiments, the visible wavelength light is about 456 nm.

[0005] In another aspect, the present disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprising: (a) incubating a molecule containing at least one tyrosine amino acid with a small molecule containing a terminal lactone and a functional handle and tyrosinase in a solution; (b) Purification of the modified molecules.

[0006] In some embodiments of these methods, the molecule containing tyrosine amino acids is an antibody. For example, the antibody can be IgG, IgA, IgD, IgE or IgM. In particular, in certain embodiments, the antibody is IgG, such as IgG1.

[0007] In certain embodiments, the tyrosine amino acid in the molecule modified with the functional handle is Y296 and / or Y300, where positions are determined according to EU numbering.

[0008] In some embodiments of these methods, prior to step (a), the method further comprises a step of incubating the molecule with a deglycosylase to remove sugar chains. In particular, in certain embodiments, the deglycosylase is peptidyl-N-glycosidase F (PNGaseF).

[0009] In some embodiments of these methods, the functional handle is an azide, an alkyne, biotin, or streptavidin. In particular, in certain embodiments, the molecule with the functional handle further comprises a detectable group (e.g., a fluorophore). In some embodiments, the molecule with the functional handle further comprises a hydrophobic group (e.g., DPSE).

[0010] In certain embodiments, the molecule with the functional handle is: Where n is an integer from 1 to 10.

[0011] In certain embodiments, the molecule with the functional handle is: Where n is an integer from 1 to 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A and 1B : The substrate scope of different peptides containing tyrosine. (A) The scope of different short peptides as starting materials. (B) The scope of different small molecules containing terminal olefins.

[0013] Figure 2A-2D: Selective modification of Trastuzumab using VE-N3 (a small molecule containing terminal olefin and azide functional handles) and DBCO-TAMRA (a cyclooctyne conjugated to a fluorophore) for live cell imaging. (A) Schematic diagram of two-step fluorescent labeling of Trastuzumab with VE-N3. (B) SDS-PAGE analysis of Trastuzumab-(VE)-TAMRA labeled by click reaction. Left, Coomassie Brilliant Blue staining; right, fluorescent image, excitation wavelength 365 nm. (C) Fluorescence signal of SK-OV-3 cells incubated with IgG-(VE)-TAMRA was analyzed by flow cytometry. (D) IgG-(VE)-TAMRA selectively stained SK-OV-3 cells.

[0014] Figure 3 : Tyrosine-selective modification of various peptides using small molecules containing terminal lactones.

[0015] Figures 4A-4C Shown is a kinetic study of a nucleophilic reaction using a small molecule containing a terminal lactone and an azide functional handle. (A) Schematic diagram of the reaction. (B) Analysis of the reaction at different times by SDS-PAGE. (C) Quantification of the reaction yield at different times.

[0016] Figures 5A-5C: Selective modification of trastuzumab using FuA-N3 (a small molecule containing terminal lactone and azide functional handles) and DBCO-TAMRA (a cyclooctyne conjugated to a fluorophore) for live cell imaging. (A) Schematic diagram of two-step fluorescent labeling of trastuzumab with FuA-N3. (B) SDS-PAGE analysis of trastuzumab-(FuA)-TAMRA labeled by click reaction. Left, Coomassie Brilliant Blue staining; right, fluorescence image, excitation wavelength 365 nm. (C) IgG-(FuA)-TAMRA selectively stains SK-OV-3 cells.

[0017] Figures 6A-6E : Synthesis of immunoliposome-labeled cells. (A) Schematic diagram of antibody-lipid conjugates. (B) SDS-PAGE analysis of antibody-lipid conjugates. (C) Schematic diagram of immunoliposome-fused cell generation. (D) Fluorescence image showing immunoliposome-fused cells. (E) Fluorescence intensity analysis of images.

[0018] Figures 7A-7G : Modification of THP1 cells and NK cells for nanobody- and antibody-directed cell-to-cell interactions. (A) Route for the synthesis of nbHER2-BPA. (B) Flow cytometric results of UV light-induced conjugation of nbHER2-BPA-AF 488 to THP1 cells. (C) Confocal image of THP1-nbHER2 interaction with HER2(+)SK-OV-3. (D) Cytotoxicity test by cell-to-cell interaction. Groups with asterisk labels (*) mean no light irradiation. (E) Route for the synthesis of Tras-BPA. (F) Flow cytometric results of UV light-induced conjugation of Tras-BPA-AF 488 to THP1 cells. (G) Cytotoxicity test by cell-to-cell interaction. Groups with asterisk labels (*) mean no light irradiation. DETAILED DESCRIPTION I. Introduction

[0019] The present disclosure relates to modifying antibodies by attaching molecules with functional handles to the antibodies. Once the antibodies are modified with the functional handles, they can be further conjugated with other molecules containing functional groups that can react with the functional handles on the antibodies to form covalent conjugates. The modified antibodies can be further attached to other proteins or cells, such as liposomes, to create proteins or cells that can target specific antigens through the attached antibodies. II. Methods

[0020] Reactions and methods described herein can attach molecules with functional handles to one or more tyrosine amino acids in antibodies (e.g., IgG1). The first reaction provides a visible light-induced cycloaddition reaction to attach a small molecule containing a terminal olefin and a functional handle to one or more tyrosine amino acids in an antibody. These methods include: (a) incubating a molecule containing at least one tyrosine amino acid (e.g., an antibody) with a small molecule containing a terminal olefin and a functional handle and tyrosinase in a solution; (b) irradiating the solution of step (a) under visible wavelength light; and (c) purifying the modified molecule. In some embodiments, the range of visible wavelength light is 380 nm to 700 nm (e.g., from 380 to 680 nm, from 380 to 660 nm, from 380 to 640 nm, from 380 to 620 nm, from 380 to 600 nm, from 380 to 580 nm, from 380 to 560 nm, from 380 to 540 nm, from 380 to 520 nm, from 380 to 500 nm, from 380 to 480 nm, from 380 to 460 nm, from 380 to 440 nm, from 380 to 420 nm). 700 nm, from 600 to 700 nm, from 620 to 700 nm, from 640 to 700 nm, from 660 to 700 nm, or from 680 to 700 nm). In particular, in some embodiments, the visible wavelength light is about 456 nm.

[0021] In this method for attaching a small molecule containing a terminal olefin and a functional handle to one or more tyrosine amino acids in a molecule via a photoinduced cycloaddition reaction, tyrosinase first oxidizes the tyrosine amino acid to o-quinone, and then reacts with the terminal olefin in the small molecule containing the functional handle under irradiation with visible wavelength light to form a covalent conjugate.

[0022] In some embodiments of this reaction, the molecule with the functional handle is: Where n is an integer from 1 to 10.

[0023] In a specific embodiment of a photoinduced cycloaddition reaction for attaching a small molecule containing a terminal olefin and a functional handle to one or more tyrosine amino acids in a molecule, the molecule with the functional handle can be:

[0024] In a specific embodiment of a photoinduced cycloaddition reaction for attaching a small molecule containing a terminal olefin and a functional handle to one or more tyrosine amino acids in a molecule, the molecule with the functional handle can be one of the following.

[0025] The present disclosure also provides a second reaction using a small molecule containing a terminal lactone and a functional handle. These methods include: (a) incubating a molecule containing at least one tyrosine amino acid with a small molecule containing a terminal lactone and a functional handle and tyrosinase in a solution; and (b) purifying the modified molecule. In this reaction, tyrosinase first oxidizes the tyrosine amino acid to o-quinone, and then reacts with the terminal lactone in the small molecule containing the functional handle to form a covalent conjugate. In some embodiments of this method, the molecule with the functional handle is: Where n is an integer from 1 to 10.

[0026] In certain embodiments, the molecule with the functional handle is:

[0027] In certain embodiments, the molecule with the functional handle is:

[0028] In some embodiments of the methods described herein, the molecule containing one or more tyrosine amino acids is an antibody, such as IgG, IgA, IgD, IgE or IgM. In certain embodiments, the antibody is an IgG, such as IgG1. In specific embodiments, the tyrosine amino acid in the modified antibody (e.g., IgG1) is Y296 and / or Y300, where the position is determined according to EU numbering. In certain embodiments, the tyrosine amino acid in the modified antibody (e.g., IgG1) is Y296. In certain embodiments, the tyrosine amino acid in the modified antibody (e.g., IgG1) is Y300. In certain embodiments, the tyrosine amino acid in the modified antibody (e.g., IgG1) is Y296 and Y300.

[0029] In some cases, prior to incubation with tyrosinase, the methods described herein also include a step of incubating the molecule with a deglycosylase to remove sugar chains in the molecule. This deglycosylation step can remove specific sugar chains in a molecule (e.g., an antibody, such as IgG1) to access tyrosine amino acids. For example, in the heavy chain of IgG1, not all tyrosine amino acids can be accessed by tyrosinase. The deglycosylase can cut, for example, the sugar chain at N297 so that Y296 and Y300 can be oxidized by tyrosinase. In a specific embodiment, the deglycosylase is peptidyl-N-glycosidase F (PNGase F).

[0030] In some embodiments of these methods, the functional handle can be an azide, an alkyne, biotin, or streptavidin. The azide functional handle can then react with an alkyne, such as a terminal alkyne or an internal alkyne (e.g., cyclooctyne). The terminal alkyne or internal alkyne (e.g., cyclooctyne) can then react with an azide. The biotin functional handle can then react with streptavidin. The streptavidin functional handle can then react with biotin. In certain embodiments, the functional handle can include a detectable group, such as a fluorophore (e.g., fluorescein, rhodamine, Texas Red, cyanine, Alexa Fluors, or coumarin). Examples of fluorophores are also described in the study of Wang et al., Adv Colloid Interface Sci 285:102286, 2020. In other embodiments, the molecule with the functional handle can further include a hydrophobic group, such as DSPE or other phospholipid derivatives. In certain embodiments, such hydrophobic groups allow the modified molecule (eg, a modified antibody) to be directly attached to a cell, for example, to a liposome by inserting the hydrophobic group into the lipid bilayer of the cell. Tyrosinase

[0031] In the present disclosure, tyrosinase is used to modify tyrosine amino acids in proteins (e.g., antibodies) to generate tyrosine amino acids modified with functional handles (e.g., azide, alkyne). In nature, tyrosinase is an oxidase and a rate-limiting enzyme that controls melanin production. The enzyme is primarily involved in two different reactions in melanin synthesis, also known as the Raper Mason pathway. First, there is the hydroxylation of monophenols, and second, the conversion of ortho-diphenols to the corresponding ortho-quinones. Finally, ortho-quinones undergo several reactions to ultimately form melanin (see, e.g., Kumar et al., Biochimie. 93(3):562–9, 2011). Tyrosinase is a copper-containing enzyme found in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine through oxidation. It is found inside melanosomes synthesized in skin melanocytes. In humans, tyrosinase is encoded by the TYR gene.

[0032] Tyrosinase catalyzes the ortho-hydroxylation of monophenols and the oxidation of ortho-diphenols to ortho-quinones. Distinguishing the enzyme activity into two different reactions simply distinguishes two reactions catalyzed by the same enzyme. Although both tyrosinase and laccase use molecular oxygen to oxidize very similar substrates, they can be distinguished. A typical property of tyrosinase is that it cannot catalyze the oxidation of p-diphenols. Tyrosinase is also able to catalyze the oxidation of the p-hydroxyphenyl group of tyrosine residues to form ortho-quinone. The ortho-quinone formed can react with functional groups such as amino, sulfhydryl, thioether, phenolic, indole and imidazole.

[0033] Tyrosinases have been isolated and studied from a variety of plant, animal, and fungal species. There is diversity in the structural properties, tissue distribution, and cellular location of tyrosinases from different species. No common tyrosinase protein structure has been found that is present in all species. The enzymes found in plant, animal, and fungal tissues often differ in their primary structure, size, glycosylation pattern, and activation properties. However, all tyrosinases share a binuclear type III copper center in their active site. In the active site, two copper atoms are each coordinated to three histidine residues.

[0034] Mammalian tyrosinase is a single-span transmembrane protein (see, e.g., Kwon et al., Proceedings of the National Academy of Sciences of the United States of America, PNAS 84(21):7473–7, 1987). In humans, tyrosinase is sorted into melanosomes, with the catalytically active domain of the protein located inside the melanosomes. Only a small portion of the protein that is enzymatically non-essential extends into the cytoplasm of melanocytes. The tyrosinase that can be used in the methods and compositions disclosed herein can have a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence shown in PDB ID: 2Y9W or EC 1.14.18.1. III. Bioorthogonal reactions

[0035] As described herein, once tyrosinase converts the p-hydroxyphenyl group of a tyrosine residue into an o-quinone, the o-quinone can react with a variety of small molecules with functional handles (e.g., photoinduced reactions or nucleophilic reactions). The small molecules with functional handles are then covalently bound to the o-quinone. The functional handles can serve as attachment points to a variety of different molecules.

[0036] In certain embodiments, the functional handle and its reaction partner constitute a bioorthogonal pair. Bioorthogonal chemical reactions are those reactions that can occur within living cells without interfering with the cellular biochemical processes while enabling the derivatization of the bioorthogonal functional handle to attach desired functional groups. Over the past decade, several such bioorthogonal reactions have been developed with varying reaction kinetics, selectivity, and chemical and biological inertness. Three bioorthogonal reactions stand out due to their speed. First, a modified version of the classic [3+2] Huisgen azide-alkyne cycloaddition reaction utilizes ring strain-activated alkynes, such as cyclooctyne and cycloheptyne, to accelerate the triazole formation reaction. This strain-promoted cycloaddition reaction can be performed at speeds up to 1 M even in the absence of Cu(I) catalysts, high temperatures, or pressures. -1 s -1 Second, the very fast inverse electron demand Diels-Alder cycloaddition between tetrazines and trans-cyclooctenes further pushed the boundaries of bioorthogonal reaction rates, achieving rates as high as 10 3 –10 4 M -1 s -1 Furthermore, a clever modification of the traditional copper-catalyzed azide-alkyne cycloaddition (CuAAC) introduced a series of water-soluble triazine ligands that can simultaneously accelerate the reaction and act as sacrificial reductants, suppressing the cytotoxicity typically associated with CuAAC. This cell-compatible CuAAC exhibits excellent reaction kinetics with pseudo-second-order rate constants as high as 10 per mole of copper. 6 M -1 s -1 While tension-promoted and Diels-Alder cycloadditions are compatible with intracellular labeling, CuAAC is currently limited to cell surface labeling because of the presence of copper-chelating thiols within cells and the difficulty of delivering all CuAAC reaction components into cells.

[0037] In some embodiments of the methods described herein, the functional handle is an azide, and its reaction partner is an alkyne (e.g., cyclooctyne (OCT), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dimethoxyazacyclooctyne (DIMAC), diphenylcyclooctyne (DIBO), diphenylazacyclooctyne (DIBAC), bis-arylazacyclooctyne (BARAC), bicyclononyne (BCN), 2,3,6,7-tetramethoxy-DIBO (TMDIBO), sulfonated DIBO (S-DIBO), carboxymethylmonophenylcyclooctyne (COMBO), and pyrrolecyclooctyne (PYRROC). Examples of cyclooctynes ​​that can react with azides are described in studies by Dommerholt et al., e.g., Top Curr Chem (Z) 374:16, 2016. IV. Application

[0038] In certain embodiments, modified molecules, such as modified antibodies (e.g., modified IgG1), can be attached to cells to provide targeting specificity to the cells. For example, the cell can be a liposome, which has been used as a drug delivery system. The modified antibody (e.g., modified IgG1) can be further modified by additional molecules containing reaction partners of the functional handle on the modified antibody. For example, if the functional handle on the modified antibody is an azide, the additional molecule can contain cyclooctyne and a hydrophobic group (e.g., DSPE). Once the additional molecule is attached to the modified antibody (e.g., modified IgG1), the hydrophobic group (e.g., DSPE) can be inserted into the lipid bilayer of the cell (e.g., liposome), ultimately fixing the antibody to the cell.

[0039] With antibodies attached to the cell surface, cells can be directly targeted to specific antigens or receptors targeted by the antibodies. For example, to further improve the specificity of liposomes, immunoliposomes can be developed by attaching modified antibodies created by the disclosed methods to the surface of liposomes. Liposomes conjugated with antibodies can achieve active tissue targeting by binding to tumor cell-specific receptors targeted by the antibodies on the surface of liposomes. Such modified antibody liposomes can be used for specific drug delivery to cancer cells, gene therapy, drug delivery across the blood-brain barrier, or molecular imaging. Example

[0040] The present disclosure will be described in more detail by specific examples. The following examples are for illustrative purposes only and are not intended to limit the present disclosure in any way. Experts in the technical field will readily identify various non-critical parameters that can be changed or modified to obtain substantially the same results. Example 1: Synthesis of small molecule reactants

[0041] Small molecules that react with o-quinone are synthesized via amide condensation reactions.

[0042] Responses induced by visible wavelength light:

[0043] In a round-bottom flask, 3-(vinyloxy)-1-propylamine (0.5mM), 2-azidoacetic acid (1.2 equivalents), EDCI (1.2 equivalents), HOAt (1.05 equivalents) and TEA (1.5 equivalents) were dissolved in DMF (5mL) in an ice bath for 1 hour. Afterwards, the reaction solution was stirred at room temperature for 12 hours. Then 30mL of water was added to the flask, and the product was extracted three times with ethyl acetate (10mL×3). The organic layer was washed twice with saturated NaCl solution (10mL) and dried over anhydrous Na2SO4. The organic layer was then concentrated and purified by column chromatography (PE / EA=2 / 1) to give 70 mg of a colorless oil (yield 76%).

[0044] Figure 1A Further shown are the reaction yields of various short peptides starting from tyrosine amino acid and modified after tyrosine amino acid was incubated with tyrosinase and small molecule terminal olefin under light irradiation. Figure 1B The reaction yields of different terminal olefin small molecules are shown. Reaction conditions: peptide (10mM) and tyrosinase (1.68μM) were incubated in PB buffer (0.2mM, pH6.5) at 4°C for 30 minutes, then VE (terminal olefin small molecule, 100mM) was added and irradiated at 456nm light for 5 minutes. The reaction yield was detected by the integrated area of ​​the product in HPLC analysis.

[0045] For nucleophilic reactions:

[0046] In a round-bottom flask, FuA-NHS (0.5 mmol), NH2-PEG3-N3 (1.2 equivalents), TEA (1.2 equivalents) were dissolved in DMF (5 mL). The reaction solution was stirred at room temperature for 12 hours. Then 30 mL of water was added to the flask, and the product was extracted three times with ethyl acetate (10 mL×3). The organic layer was washed twice with saturated NaCl solution (10 mL) and dried over anhydrous Na2SO4. The organic layer was then concentrated and purified by reverse phase high performance liquid chromatography (RP-HPLC) with a yield of 33%.

[0047] Figure 3 The selective modification of tyrosine with small molecules containing terminal lactones on various peptides and their yields are further demonstrated.Conditions: Peptides (1 mM) and FuA (small molecules containing terminal lactones, 2.0 mM) were incubated with tyrosinase (1.4 μM) at room temperature for 1 hour. Example 2: Photoinduced cycloaddition reaction for protein modification (VE-tyrosine reaction)

[0048] Deglycosylated trastuzumab (20 μL, 2.0 mg / mL, 40 μg in PBS, pH 6.5) was diluted with 20 μL PBS, pH 6.5, and incubated with VE-N3 (2 μL, 5 mM in DMSO) and mushroom tyrosinase (5.0 μL, 1.7 mg / mL in phosphate buffer, pH 6.0) at 4 °C and illuminated with 456 nm light (20 mW / cm 2 ) for 8 hours. After completion, the product was concentrated and small molecules were removed by a 30k MWCO filter. Afterwards, DBCO-TAMRA (5 μL, 2 mM in DMSO) was added and incubated at room temperature for 2 hours. FIG. 2A shows a schematic diagram of two-step fluorescent labeling of trastuzumab by VE-N3.

[0049] Trastuzumab-(VE)-TAMRA labeled by click reaction was analyzed by SDS-PAGE (Figure 2B). In addition, SK-OV-3 cells incubated with IgG-(VE)-TAMRA were analyzed by flow cytometry (Figure 2C). SK-OV-3 cells were incubated with PBS solution containing 200nM modified IgG at room temperature for 30 minutes. Figure 2D Further demonstrated selective staining of SK-OV-3 cells by IgG-(VE)-TAMRA. Conditions: SK-OV-3 cells were incubated with trastuzumab-(VE)-TAMRA at 37°C for 1 hour, then washed three times with PBS, and then imaged using confocal fluorescence microscopy (upper panel), and MDA-MB-231 was imaged under the same conditions (lower panel), scale bar, 20 μm. Example 3: Spontaneous nucleophilic addition reaction for antibody modification (FuA-Tyrosine reaction)

[0050] Deglycosylated trastuzumab (20 μL, 2.0 mg / mL, 40 μg in PBS at pH 6.5) was diluted with 20 μL PBS at pH 6.5 and incubated with FuA-PEG-N3 (2 μL, 5 mM in DMSO) and mushroom tyrosinase (5.0 μL, 1.7 mg / mL in phosphate buffer at pH 6.0) at 4 ° C for 8 hours. After completion, the product was concentrated and small molecules were removed by a 30k MWCO filter. Afterwards, DBCO-TAMRA (5 μL, 2 mM in DMSO) was added and incubated for 2 hours at room temperature.

[0051] Trastuzumab-(FuA)-TAMRA ( Figure 5A ) by SDS-PAGE( Figure 5A ) analysis. IgG-(FuA)-TAMRA selectively stained SK-OV-3 cells ( Figure 5BConditions: SK-OV-3 cells were incubated with trastuzumab-(FuA)-TAMRA at 37°C for 1 hour, then washed three times with PBS and imaged using confocal fluorescence microscopy (upper panel), MDA-MB-231 was imaged under the same conditions (lower panel), scale bar, 20 μm ( Figure 5C ). Example 4: Kinetic study of nucleophilic reactions

[0052] The antibody (Atezolizumab) was incubated with FuA-PEG-N3 (a small molecule containing terminal lactone and azide functional handles, 100 equivalents) in 1X PBS buffer (pH 7.4) at room temperature for different times. Figure 4A A schematic diagram of the reaction is shown. Figure 4B Analysis of the reactions at different times by SDS-PAGE is shown. Figure 4C It was shown that after about 16 hours, the conversion or labeling was nearly complete. Example 5: Liposome labeling

[0053] Synthetic immunoliposomes ( Fig. 6A ): 10 μM azide-labeled trastuzumab and 100 μM DSPE-PEG2000-DBCO (cyclooctyne labeled with lipid group DSPE attached via PEG) were incubated in PBS buffer (pH 7.4) at 37°C for 2 hours to prepare antibody-lipid conjugates. The reaction was analyzed by SDS-PAGE ( Figure 6B )monitor. Figure 6C A schematic diagram of immunoliposome-fused cells is shown. Figure 6C As shown, immunoliposomes were fused with SK-OV-3 cells to obtain labeled cells. SK-OV-3 cells were incubated with immunoliposomes (10 μM) in DMEM medium at 37°C for 10, 30 minutes. Cells were washed, fixed and imaged ( Fig.6D ). Fig. 6E Fluorescence intensity analysis of the experimental and control groups is shown. Example 6: Method

[0054] Antibody-cell conjugation. The cells (THP1 cells or NK 92 cells) to be conjugated were centrifuged at 300 g for 2 minutes. The supernatant was then removed and the cells were resuspended in approximately 1 mL of DPBS. The centrifugation step was repeated and the supernatant was removed to wash the cells. This was repeated until a total of three washes were completed. In the last wash, approximately 1×10 6 The cells were resuspended in 1 mL of DPBS and incubated with 20 μM nbHER2-BPA or Tras-BPA at 37°C for 10 min. Fig. 7A A schematic diagram of the synthesis of nbHER2-BPA is shown. Fig. 7E A schematic diagram of the synthesis of Tras-BPA is shown. Tubes containing cells and nbHER2-BPA or Tras-BPA were placed on ice and illuminated with 365 nm light at 20 mW / cm 2 Irradiation intensity for 20 minutes ( Fig. 7A ). After irradiation, add 1 mL of DPBS to each tube and centrifuge at approximately 300 g for 2 minutes. Wash cells for a total of two washes before proceeding to any downstream applications.

[0055] Flow cytometric analysis. nbHER2-BPA or Tras-BPA was labeled with AF488-NHS ester (10 μM) at room temperature for 60 min. Excess AF488-NHS ester was removed by desalting column. THP1 cells were then irradiated with 20 μM nbHER2-BPA / AF488 or Tras-BPA / AF488 under the above-mentioned 365 nm light. The group without nbHER2-BPA / AF488 and light irradiation and the group with nbHER2-BPA / AF488 but without light irradiation were set as control groups. After the reaction, the cells were washed three times with DPBS and counted 1×10 5 Cells were analyzed by flow cytometry ( Figure 7B and 7F ).

[0056] Fluorescence staining. SK-OV-3 cells and THP1 cells were stained with Mito Trakcer Red (Invitrogen) and DiO (dinonadecanediocyanate, Sigma-Aldrich), respectively, following the manufacturer's protocol. THP1 cells were conjugated with nbHER2-BPA as described above. Unlabeled THP1 cells were used as negative control. After washing with PBS, THP1 cells were resuspended in RPMI medium containing 10% FBS and then mixed with SK-OV-3 cells in the same medium and incubated at 37°C and 5% CO2. After 8 h, the cells were gently washed 3 times with PBS and imaged on a confocal microscope (SP8, Leica) ( Figure 7C ).

[0057] Cytotoxicity assay. Target cell cytotoxicity was assessed as described previously. Adherent tumor cells (SK-OV-3, MDA-MB-231) were cultured at 2.5 × 10 4The cells were seeded at a density of 10 cells / well in a 96-well flat-bottom plate and incubated overnight in complete RPMI medium at 37°C in a humidified atmosphere of 5% CO2. Target cells were incubated with nbHER2-NK-92 conjugate or Tras-NK-92 in NK 92 medium containing 0.2% interleukin-2 (IL-2) (purchased from PROCELL) at a 2:1 effector cell / target cell (E / T) ratio (1×10 5 cells / well) for 48 hours. TM LDH cytotoxicity assay kit (Invitrogen TM Cytotoxicity was measured by lactate dehydrogenase (LDH) released from dying target cells according to the manufacturer's instructions. Figure 7C ). Spontaneous LDH release was assessed using target cells and effector cells without antibody. Maximum target cell lysis was achieved by incubating the target cells with lysis buffer. The percentage of cytotoxicity to the target cells was calculated according to the following formula and Figure 7C Display:

Claims

1. A method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprising: (a) incubating a molecule containing at least one tyrosine amino acid with a small molecule containing a terminal olefin and a functional handle and tyrosinase in a solution; (b) irradiating the solution of step (a) with visible wavelength light; (c) Purification of the modified molecules.

2. The method of claim 1, wherein the visible wavelength light ranges from 380 nm to 700 nm.

3. The method of claim 2, wherein the visible wavelength light is approximately 456 nm.

4. A method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprising: (a) incubating a molecule containing at least one tyrosine amino acid with a small molecule containing a terminal lactone and a functional handle and tyrosinase in a solution; (b) Purification of the modified molecules.

5. The method according to any one of claims 1 to 4, wherein the molecule containing tyrosine amino acids is an antibody.

6. The method according to claim 5, wherein the antibody is IgG, IgA, IgD, IgE or IgM.

7. The method of claim 6, wherein the antibody is IgG.

8. The method of claim 7, wherein the antibody is IgG1.

9. The method according to claim 7 or 8, wherein the tyrosine amino acid in the molecule modified with the functional handle is Y296 and / or Y300, wherein the positions are determined according to EU numbering.

10. The method according to any one of claims 1 to 9, wherein before step (a), the method further comprises the step of incubating the molecule with a deglycosylation enzyme to remove sugar chains.

11. The method according to claim 10, wherein the deglycosylation enzyme is peptidyl-N-glycosidase F (PNGase F).

12. The method according to any one of claims 1 to 11, wherein the functional handle is an azide, an alkyne, biotin or streptavidin.

13. The method according to any one of claims 1 to 12, wherein the molecule with the functional handle further comprises a detectable group.

14. The method of claim 13, wherein the detectable group is a fluorophore.

15. The method according to any one of claims 1 to 14, wherein the molecule carrying the functional handle further comprises a hydrophobic group.

16. The method according to any one of claims 1 to 3 and 5 to 15, wherein the molecule with the functional handle is: Where n is an integer from 1 to 10.

17. A method according to any one of claims 4 to 16, wherein the molecule with the functional handle is: Where n is an integer from 1 to 10.

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