WO2024078487A1 - Site-specific tyrosine modification of immunoglobins - Google Patents
Site-specific tyrosine modification of immunoglobins Download PDFInfo
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- WO2024078487A1 WO2024078487A1 PCT/CN2023/123757 CN2023123757W WO2024078487A1 WO 2024078487 A1 WO2024078487 A1 WO 2024078487A1 CN 2023123757 W CN2023123757 W CN 2023123757W WO 2024078487 A1 WO2024078487 A1 WO 2024078487A1
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- molecule
- functional handle
- antibody
- cells
- amino acid
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 title claims description 38
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- 238000012986 modification Methods 0.000 title description 8
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- 238000000034 method Methods 0.000 claims abstract description 45
- -1 tyrosine amino acid Chemical class 0.000 claims description 35
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- 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
- C07K1/113—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 without change of the primary structure
Definitions
- Functionalized antibodies can be used to create conjugates with small organic molecules, such as small molecule drugs, and biologics, such as proteins and peptides, as well as cells and tissues. Techniques that can functionalize antibodies in a selective manner are much needed in the fields of biotechnology and pharmaceuticals.
- the disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:
- the visible wavelength light is from 380 nm to 700 nm. In particular embodiments, the visible wavelength light is about 456 nm.
- the disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:
- the molecule containing the tyrosine amino acid is an antibody.
- the antibody can be an IgG, IgA, IgD, IgE, or IgM.
- the antibody is an IgG, such as an IgG1.
- the tyrosine amino acid in the molecule being modified with the functional handle is Y296 and/or Y300, wherein the positions are determined according to EU numbering.
- the method further comprises a step of removing glycans by incubating the molecule with a deglycosylation enzyme.
- the deglycosylation enzyme is a peptide: N-glycosidase F (PNGase F) .
- the functional handle is an azide, an alkyne, a biotin, or a streptavidin.
- the molecule with the functional handle further comprises a detectable moiety (e.g., a fluorophore.
- the molecule with the functional handle further comprises a hydrophobic moiety (e.g., DPSE) .
- the molecule with the functional handle is: wherein n is an integer from 1 to 10.
- the molecule with the functional handle is: wherein n is an integer from 1 to 10.
- FIGS. 1A and 1B Substrate scope of different tyrosine containing peptides.
- A scope of different short peptides as starting material.
- B Scope of different small molecules containing a terminal alkene.
- FIGS. 2A-2D Selective modification of Trastuzumab by VE-N3 (small molecule with terminal alkene and azide functional handle) and DBCO-TAMRA (cyclooctyne conjugated to a fluorophore) for living cell imaging.
- A Schematic demonstration of the two-step fluorescent labelling of trastuzumab by VE-N3.
- B SDS-PAGE analysis of labelled trastuzumab- (VE) -TAMRA by click reaction. Left, Coomassie stain; right, fluorescent image, Ex, 365 nm.
- C Fluorescence signal analysis of SKOV3 cells incubated with IgG- (VE) -TAMRA by flow cytometry.
- D IgG- (VE) -TAMRA selectively stained SKOV3 cells.
- FIG. 3 Tyrosine selective modification of various peptides using small molecule with terminal lactone.
- FIGS. 4A-4C show kinetic study of the nucleophilic reaction using a small molecule with a terminal lactone and an azide functional handle.
- A Schematics of the reaction.
- B Analysis of the reaction at different times by SDS-PAGE.
- C Quantitative analysis of the reaction yield at different times.
- FIGS. 5A-5C Selective modification of Trastuzumab by FuA-N3 (small molecule with terminal lactone and azide functional handle) and DBCO-TAMRA (cyclooctyne conjugated to a fluorophore) for living cell imaging.
- A Schematic demonstration of the two-step fluorescent labelling of trastuzumab by FuA-N3.
- B SDS-PAGE analysis of labelled trastuzumab- (FuA) -TAMRA by click reaction. Left, Coomassie stain; right, fluorescent image, Ex, 365 nm.
- C IgG- (FuA) -TAMRA selectively stained SKOV3 cells.
- FIGS. 6A-6E Synthesis of immunoliposome labeled cells.
- A Schematic demonstration of antibody-lipid conjugate.
- B SDS-PAGE analysis of the antibody-lipid conjugate.
- C Schmatic demonstration of the generation of immunoliposome fused cells.
- D Fluorescent images showing immunoliposome fused cells.
- E Fluorescene intensity analysis of the images.
- FIGS. 7A-7G Decoration of THP1 cells and NK cells for nanobody and antibody directed cell cell interaction.
- A Synthetic route towards nbHER2-BPA.
- B Flowcytometry results of UV light induced conjugation of nbHER2-BPA-AF 488 to THP1 cells.
- C Confocal images of interaction of THP1-nbHER2 towards HER2 (+) SKOV3.
- D Cell cytotoxicity test via cell-cell interaction. Groups with star label (*) mean no light irradiation.
- E Synthetic route towards Tras-BPA.
- F Flowcytometry results of UV light-induced conjugation of Tras-BPA-AF 488 to THP1 cells.
- G Cell cytotoxicity test via cell-cell interaction. Groups with star label (*) mean no light irradiation.
- the present disclosure is directed to modifying an antibody by attaching a molecule with a functional handle to the antibody.
- the antibody once modified with the functional handle, can be further conjugated to other molecules containing a functional group that can react with the functional handle on the antibody to form a covalent conjugate.
- the modified antibody can further be attached to other proteins or cells, such as liposomes, to create proteins or cells that can target specific antigens via the attached antibody.
- the first reaction provides a visible-light induced cycloaddition that attaches a small molecule with a terminal alkene and a functional handle to one or more tyrosine amino acids in an antibody.
- the methods comprise: (a) incubating a molecule (e.g., an antibody) comprising at least one tyrosine amino acid with a small molecule comprising a terminal alkene and a functional handle and a tyrosinase in a solution; (b) irradiate the solution of (a) under visible wavelength light; and (c) purify the modified molecule.
- a molecule e.g., an antibody
- the visible wavelength light is from 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, from 380 to 400 nm, from 400 to 700 nm, from 420 to 700 nm, from 440 to 700 nm, from 460 to 700 nm, from 480 to 700 nm, from 500 to 700 nm, from 520 to 700 nm, from 540 to 700 nm, from 560 to 700 nm, from 5
- the tyrosinase enzyme first oxidizes the tyrosine amino acids to for o-quinone, which then reacts with the terminal alkene in the small molecule containing the functional handle under the irradiation from visible-wavelength light to form a covalent conjugate.
- the molecule with the functional handle is: in which n is an integer from 1 to 10.
- the molecule with the functional handle can be:
- the molecule with the functional handle can be one of the following.
- the disclosure also provides a second reaction that uses a small molecule containing a terminal lactone and a functional handle.
- the methods comprise: (a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal lactone and the functional handle and a tyrosinase in a solution; and (b) purify the modified molecule.
- the tyrosinase enzyme first oxidizes the tyrosine amino acids to for o-quinone, which then reacts with the terminal lactone in the small molecule containing the functional handle to form a covalent conjugate.
- the molecule with the functional handle is: in which n is an integer from 1 to 10.
- the molecule with the functional handle is:
- the molecule with the functional handle is:
- the molecule containing one or more tyrosine amino acids is an antibody, such as an IgG, IgA, IgD, IgE, or IgM.
- the antibody is an IgG, such as an IgG1.
- the tyrosine amino acid being modified in an antibody e.g., IgG1 is Y296 and/or Y300, wherein the positions are determined according to EU numbering.
- the tyrosine amino acid being modified in an antibody is Y296.
- the tyrosine amino acid being modified in an antibody is Y300. In certain embodiments, the tyrosine amino acids being modified in an antibody (e.g., IgG1) are Y296 and Y300.
- the methods described herein further comprise a step of removing glycans in the molecule by incubating the molecule with a deglycosylation enzyme.
- This step of deglycosylation removes the certain glycans in the molecule (e.g., an antibody, e.g., IgG1) to provide access to tyrosine amino acids.
- an antibody e.g., IgG1
- not all tyrosine amino acids are accessible by the tyrosinase enzyme.
- a deglycosylation enzyme can cleave, for example, the glycan group at N297, which provides access to Y296 and Y300 to be oxidized by the tyrosinase enzyme.
- the deglycosylation enzyme is a peptide: N-glycosidase F (PNGase F) .
- a functional handle can be an azide, an alkyne, a biotin, or a streptavidin.
- An azide functional handle can subsequently react with an alkyne, such as a terminal alkyne or an internal alkyne (e.g., a cyclooctyne) .
- An alkyne e.g., a terminal alkyne or an internal alkyne (e.g., a cyclooctyne)
- a biotin functional handle can subsequently react with a streptavidin.
- a streptavidin functional handle can subsequently reaction with a biotin.
- the functional handle can contain a detectable moiety, such as a fluorophore (e.g., fluorescein, rhodamine, Texas Red, cyanine dyes, Alexa Fluors, or courmarin) .
- a fluorophore e.g., fluorescein, rhodamine, Texas Red, cyanine dyes, Alexa Fluors, or courmarin
- fluorophores are also described in, e.g., Wang et al., Adv Colloid Interface Sci 285: 102286, 2020.
- the molecule with the functional handle can further contain a hydrophobic moiety, such as DSPE or other phospholipid derivatives.
- such hydrophobic moieties can allow the attachment of the modified molecule (e.g., modified antibody) directly to a cell, e.g., a liposome, by way of insertion of the hydrophobic moiety into the lipid bilayer of the cell.
- modified molecule e.g., modified antibody
- tyrosinase is used to modify tyrosine amino acids in a protein (e.g., an antibody) to generate tyrosine amino acids that are modified with a functional handle (e.g., azide, alkyne) .
- a functional handle e.g., azide, alkyne
- tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin.
- the enzyme is mainly involved in two distinct reactions of melanin synthesis, also known as the Raper Mason pathway. Firstly, the hydroxylation of a monophenol, and secondly, the conversion of an o-diphenol to the corresponding o-quinone.
- Tyrosinase is a copper-containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation. It is found inside melanosomes which are synthesized in the skin melanocytes. In humans, the tyrosinase enzyme is encoded by the TYR gene.
- Tyrosinase catalyses both the o-hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones.
- the differentiation of the enzymatic activity into two distinct reactions only distinguishes between two reactions catalyzed by the same enzyme.
- Tyrosinases can be distinguished from laccases although both use molecular oxygen to oxidize quite similar substrates.
- a typical property of tyrosinase is inability to catalyse the oxidation of p-diphenols.
- Tyrosinase is also able to catalyze the oxidation of the p-hydroxyphenyl group of tyrosine residues, generating o-quinones.
- the result o-quinones can react with the, e.g., amino, sulfhydryl, thioether, phenolic, indole, and imidazole functional groups.
- Tyrosinases have been isolated and studied from a wide variety of plant, animal, and fungal species. Tyrosinases from different species are diverse in terms of their structural properties, tissue distribution, and cellular location. No common tyrosinase protein structure occurring across all species has been found. The enzymes found in plant, animal, and fungal tissue frequently differ with respect to their primary structure, size, glycosylation pattern, and activation characteristics. However, all tyrosinases have in common a binuclear, type 3 copper centre within their active sites. In the active site, two copper atoms are each coordinated with three histidine residues.
- Mammalian tyrosinase is a single membrane-spanning transmembrane protein (see, e.g., Kwon et al., Proceedings of the National Academy of Sciences, PNAS 84 (21) : 7473–7, 1987.
- tyrosinase is sorted into melanosomes and the catalytically active domain of the protein resides within melanosomes. Only a small, enzymatically inessential part of the protein extends into the cytoplasm of the melanocyte.
- Tyrosinases that can be used in methods and compositions disclosed herein can have a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence as shown in PDB ID.: 2Y9W or EC 1.14.18.1.
- the o-quinone can react with a number of small molecules with a functional handle (e.g., light induced reaction or nucleophilic reaction) .
- a functional handle e.g., light induced reaction or nucleophilic reaction
- the small molecule with the functional handle is then covalently joined to the o-quinone.
- the functional handle can serve as a point of attachment to a large variety of different molecules.
- the functional handle and its reactive partner form a bioorthgonal pair.
- Bioorthogonal chemical reactions are those that can occur in a living cell without interfering with the cell’s biochemical processes while enabling the derivatization of a bioorthogonal functional handle with a desired moiety.
- bioorthogonal reactions have been developed in the past decade with varying kinetics, selectivity, and chemical and biological inertness. Three bioorthogonal reactions stand out due to their speed.
- Such cell-compatible CuAAC has excellent reaction kinetics with pseudosecond-order rate constants of up to 10 6 M -1 s -1 per molar Cu. Both strain-promoted and Diels–Alder cycloadditions are compatible with intracellular labeling whereas CuAAC is currently limited to labeling at the cell surface due to the presence of Cu-chelating thiols inside cells and the difficulty in delivering all of the CuAAC reaction components into cells.
- the functional handle is an azide and its reactive partner is an alkyne (e.g., cyclooctyne (OCT) , monofluorinated cyclooctyne (MOFO) , difluorocyclooctyne (DIFO) , dimethoxyazacyclooctyne (DIMAC) , dibenzocyclooctyne (DIBO) , dibenzoazacyclooctyne (DIBAC) , biarylazacyclooctynone (BARAC) , bicyclononyne (BCN) , 2, 3, 6, 7-tetramethoxy-DIBO (TMDIBO) , sulfonylated DIBO (S-DIBO) , carboxymethylmonobenzocyclooctyne (COMBO) , and pyrrolocyclooctyne (PY) cyclooctyne
- the modified molecule such as a modified antibody (e.g., a modified IgG1)
- a modified antibody e.g., a modified IgG1
- the cell can be a liposome, which has bee used as a drug delivery system.
- a modified antibody e.g., a modified IgG1
- the additional molecule can contain a cyclooctyne and a hydrophobic moiety (e.g., DSPE) .
- the hydrophobic moiety e.g., DSPE
- the hydrophobic moiety can insert itself into the lipid bilayer of the cell (e.g., a liposome) to finally attach the antibody onto the cell.
- the cell can be directly targeted to specific antigens or receptors targeted by the antibody.
- immunoliposomes can be developed by attaching modified antibodies created by the methods described herein to the surface of the liposomes.
- Antibody-conjugated liposomes can allow for an active tissue targeting through binding to tumor cell-specific receptors targeted by the antibody on the surface of the liposome.
- Such antibody modified liposomes can be used in specific drug delivery to cancer cells, gene therapy, drug delivery through blood brain barrier, or molecular imaging.
- the small molecules used to react with o-quinone is synthesized via amide condensation reaction.
- FIG. 1A further shows a variety of short peptides with a tyrosine amino acid as the starting material and the reaction yields after the tyrosine amino acid was modified by incubating with tyrosinase enzyme and a small molecule containing a terminal alkene under light irradiation.
- FIG. 1B shows the reaction yields with different small molecules with a terminal alkene.
- reaction conditions the peptide (10 mM) was incubated with tyrosinase (1.68 ⁇ M) in PB buffer (0.2 mM, pH 6.5) at 4 °C for 30 min, then VE (small molecule with the terminal alkene, 100 mM) was added and irradiated at 456 nm light for 5 min.
- VE small molecule with the terminal alkene, 100 mM
- FIG. 3 further shows tyrosine selective modification of various peptides using small molecule with a terminal lactone and their yields.
- Deglycosylated trastuzumab (20 ⁇ L, 2.0 mg/mL, 40 ⁇ g in PBS pH 6.5) was diluted with 20 ⁇ L PBS pH 6.5 and incubated with VE-N 3 1 (2 ⁇ L, 5 mM in DMSO) and mushroom tyrosinase (5.0 ⁇ L, 1.7 mg/mL in phosphate buffer pH 6.0) and irradiated under 456nm light (20 mW/cm 2 ) for 8 h at 4 °C. After completion, the product was concentrated, and small molecules were removed by 30k MWCO filters.
- FIG. 2A shows this schematic demonstration of the two-step fluorescent labelling of trastuzumab by VE-N3.
- FIG. 2B Labelled trastuzumab- (VE) -TAMRA by click reaction was analyzed by SDS-PAGE (FIG. 2B) . Further, SKOV3 cells incubated with IgG- (VE) -TAMRA was analyzed by flow cytometry (FIG. 2C) . The SKOV3 cells were incubated with the PBS solution containing 200 nM modified IgG for 30 min at room temperature. FIG. 2D further shows that IgG- (VE) -TAMRA selectively stained SKOV3 cells.
- SKOV3 cells were incubated with trastuzumab- (VE) -TAMRA at 37 °C for 1 h, followed by PBS washing for three times and then imaging using confocal fluorescence microscope (top) , MDA-MB-231 imaging using identical conditions (bottom) , scale bar, 20 ⁇ m.
- trastuzumab- (VE) -TAMRA trastuzumab- (VE) -TAMRA
- Deglycosylated trastuzumab (20 ⁇ L, 2.0 mg/mL, 40 ⁇ g in PBS pH 6.5) was diluted with 20 ⁇ L PBS pH 6.5 and incubated with FuA-PEG-N3 (2 ⁇ L, 5 mM in DMSO) and mushroom tyrosinase (5.0 ⁇ L, 1.7 mg/mL in phosphate buffer pH 6.0) for 8 h at 4 °C. After completion, the product was concentrated, and small molecules were removed by 30k MWCO filters. After that, DBCO-TAMRA (5 ⁇ L, 2 mM in DMSO) was added and incubated at room temperature for 2h.
- FIG. 5A Labelled trastuzumab- (FuA) -TAMRA by click reaction (FIG. 5A) was analyzed by SDS-PAGE (FIG. 5A) .
- IgG- (FuA) -TAMRA selectively stained SKOV3 cells (FIG. 5B) .
- FIG. 4A shows the schematics of the reaction.
- FIG. 4B shows analysis of the reaction at different times by SDS-PAGE.
- FIG. 4C shows that the conversion or labeling is near complete at around 16 hours.
- FIG. 6A To synthesize immunoliposomes (FIG. 6A) : 10 ⁇ M azide labeled trastuzumab and 100 ⁇ M DSPE-PEG2000-DBCO (cyclooctyne labeled with a lipid group DSPE via a PEG linker) were incubated in PBS buffer (pH 7.4) at 37 °C for 2 h to make the antibody-lipid conjugate. The reaction was monitored by SDS-PAGE (FIG. 6B) . The schmatics to show the generation of immunoliposome fused cells is shown in FIG. 6C. As shown in FIG. 6C, the immunoliposomes were fused with SK-OV-3 cells, resulting in labeled cells.
- FIG. 6E shows the fluorescene intensity analysis of experiment groups and control groups.
- FIG. 7A shows a schematic illustrating the synthesis of nbHER2-BPA.
- FIG. 7E shows a schematic illustrating the synthesis of Tras-BPA.
- the tube containing cells and nbHER2-BPA or Tras-BPA was put on ice and irradiated by 365 nm light 20 mW/cm 2 for 20 min (FIG. 7A) .
- After the irradiation add 1 mL of DPBS to each tube and spin the tube at ⁇ 300 g for 2 min. Wash the cells until a total of two washes have been performed and move on to any downstream applications.
- nbHER2-BPA or Tras-BPA were labeled with AF488-NHS ester (10 ⁇ M) at room temperature for 60 min. And the extra AF488-NHS ester was excluded by desalting columns. Then THP1 cells were incubated with 20 ⁇ M nbHER2-BPA/AF488 or Tras-BPA/AF488 and irradiated under 365 nm light as described above. The group without both 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 with DPBS 3 times and analyzed by flow cytometry in AF 488 channel by counting 1 ⁇ 10 5 cells (FIGS. 7B and 7F) .
- SKOV3 and THP1cells were stained with Mito Trakcer Red (Invitrogen) and Dio (Dioctadecyloxacarbocyanine perchlorate, Sigma-Aldrich) , respectively, following the manufacturer’s protocol.
- THP1 cells were conjugated with nbHER2-BPA as described above. Non-labeled THP1 cells were used as negative controls. After washing with PBS, the THP1 cells were resuspended with RPMI media with 10 %FBS, then mixed with SKOV3 cells in the same media and incubated at 37 °C and 5 %CO 2 . After 8 hours, cells were gently washed with PBS 3 times and imaged on a confocal microscope (SP8, Leica) (FIG. 7C) .
- SP8 Leica confocal microscope
- Target cell cytotoxicity was evaluated as previously described.
- Adherent tumor cells (SKOV3, MDAMB231) were seeded at 2.5 ⁇ 10 4 cells/well in 96-well flat-bottom plates in the complete RPMI medium and incubated overnight at 37°C in a humidified 5%CO 2 atmosphere.
- Target cells were incubated with nbHER2-NK-92 conjugate or Tras-NK-92 for 48 h in NK 92 culture medium containing 0.2%interleukin-2 (IL-2) (Purchasing from PROCELL) at 37°C, 5%CO 2 in a 2: 1 Effector cells/Target cells (E/T) ratio (1 ⁇ 10 5 cells/well) .
- IL-2 interleukin-2
- LDH lactate dehydrogenase
- FIG. 7C Cellular cytotoxicity was measured via the release of lactate dehydrogenase (LDH) from dead target cells by using CyQUANT TM LDH Cytotoxicity Assay (Invitrogen TM ) according to the manufacturer’s instructions (FIG. 7C) . Spontaneous LDH release was assessed using target and effector cells without antibodies. Maximal target cell lysis was achieved by incubation of target cells with lysis buffer. The percentage of cytotoxicity towards target cells was calculated based on the following formula and shown in FIG. 7C:
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Abstract
Provided herein are methods for modifying an antibody by attaching a molecule with a functional handle to the antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application No. 63/416,373, filed October 14, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE DISCLOSURE
Functionalized antibodies can be used to create conjugates with small organic molecules, such as small molecule drugs, and biologics, such as proteins and peptides, as well as cells and tissues. Techniques that can functionalize antibodies in a selective manner are much needed in the fields of biotechnology and pharmaceuticals.
BRIEF SUMMARY OF THE DISCLOSURE
In one aspect, the disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:
(a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal alkene and the functional handle and a tyrosinase in a solution;
(b) irradiate the solution of (a) under visible wavelength light; and
(c) purify the modified molecule.
In some embodiments, the visible wavelength light is from 380 nm to 700 nm. In particular embodiments, the visible wavelength light is about 456 nm.
In another aspect, the disclosure provides a method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:
(a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal lactone and the functional handle and a tyrosinase in a solution; and
(b) purify the modified molecule.
In some embodiments of the methods, the molecule containing the tyrosine amino acid is an antibody. For example, the antibody can be an IgG, IgA, IgD, IgE, or IgM. In particular embodiments, the antibody is an IgG, such as an IgG1.
In some embodiments, the tyrosine amino acid in the molecule being modified with the functional handle is Y296 and/or Y300, wherein the positions are determined according to EU numbering.
In some embodiments of the methods, wherein, prior to step (a) , the method further comprises a step of removing glycans by incubating the molecule with a deglycosylation enzyme. In particular embodiments, the deglycosylation enzyme is a peptide: N-glycosidase F (PNGase F) .
In some embodiments of the methods, the functional handle is an azide, an alkyne, a biotin, or a streptavidin. In particular embodiments, the molecule with the functional handle further comprises a detectable moiety (e.g., a fluorophore. In some embodiments, the molecule with the functional handle further comprises a hydrophobic moiety (e.g., DPSE) .
In particular embodiments, the molecule with the functional handle is: wherein n is an integer from 1 to 10.
In particular embodiments, the molecule with the functional handle is: wherein n is an integer from 1 to 10.
FIGS. 1A and 1B: Substrate scope of different tyrosine containing peptides. (A) scope of different short peptides as starting material. (B) Scope of different small molecules containing a terminal alkene.
FIGS. 2A-2D: Selective modification of Trastuzumab by VE-N3 (small molecule with terminal alkene and azide functional handle) and DBCO-TAMRA (cyclooctyne conjugated to a fluorophore) for living cell imaging. (A) Schematic demonstration of the two-step fluorescent labelling of trastuzumab by VE-N3. (B) SDS-PAGE analysis of labelled trastuzumab- (VE) -TAMRA by click reaction. Left, Coomassie stain; right, fluorescent image, Ex, 365 nm. (C) Fluorescence signal analysis of SKOV3 cells incubated with IgG- (VE) -TAMRA by flow cytometry. (D) IgG- (VE) -TAMRA selectively stained SKOV3 cells.
FIG. 3: Tyrosine selective modification of various peptides using small molecule with terminal lactone.
FIGS. 4A-4C show kinetic study of the nucleophilic reaction using a small molecule with a terminal lactone and an azide functional handle. (A) Schematics of the reaction. (B) Analysis of the reaction at different times by SDS-PAGE. (C) Quantitative analysis of the reaction yield at different times.
FIGS. 5A-5C: Selective modification of Trastuzumab by FuA-N3 (small molecule with terminal lactone and azide functional handle) and DBCO-TAMRA (cyclooctyne conjugated to a fluorophore) for living cell imaging. (A) Schematic demonstration of the two-step fluorescent labelling of trastuzumab by FuA-N3. (B) SDS-PAGE analysis of labelled trastuzumab- (FuA) -TAMRA by click reaction. Left, Coomassie stain; right, fluorescent image, Ex, 365 nm. (C) IgG- (FuA) -TAMRA selectively stained SKOV3 cells.
FIGS. 6A-6E: Synthesis of immunoliposome labeled cells. (A) Schematic demonstration of antibody-lipid conjugate. (B) SDS-PAGE analysis of the antibody-lipid conjugate. (C) Schmatic demonstration of the generation of immunoliposome fused cells. (D) Fluorescent images showing immunoliposome fused cells. (E) Fluorescene intensity analysis of the images.
FIGS. 7A-7G: Decoration of THP1 cells and NK cells for nanobody and antibody directed cell cell interaction. (A) Synthetic route towards nbHER2-BPA. (B) Flowcytometry results of UV light induced conjugation of nbHER2-BPA-AF 488 to THP1 cells. (C) Confocal images of interaction of THP1-nbHER2 towards HER2 (+) SKOV3. (D) Cell cytotoxicity test via cell-cell interaction. Groups with star label (*) mean no light irradiation. (E) Synthetic route towards Tras-BPA. (F) Flowcytometry results of UV light-induced conjugation of Tras-BPA-AF 488 to THP1 cells. (G) Cell cytotoxicity test via cell-cell interaction. Groups with star label (*) mean no light irradiation.
DETAILED DESCRIPTION OF THE DISCLOSURE
I. Introduction
The present disclosure is directed to modifying an antibody by attaching a molecule with a functional handle to the antibody. The antibody, once modified with the functional handle, can be further conjugated to other molecules containing a functional group that can react with the functional handle on the antibody to form a covalent conjugate. The modified
antibody can further be attached to other proteins or cells, such as liposomes, to create proteins or cells that can target specific antigens via the attached antibody.
II. Methods
Described herein are reactions and methods that can attach a molecule with a functional handle to one or more tyrosine amino acids in an antibody (e.g., IgG1) . The first reaction provides a visible-light induced cycloaddition that attaches a small molecule with a terminal alkene and a functional handle to one or more tyrosine amino acids in an antibody. The methods comprise: (a) incubating a molecule (e.g., an antibody) comprising at least one tyrosine amino acid with a small molecule comprising a terminal alkene and a functional handle and a tyrosinase in a solution; (b) irradiate the solution of (a) under visible wavelength light; and (c) purify the modified molecule. In certain embodiments, the visible wavelength light is from 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, from 380 to 400 nm, from 400 to 700 nm, from 420 to 700 nm, from 440 to 700 nm, from 460 to 700 nm, from 480 to 700 nm, from 500 to 700 nm, from 520 to 700 nm, from 540 to 700 nm, from 560 to 700 nm, from 580 to 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 embodiments, the visible wavelength light is about 456 nm.
In this method of light-induced cycloaddition to attach a small molecule containing a terminal alkene and a functional handle to one or more tyrosine amino acids in a molecule, the tyrosinase enzyme first oxidizes the tyrosine amino acids to for o-quinone, which then reacts with the terminal alkene in the small molecule containing the functional handle under the irradiation from visible-wavelength light to form a covalent conjugate.
In some embodiments of this reaction, the molecule with the functional handle is: in which n is an integer from 1 to 10.
In particular embodiments of the method of light-induced cycloaddition to attach a small molecule containing a terminal alkene and a functional handle to one or more tyrosine amino acids in a molecule, the molecule with the functional handle can be:
In particular embodiments of the method of light-induced cycloaddition to attach a small molecule containing a terminal alkene 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.
The disclosure also provides a second reaction that uses a small molecule containing a terminal lactone and a functional handle. The methods comprise: (a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal lactone and the functional handle and a tyrosinase in a solution; and (b) purify the modified molecule. In this reaction, the tyrosinase enzyme first oxidizes the tyrosine amino acids to for o-quinone, which 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: in which n is an integer from 1 to 10.
In particular embodiments, the molecule with the functional handle is:
In particular embodiments, the molecule with the functional handle is:
In some embodiments of the methods described herein, the molecule containing one or more tyrosine amino acids is an antibody, such as an IgG, IgA, IgD, IgE, or IgM. In certain embodiments, the antibody is an IgG, such as an IgG1. In particular embodiments, the tyrosine amino acid being modified in an antibody (e.g., IgG1) is Y296 and/or Y300, wherein the positions are determined according to EU numbering. In certain embodiments, the tyrosine amino acid being modified in an antibody (e.g., IgG1) is Y296. In certain embodiments, the tyrosine amino acid being modified in an antibody (e.g., IgG1) is Y300. In certain embodiments, the tyrosine amino acids being modified in an antibody (e.g., IgG1) are Y296 and Y300.
In some cases, prior to incubating the molecule with the tyrosinase enzyme, the methods described herein further comprise a step of removing glycans in the molecule by incubating the molecule with a deglycosylation enzyme. This step of deglycosylation removes the certain glycans in the molecule (e.g., an antibody, e.g., IgG1) to provide access to tyrosine amino acids. For example, in the heavy chain of IgG1, not all tyrosine amino acids are accessible by the tyrosinase enzyme. A deglycosylation enzyme can cleave, for example, the glycan group at N297, which provides access to Y296 and Y300 to be oxidized by the tyrosinase enzyme. In particular embodiments, the deglycosylation enzyme is a peptide: N-glycosidase F (PNGase F) .
In some embodiments of the methods, a functional handle can be an azide, an alkyne, a biotin, or a streptavidin. An azide functional handle can subsequently react with an alkyne, such as a terminal alkyne or an internal alkyne (e.g., a cyclooctyne) . An alkyne (e.g., a terminal alkyne or an internal alkyne (e.g., a cyclooctyne) ) can subsequently react with an azide. A
biotin functional handle can subsequently react with a streptavidin. A streptavidin functional handle can subsequently reaction with a biotin. In certain embodiments, the functional handle can contain a detectable moiety, such as a fluorophore (e.g., fluorescein, rhodamine, Texas Red, cyanine dyes, Alexa Fluors, or courmarin) . Examples of fluorophores are also described in, e.g., Wang et al., Adv Colloid Interface Sci 285: 102286, 2020. In yet other embodiments, the molecule with the functional handle can further contain a hydrophobic moiety, such as DSPE or other phospholipid derivatives. In certain embodiments, such hydrophobic moieties can allow the attachment of the modified molecule (e.g., modified antibody) directly to a cell, e.g., a liposome, by way of insertion of the hydrophobic moiety into the lipid bilayer of the cell.
Tyrosinase
In the present disclosure, tyrosinase is used to modify tyrosine amino acids in a protein (e.g., an antibody) to generate tyrosine amino acids that are modified with a functional handle (e.g., azide, alkyne) . In nature, tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin. The enzyme is mainly involved in two distinct reactions of melanin synthesis, also known as the Raper Mason pathway. Firstly, the hydroxylation of a monophenol, and secondly, the conversion of an o-diphenol to the corresponding o-quinone. Finally, o-quinone undergoes several reactions to eventually form melanin (see, e.g., Kumar et al., Biochimie. 93 (3) : 562–9, 2011) . Tyrosinase is a copper-containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation. It is found inside melanosomes which are synthesized in the skin melanocytes. In humans, the tyrosinase enzyme is encoded by the TYR gene.
Tyrosinase catalyses both the o-hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones. The differentiation of the enzymatic activity into two distinct reactions only distinguishes between two reactions catalyzed by the same enzyme. Tyrosinases can be distinguished from laccases although both use molecular oxygen to oxidize quite similar substrates. A typical property of tyrosinase is inability to catalyse the oxidation of p-diphenols. Tyrosinase is also able to catalyze the oxidation of the p-hydroxyphenyl group of tyrosine residues, generating o-quinones. The result o-quinones can react with the, e.g., amino, sulfhydryl, thioether, phenolic, indole, and imidazole functional groups.
Tyrosinases have been isolated and studied from a wide variety of plant, animal, and fungal species. Tyrosinases from different species are diverse in terms of their structural
properties, tissue distribution, and cellular location. No common tyrosinase protein structure occurring across all species has been found. The enzymes found in plant, animal, and fungal tissue frequently differ with respect to their primary structure, size, glycosylation pattern, and activation characteristics. However, all tyrosinases have in common a binuclear, type 3 copper centre within their active sites. In the active site, two copper atoms are each coordinated with three histidine residues.
Mammalian tyrosinase is a single membrane-spanning transmembrane protein (see, e.g., Kwon et al., Proceedings of the National Academy of Sciences, PNAS 84 (21) : 7473–7, 1987. In humans, tyrosinase is sorted into melanosomes and the catalytically active domain of the protein resides within melanosomes. Only a small, enzymatically inessential part of the protein extends into the cytoplasm of the melanocyte. Tyrosinases that can be used in methods and compositions disclosed herein can have a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence as shown in PDB ID.: 2Y9W or EC 1.14.18.1.
III. Bioorthogonal Reactions
As described herein, once the tyrosinase converts the p-hydroxyphenyl group of tyrosine residues into o-quinone, the o-quinone can react with a number of small molecules with a functional handle (e.g., light induced reaction or nucleophilic reaction) . The small molecule with the functional handle is then covalently joined to the o-quinone. The functional handle can serve as a point of attachment to a large variety of different molecules.
In certain embodiments, the functional handle and its reactive partner form a bioorthgonal pair. Bioorthogonal chemical reactions are those that can occur in a living cell without interfering with the cell’s biochemical processes while enabling the derivatization of a bioorthogonal functional handle with a desired moiety. Several such bioorthogonal reactions have been developed in the past decade with varying kinetics, selectivity, and chemical and biological inertness. Three bioorthogonal reactions stand out due to their speed. First, an adaptation of the classic [3+2] Huisgen azide-alkyne cycloaddition makes use of alkynes activated by ring strain, such as cyclooctynes and cycloheptynes, to accelerate the triazole-forming reaction. Such strain-promoted cycloadditions, even without catalysts such as Cu (I) , high temperature, or pressure, can proceed quite efficiently with second-order rate constants of up to 1 M-1 s-1. Second, a very fast inverse-electron demand Diels-Alder cycloaddition between tetrazines and trans cyclooctenes further pushes the boundary of a bioorthogonal reaction rates,
achieving second-order rate constants of up to 103–104 M-1 s-1. Further, a clever modification of the traditional copper-catalyzed azide-alkyne cycloaddition (CuAAC) introduces a series of water-soluble tris-triazole ligands that can simultaneously accelerate the reaction and act as sacrificial reductants, quelling cytotoxicity generally associated with CuAAC. Such cell-compatible CuAAC has excellent reaction kinetics with pseudosecond-order rate constants of up to 106 M-1 s-1 per molar Cu. Both strain-promoted and Diels–Alder cycloadditions are compatible with intracellular labeling whereas CuAAC is currently limited to labeling at the cell surface due to the presence of Cu-chelating thiols inside cells and the difficulty in delivering all of the CuAAC reaction components into cells.
In some embodiments of the methods described herein, the functional handle is an azide and its reactive partner is an alkyne (e.g., cyclooctyne (OCT) , monofluorinated cyclooctyne (MOFO) , difluorocyclooctyne (DIFO) , dimethoxyazacyclooctyne (DIMAC) , dibenzocyclooctyne (DIBO) , dibenzoazacyclooctyne (DIBAC) , biarylazacyclooctynone (BARAC) , bicyclononyne (BCN) , 2, 3, 6, 7-tetramethoxy-DIBO (TMDIBO) , sulfonylated DIBO (S-DIBO) , carboxymethylmonobenzocyclooctyne (COMBO) , and pyrrolocyclooctyne (PYRROC) ) . Examples of cyclooctynes that can react with an azide are described in, e.g., Dommerholt et al., Top Curr Chem (Z) 374: 16, 2016.
IV. Applications
In certain embodiments, the modified molecule, such as a modified antibody (e.g., a modified IgG1) , can be attached to a cell to provide target specificity to the cell. For example, the cell can be a liposome, which has bee used as a drug delivery system. A modified antibody (e.g., a modified IgG1) can be further modified by an additional molecule containing the reactive partner 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 a cyclooctyne and a hydrophobic moiety (e.g., DSPE) . Once the additional molecule is attached to the modified antibody (e.g., modified IgG1) , the hydrophobic moiety (e.g., DSPE) can insert itself into the lipid bilayer of the cell (e.g., a liposome) to finally attach the antibody onto the cell.
With the antibody attached to the surface of the cell, the cell can be directly targeted to specific antigens or receptors targeted by the antibody. For example, to further improve the specificity of liposomes, immunoliposomes can be developed by attaching modified antibodies created by the methods described herein to the surface of the liposomes. Antibody-conjugated
liposomes can allow for an active tissue targeting through binding to tumor cell-specific receptors targeted by the antibody on the surface of the liposome. Such antibody modified liposomes can be used in specific drug delivery to cancer cells, gene therapy, drug delivery through blood brain barrier, or molecular imaging.
The present Disclosure will be described in greater detail by way of a specific example. The following example is offered for illustrative purposes, and is not intended to limit the Disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
Example 1. Synthesis of small molecule reactants
The small molecules used to react with o-quinone is synthesized via amide condensation reaction.
For visible-wavelength light induced reaction:
In a round bottle flask, 3- (Vinyloxy) -1-propylamine (0.5 mmol) , 2-azidoacetic acid (1.2 eq) , EDCI (1.2 eq) , HOAt (1.05 eq) and TEA (1.5 eq) were dissolved in DMF (5 mL) on ice bath for one hour. After that, the reaction solution was stirred at room temperature for 12 hours. Then 30 mL water was added to the flask and ethyl acetate (10 mL X3) was used to extracat the product for three times. The organic layer was washed by NaCl saturated solution (10 mL ) for two times and dried by anhydrous Na2SO4. Then the organic layer was concentrated and purified by column chromatography (PE/EA=2/1) to get colorless oil 70 mg (yield 76%) .
FIG. 1A further shows a variety of short peptides with a tyrosine amino acid as the starting material and the reaction yields after the tyrosine amino acid was modified by incubating with tyrosinase enzyme and a small molecule containing a terminal alkene under light irradiation. FIG. 1B shows the reaction yields with different small molecules with a terminal alkene. Reaction conditions: the peptide (10 mM) was incubated with tyrosinase (1.68 μM) in PB buffer (0.2 mM, pH 6.5) at 4 ℃ for 30 min, then VE (small molecule with the
terminal alkene, 100 mM) was added and irradiated at 456 nm light for 5 min. The reaction yield was detected by integrated area of product in HPLC analysis.
For nucleophilic reaction:
In a round bottle flask, FuA-NHS (0.5 mmol) , NH2-PEG3-N3 (1.2 eq) , TEA (1.2 eq) were dissolved in DMF (5 mL) . The reaction solution was stirred at room temperature for 12 hours. Then 30 mL water was added to the flask and ethyl acetate (10 mL X3) was used to extract the product for three times. The organic layer was washed by NaCl saturated solution (10 mL ) for two times and dried by anhydrous Na2SO4. Then the organic layer was concentrated and purified by RP-HPLC (33%yield) .
FIG. 3 further shows tyrosine selective modification of various peptides using small molecule with a terminal lactone and their yields. Conditions: peptide (1 mM) and FuA (small molecule with terminal lactone, 2.0 mM) were incubated with tyrosinase (1.4 μM) at room temperature for 1h.
Example 2. Light induced cycloaddition reaction used for protein modification (VE-tyrosine reaction)
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 1 (2 μL, 5 mM in DMSO) and mushroom tyrosinase (5.0 μL, 1.7 mg/mL in phosphate buffer pH 6.0) and irradiated under 456nm light (20 mW/cm2) for 8 h at 4 ℃. After completion, the product was concentrated, and small molecules were removed by 30k MWCO filters. After that, DBCO-TAMRA (5 μL, 2 mM in DMSO) was added and incubated at room temperature for 2h. FIG. 2A shows this schematic demonstration of the two-step fluorescent labelling of trastuzumab by VE-N3.
Labelled trastuzumab- (VE) -TAMRA by click reaction was analyzed by SDS-PAGE (FIG. 2B) . Further, SKOV3 cells incubated with IgG- (VE) -TAMRA was analyzed by flow cytometry (FIG. 2C) . The SKOV3 cells were incubated with the PBS solution containing 200 nM modified IgG for 30 min at room temperature. FIG. 2D further shows that IgG- (VE) -TAMRA selectively stained SKOV3 cells. Conditions: SKOV3 cells were incubated with trastuzumab- (VE) -TAMRA at 37 ℃ for 1 h, followed by PBS washing for three times and then
imaging using confocal fluorescence microscope (top) , MDA-MB-231 imaging using identical conditions (bottom) , scale bar, 20 μm.
Example 3. Spontaneous nucleophile addition reaction used for antibody modification (FuA-tyrosine reaction)
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 FuA-PEG-N3 (2 μL, 5 mM in DMSO) and mushroom tyrosinase (5.0 μL, 1.7 mg/mL in phosphate buffer pH 6.0) for 8 h at 4 ℃. After completion, the product was concentrated, and small molecules were removed by 30k MWCO filters. After that, DBCO-TAMRA (5 μL, 2 mM in DMSO) was added and incubated at room temperature for 2h.
Labelled trastuzumab- (FuA) -TAMRA by click reaction (FIG. 5A) was analyzed by SDS-PAGE (FIG. 5A) . IgG- (FuA) -TAMRA selectively stained SKOV3 cells (FIG. 5B) . Conditions: SKOV3 cells were incubated with trastuzumab- (FuA) -TAMRA at 37 ℃ for 1 h, followed by PBS washing for three times and then imaging using confocal fluorescence microscope (top) , MDA-MB-231 imaging using identical conditions (bottom) , scale bar, 20 μm (FIG. 5C) .
Example 4. Kinetic study of nucleophilic reaction
Antibody (Atezolizumab) was incubated with FuA-PEG-N3 (small molecule with terminal lactone and azide functional handle, 100 eq) at room temperature for different times in 1X PBS buffer (pH 7.4) . FIG. 4A shows the schematics of the reaction. FIG. 4B shows analysis of the reaction at different times by SDS-PAGE. FIG. 4C shows that the conversion or labeling is near complete at around 16 hours.
Example 5. Liposome labeling
To synthesize immunoliposomes (FIG. 6A) : 10 μM azide labeled trastuzumab and 100 μM DSPE-PEG2000-DBCO (cyclooctyne labeled with a lipid group DSPE via a PEG linker) were incubated in PBS buffer (pH 7.4) at 37 ℃ for 2 h to make the antibody-lipid conjugate. The reaction was monitored by SDS-PAGE (FIG. 6B) . The schmatics to show the generation of immunoliposome fused cells is shown in FIG. 6C. As shown in FIG. 6C, the immunoliposomes were fused with SK-OV-3 cells, resulting in labeled cells. The SK-OV-3 cells were incubated with immunoliposomes (10 μM) in a DMEM medium at 37 ℃ for 10, 30 min. Cells were fixed after washing and then imaged (FIG. 6D) . FIG. 6E shows the fluorescene intensity analysis of experiment groups and control groups.
Example 6. Methods
Antibody-cell conjugation. The cell used for conjugation (THP1 cell or NK 92 cell) was spun down at 300 g for 2 min. Then remove supernatant and resuspend cells in ~ 1 mL of DPBS. Repeat the centrifugation step and remove the supernatant to wash the cells. Repeat until a total of three washes have been completed. On the final wash, resuspend about 1×106 cells in 1 mL of DPBS. Cells were incubated with 20 μM nbHER2-BPA or Tras-BPA for 10 min at 37℃. FIG. 7A shows a schematic illustrating the synthesis of nbHER2-BPA. FIG. 7E shows a schematic illustrating the synthesis of Tras-BPA. The tube containing cells and nbHER2-BPA or Tras-BPA was put on ice and irradiated by 365 nm light 20 mW/cm2 for 20 min (FIG. 7A) . After the irradiation, add 1 mL of DPBS to each tube and spin the tube at ~300 g for 2 min. Wash the cells until a total of two washes have been performed and move on to any downstream applications.
Flow cytometry analysis. nbHER2-BPA or Tras-BPA were labeled with AF488-NHS ester (10 μM) at room temperature for 60 min. And the extra AF488-NHS ester was excluded by desalting columns. Then THP1 cells were incubated with 20 μM nbHER2-BPA/AF488 or Tras-BPA/AF488 and irradiated under 365 nm light as described above. The group without both 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 with DPBS 3 times and analyzed by flow cytometry in AF 488 channel by counting 1×105 cells (FIGS. 7B and 7F) .
Fluorescent staining. SKOV3 and THP1cells were stained with Mito Trakcer Red (Invitrogen) and Dio (Dioctadecyloxacarbocyanine perchlorate, Sigma-Aldrich) , respectively, following the manufacturer’s protocol. THP1 cells were conjugated with nbHER2-BPA as described above. Non-labeled THP1 cells were used as negative controls. After washing with PBS, the THP1 cells were resuspended with RPMI media with 10 %FBS, then mixed with SKOV3 cells in the same media and incubated at 37 ℃ and 5 %CO2. After 8 hours, cells were gently washed with PBS 3 times and imaged on a confocal microscope (SP8, Leica) (FIG. 7C) .
Cytotoxicity assay. Target cell cytotoxicity was evaluated as previously described. Adherent tumor cells (SKOV3, MDAMB231) were seeded at 2.5×104 cells/well in 96-well flat-bottom plates in the complete RPMI medium and incubated overnight at 37℃ in a humidified 5%CO2 atmosphere. Target cells were incubated with nbHER2-NK-92 conjugate or Tras-NK-92 for 48 h in NK 92 culture medium containing 0.2%interleukin-2 (IL-2)
(Purchasing from PROCELL) at 37℃, 5%CO2 in a 2: 1 Effector cells/Target cells (E/T) ratio (1×105 cells/well) . Cellular cytotoxicity was measured via the release of lactate dehydrogenase (LDH) from dead target cells by using CyQUANTTM LDH Cytotoxicity Assay (InvitrogenTM) according to the manufacturer’s instructions (FIG. 7C) . Spontaneous LDH release was assessed using target and effector cells without antibodies. Maximal target cell lysis was achieved by incubation of target cells with lysis buffer. The percentage of cytotoxicity towards target cells was calculated based on the following formula and shown in FIG. 7C:
Claims (17)
- A method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:(a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal alkene and the functional handle and a tyrosinase in a solution;(b) irradiate the solution of (a) under visible wavelength light; and(c) purify the modified molecule.
- The method of claim 1, wherein the visible wavelength light is from 380 nm to 700 nm.
- The method of claim 2, wherein the visible wavelength light is about 456 nm.
- A method for modifying a tyrosine amino acid in a molecule with a functional handle, the method comprises:(a) incubate a molecule comprising at least one tyrosine amino acid with a small molecule comprising a terminal lactone and the functional handle and a tyrosinase in a solution; and(b) purify the modified molecule.
- The method of any one of claims 1 to 4, wherein the molecule containing the tyrosine amino acid is an antibody.
- The method of claim 5, wherein the antibody is an IgG, IgA, IgD, IgE, or IgM.
- The method of claim 6, wherein the antibody is an IgG.
- The method of claim 7, wherein the antibody is an IgG1.
- The method of claim 7 or 8, wherein the tyrosine amino acid in the molecule being modified with the functional handle is Y296 and/or Y300, wherein the positions are determined according to EU numbering.
- The method of any one of claims 1 to 9, wherein, prior to step (a) , the method further comprises a step of removing glycans by incubating the molecule with a deglycosylation enzyme.
- The method of claim 10, wherein the deglycosylation enzyme is a peptide: N-glycosidase F (PNGase F) .
- The method of any one of claims 1 to 11, wherein the functional handle is an azide, an alkyne, a biotin, or a streptavidin.
- The method of any one of claims 1 to 12, wherein the molecule with the functional handle further comprises a detectable moiety.
- The method of claim 13, wherein the detectable moiety is a fluorophore.
- The method of any one of claims 1 to 14, wherein the molecule with the functional handle further comprises a hydrophobic moiety.
- The method of any one of claims 1 to 3 and 5 to 15, wherein the molecule with the functional handle is:wherein n is an integer from 1 to 10.
- The method of any one of claims 4 to 16, wherein the molecule with the functional handle is:wherein n is an integer from 1 to 10.
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