Patent Application
20240424148
The present invention relates to proximity-based labeling systems and, in particular, to compositions and methods permitting high resolution labeling of in-vivo biological environments.
Protein proximity labeling has emerged as a powerful approach for profiling protein inter-action networks. The ability to label associated or bystander proteins through proximity labeling can have important implications on further understanding the cellular environment and biological role of a protein or biomolecular species of interest. Current proximity labeling methods all involve the use of enzyme-based generation of reactive intermediates that label neighboring proteins on a few select amino acid residues through diffusion or physical contact. Despite the transformative impact of this technology, the inherent stability of these reactive intermediates such as phenoxy radicals (t1/2>100 μs) through peroxidase activation or biotin-AMP (t1/2>60 s) through biotin ligases can promote diffusion far from their point of origin. As a result, these enzyme-generated reactive intermediates pose a challenge to profiling within tight micro-environments. Furthermore, the large enzyme size, the dependency on certain amino acids for labeling, and the inability to temporally control these labeling systems present additional challenges for profiling within confined spatial regions. Given these limitations, new approaches for proximity-based labeling are needed.
In one aspect, compositions and methods are described herein for providing a microenvironment mapping platform operable to selectively identify various features, including in-vivo protein-protein interactions on cellular membranes. In some embodiments, a composition comprises a tetrapyrrole photocatalyst, and a protein labeling agent, wherein the tetrapyrrole photocatalyst has electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If a protein or other biomolecule is not within the diffusion radius, the reactive intermediate is quenched by the surrounding aqueous or aqueous-based environment. As described further herein, the diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping considerations, and can be limited to the nanometer scale. In some embodiments, for example, the diffusion radius can be less than 100 nm, less than 50 nm, less than 20 nm, less than 10 nm or less than 5 nm, such as 1-5 nm. Moreover, in some embodiments, a protein labeling agent can be functionalized with a marker, such as biotin or luminescent markers for aiding in analysis.
As described herein, the tetrapyrrole photocatalyst includes a metal center that can be placed in an excited state for activating the protein labeling agent to a reactive intermediate via energy transfer. In some embodiments, the excited state of the tetrapyrrole photocatalyst can be quenched by a reductant, thereby returning the metal center to the ground state. The energy transfer to the reactive intermediate can subsequently occur from the ground state of the tetrapyrrole photocatalyst. In some embodiments, the tetrapyrrole photocatalyst absorbs electromagnetic radiation having wavelength longer than 600 nm or 650 nm to achieve an excited state. The tetrapyrrole photocatalyst, for example, may absorb radiation having a wavelength in the range of 650-1100 nm to achieve an excited state. Use of longer wavelength radiation can permit the radiation to penetrate tissue, thereby enabling the tetrapyrrole photocatalyst to interact with the radiation in a variety of in-vivo environments. Energy transfer from the catalyst to the protein labeling agent can occur via a variety of mechanisms described further herein, including Dexter energy transfer or single electron transfer. The energy transfer can occur from an excited state or ground state of the tetrapyrrole photocatalyst.
In some embodiments, the tetrapyrrole photocatalyst comprises a metal center. The metal center can comprise a transition metal or silicon, in some embodiments.
In another aspect, a composition for proximity-based labeling comprises a catalyst, and a protein labeling agent selected from the group consisting of thiatriazoles, sulfoximines, sulfilimines, anilines, acyl azides, ylides and dizo compounds. The catalyst has electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate, as described herein. The catalyst can comprise any catalyst operable to activate the protein labeling agent to the reactive intermediate. In some embodiments, the catalyst is a tetrapyrrole photocatalyst detailed herein.
In another aspect, conjugates for proximity-based labeling are described herein. A conjugate comprises a catalyst coupled to a biomolecular binding agent. The catalyst can have electronic structure for energy transfer to a protein labeling agent for generation of a reactive intermediate as described above. In some embodiments, the catalyst comprises a tetrapyrrole photocatalyst described herein. The biomolecular binding agent, in some embodiments, can be used to selectively locate or target the catalyst to a specific environment for mapping. The biomolecular binding agent, for example, locate the catalyst in the desired cellular environment for proximity labeling and associated analysis. As described herein, the cellular environment can be in vivo. The biomolecular binding agent can comprise a protein, polysaccharide, nucleic acid, or lipid, in some embodiments. In some instances, the biomolecular binding agent can comprise a multivalent display system comprising a protein, polysaccharide, nucleic acid, or lipid. Moreover, the biomolecular binding agent can also be a small molecule ligand with a specific binding affinity for a target protein.
In a further aspect, methods of proximity-based labeling are described herein. A method of proximity-based labeling comprises providing a catalyst, and activating a protein labeling agent to a reactive intermediate with the catalyst. The reactive intermediate couples or bonds to a protein. In some embodiments, the catalyst is coupled to a biomolecular binding agent to selectively locate or target the catalyst to a specific environment for protein mapping in conjunction with the protein labeling agent. The catalyst, conjugate, and protein labeling agent can have composition and/or properties described above, including tetrapyrrole photocatalysts, and in the following detailed description and Appendix attached hereto.
These and other embodiments are further described in the following detailed description.
Embodiments described herein can be understood more readily by reference to the following detailed description, examples, and Appendix and their previous and following descriptions. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.
The term “alkyl” as used herein, alone or in combination, refers to a straight or branched saturated hydrocarbon group optionally substituted with one or more substituents. For example, an alkyl can be C1-C30 or C1-C18.
The term “aryl” as used herein, alone or in combination, refers to an aromatic monocyclic or multicyclic ring system optionally substituted with one or more ring substituents.
The term “heteroaryl” as used herein, alone or in combination, refers to an aromatic monocyclic or multicyclic ring system in which one or more of the ring atoms is an element other than carbon, such as nitrogen, boron, oxygen and/or sulfur.
The term “heterocycle” as used herein, alone or in combination, refers to an mono- or multicyclic ring system in which one or more atoms of the ring system is an element other than carbon, such as boron, nitrogen, oxygen, and/or sulfur or phosphorus and wherein the ring system is optionally substituted with one or more ring substituents. The heterocyclic ring system may include aromatic and/or non-aromatic rings.
The term “alkoxy” as used herein, alone or in combination, refers to the moiety RO—, where R is alkyl, alkenyl, or aryl defined above.
The term “halo” as used herein, alone or in combination, refers to elements of Group VIIA of the Periodic Table (halogens). Depending on chemical environment, halo can be in a neutral or anionic state.
Terms not specifically defined herein are given their normal meaning in the art.
In one aspect, compositions and methods are described herein for providing a microenvironment mapping platform operable to selectively identify various features, including in vivo protein-protein interactions on cellular membranes. In some embodiments, a composition comprises a tetrapyrrole photocatalyst, and a protein labeling agent, wherein the tetrapyrrole photocatalyst has electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. As set forth herein, tetrapyrrole catalyst comprises a metal center participating in energy transfer to the protein labeling agent. In some embodiments, for example, the catalyst engages in Dexter energy transfer with the protein labeling agent. The energy transfer can proceed via single electron transfer, in some embodiments.
The energy transfer to the protein labeling agent can originate from an excited state of the tetrapyrrole photocatalyst electronic structure, in some embodiments. The excited state of the catalyst, for example, can be a singlet excited state or triplet excited state. The excited state of the tetrapyrrole photocatalyst can be generated by one or more mechanisms, including energy absorption by the photocatalyst. In some embodiments, the excited state is induced by absorption of one or more photons. In other embodiments, the catalyst may be placed in an excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer to the protein labeling agent, including electron transfer, may originate from a ground state of the catalyst electronic structure. The excited state of the tetrapyrrole photocatalyst can be quenched by reductant, returning the tetrapyrrole photocatalyst to the ground state. The energy transfer, including single electron transfer, can then proceed from the ground state of the tetrapyrrole photocatalyst to the protein labeling agent, resulting in the formation of the reactive intermediate.
In some embodiments, the tetrapyrrole photocatalyst absorbs electromagnetic radiation having wavelength longer than 600 nm to achieve an excited state. The tetrapyrrole photocatalyst, for example, may absorb radiation having a wavelength in the range of 600-1100 nm to achieve an excited state. Use of longer wavelength radiation can permit the radiation to penetrate tissue, thereby enabling the tetrapyrrole photocatalyst to interact with the radiation in a variety of in vivo environments. As described herein, the tetrapyrrole photocatalyst comprises a metal center. The metal center can be a transition metal or silicon, in some embodiments.
The tetrapyrrole photocatalyst, in some embodiments, can be modified with one or more functionalities for altering solubility of the tetrapyrrole photocatalyst in various media. The tetrapyrrole photocatalyst, for example, can have one or more polar or ionizable functionalities on the pyrole or pyrole-like rings for enhancing solubility in water or aqueous-based cellular environments. In some embodiments, the tetrapyrrole photocatalyst have one or more carboxyl, hydroxyl, and/or amine functionalities. Alternatively, the tetrapyrrole photocatalyst can exhibit one or more hydrophobic constituents.
Energy transfer, including electron transfer, to the protein labeling agent forms a reactive intermediate of the protein labeling agent. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If a protein or other biomolecule is not within the diffusion radius, the reactive intermediate is quenched by the surrounding environment, which may be an aqueous or aqueous-based environment. The diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping (proximity-based labeling) considerations, and can be limited to the nanometer scale. In some embodiments, for example, the diffusion radius of the reactive intermediate can be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm prior to quenching in the surrounding environment. The diffusion radius can be 0.5 nm to 10 nm, in some embodiments. Accordingly, the reactive intermediate will react or crosslink with a protein or other biomolecule within the diffusion radius or be quenched by the surrounding environment if no protein or biomolecule is present. In this way, high resolution of the local environment can be mapped via concerted effort between the catalyst and protein labeling agent. Additionally, the reactive intermediate can exhibit a t1/2 less than 5 ns, less than 4 ns, or less than 2 ns prior to quenching, in some embodiments. The reactive intermediate, for example, can exhibit a t1/2 less of 1-5 ns. In additional embodiments, the diffusion radius can be extended to between 5-500 nm though extension of the reactive intermediate half-life. For example, in some embodiments, the reactive intermediate can have a half-life of 1-100 μs, or greater.
In some embodiments, tetrapyrrole photocatalysts can be coupled to a biomolecular binding agent to provide a conjugate. The biomolecular binding agent, in some embodiments, can be used to selectively locate or target the catalyst to a specific environment for mapping. The biomolecular binding agent, for example, locate the catalyst in the desired cellular environment for proximity labeling and associated analysis. The biomolecular binding agent can comprise a protein, polysaccharide, nucleic acid, or lipid, in some embodiments. In some instances, the biomolecular binding agent can comprise a multivalent display system comprising a protein, polysaccharide, nucleic acid, or lipid. Moreover, the biomolecular binding agent can also be a small molecule ligand with a specific binding affinity for a target protein.
The protein labeling agent forming the reactive intermediate upon energy transfer from the tetrapyrrole photocatalyst, in some embodiments, can comprise an azide, diazirine, phenol, thiatriazole, sulfilimine, sulfoximine, ylide, diazo, aniline, or mixtures thereof. In some embodiments, the protein labeling agent can be functionalized with a marker, such as biotin. In some embodiments, the marker is desthiobiotin. The marker can assist in identification of proteins labeled by the protein labeling agent. The marker, for example, can be useful in assay results via western blot and/or other analytical techniques. Markers can include alkyne, azide, FLAG tag, fluorophore, and chloroalkane functionalities, in addition to biotin and desthiobiotin.
In some embodiments, the tetrapyrrole photocatalysts can be substituted by one or more differing transition metal catalysts for activating the protein labeling agent to the reactive intermediate via energy transfer. For example, a transition metal catalyst may comprise one or more tridentate ligands, such as terpyridine (terpy).
As described above, in some embodiments, tetrapyrrole photocatalysts, transition metal catalysts, and/or organic catalysts can be functionalized with one or more moieties to enhance the hydrophilic character or hydrophobic character of the catalysts. In some embodiments, the catalysts are functionalized to render the catalysts soluble in aqueous or aqueous-based environments. Alternatively, the catalysts are functionalized to render the catalysts cell permeable.
In another aspect, a composition for proximity-based labeling comprises a catalyst, and a protein labeling agent selected from the group consisting of thiatriazoles, sulfoximines, sulfilimines, anilines, acyl azides, ylides and dizo compounds. The catalyst has electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate, as described herein. The catalyst can comprise any catalyst operable to activate the protein labeling agent to the reactive intermediate. In some embodiments, the catalyst is a photocatalyst detailed herein, including tetrapyrrole photocatalyst.
In another aspect, conjugates for proximity-based labeling are described herein. A conjugate comprises a catalyst coupled to a biomolecular binding agent. The catalyst coupled to the biomolecular binding agent can comprise any catalyst described herein, including the tetrapyrrole photocatalysts, transition metal catalysts, and organocatalysts detailed in Section I above. Moreover, the biomolecular binding agent can comprise a protein, polysaccharide, nucleic acid, or lipid, in some embodiments. In some instances, the biomolecular binding agent can comprise a multivalent display system comprising a protein, polysaccharide, nucleic acid, or lipid. In some embodiments, the biomolecular binding agent can be a small molecule ligand with a specific binding affinity for a target protein. The biomolecular binding agent can be employed to locate the catalyst in the desired extracellular environment for proximity labeling and associated analysis. Accordingly, specific identity of the biomolecular binding agent can be selected according to the chemical and/or steric requirements of the desired target site for placement of the catalyst in the proximity based labeling process. Any biomolecular target site can be chosen, and target sites are not limited in the present disclosure. In some embodiments, target sites can be proteins for studying protein-protein interactions, including interaction with cellular membrane receptors. In some embodiments, for example, the biomolecular binding agent is an antibody, such as a secondary antibody for interacting with a primary antibody bound to the desired antigen. In other embodiments, the biomolecular binding agent is a ligand with specificity for a protein receptor of the cellular membrane, such as epidermal growth factor receptor (EGFR) or G protein-coupled receptor.
The biomolecular binding agent can be bonded to the catalyst. In some embodiments, the catalyst comprises a reactive handle or functionality for coupling the biomolecular binding agent. In some embodiments, for example, a catalyst can comprise one or more click chemistry moieties including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide.
In another aspect, systems for proximity-based labeling are described herein. A system, for example, comprises a conjugate including a catalyst coupled to a biomolecular binding agent, and a protein labeling agent activated by the catalyst for binding to a protein. The conjugate can comprise any catalyst and biomolecular binding agent described herein, including the embodiments detailed in Section II above, and the associated including the tetrapyrrole photocatalysts, transition metal catalysts, and organocatalysts detailed herein. The catalyst, for example, can have electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. Moreover, the protein labeling agent can comprise any of the labeling agents described herein, including the protein labeling agents set forth in Section I above. Specific identity of the conjugate and associated protein labeling agent can be selected according to several considerations, such as the chemical nature and/or steric requirements of the biological environment to be mapped with the proximity-based labeling system.
Systems for proximity-based labeling described herein can be employed in various applications. In some embodiments, the systems enable target identification, wherein the conjugate and associated protein labeling agent permit identification of one or more molecules in a biological context by proteomics. Additionally, systems comprising the conjugate and protein labeling agent facilitate interactome mapping. Targeting a conjugate and protein labeling agent allows detection and identification of one or more molecules and neighboring interactors in a biological context by proteomics. Identification of such molecules by systems described herein can permit enrichment and/or purification of such molecules and neighboring interactors.
Additionally, systems comprising a conjugate and protein labeling agent further enable detection and identification of one or more molecules in a biological context via microscopy.
In another aspect, methods of proximity-based labeling are described herein. A method of proximity-based labeling comprises providing a conjugate comprising a catalyst coupled to a biomolecular binding agent, activating a protein labeling agent to a reactive intermediate with the catalyst, and coupling the reactive intermediate to a protein. The conjugate can comprise any catalyst, including tetrapyrrole photocatalyst, and biomolecular binding agent described herein, including the embodiments detailed in Section II above. Moreover, the protein labeling agent can comprise any of the labeling agents described herein, including the protein labeling agents set forth in Section I above. The protein labeling agent forming the reactive intermediate upon energy transfer from the tetrapyrrole photocatalyst, in some embodiments, can comprise an azide, diazirine, phenol, thiatriazole, sulfilimine, sulfoximine, ylide, diazo, aniline, or mixtures thereof. Specific identity of the conjugate and associated protein labeling agent can be selected according to several considerations, such as the chemical nature and/or steric requirements of the biological environment to be mapped with the proximity-based labeling system. In some embodiments of proximity-based labeling, the catalyst can be provided in the absence of a biomolecular binding agent.
Methods described herein can be employed to map various in vivo biological environments, including local areas of cellular membranes and/or the local extracellular environment. As described herein, the ability of tetrapyrrole photocatalysts to be activated with electromagnetic radiation greater than 600 nm enables mapping of in vivo biological environments well below tissue exteriors, such as the skin. In some embodiments, environments can be mapped at tissue depths of greater than 5 mm or greater than 10 mm. For example, in vivo mapping can occur at tissue depths of 5 mm to 50 cm, in some embodiments.
The conjugate comprising the catalyst and biomolecular binding agent may be targeted to a specific local region of a cellular membrane, such as a receptor of interest. Activation of the protein labeling agent can identify protein(s) and/or other molecules in the targeted local region. Notably, the activated protein labeling agent can also identify or label molecules associated with another cell in contact with the targeted cellular region. Therefore, intercellular interactions and intercellular environments can be elucidated and mapped with systems and methods described herein. The foregoing methods enable interactome mapping, and the identification of one or more molecules and neighboring interactors in a biological context by proteomics. Identification of such molecules by methods described herein can permit enrichment and/or purification of such molecules and neighboring interactors.
In some embodiments, multiple photocatalysts can be employed in proximity-based labeling systems and methods described herein. The photocatalysts can exhibit differing absorption profiles, thereby enabling selective proximity-based labeling dependent on the wavelength of excitation radiation provided. In some embodiments, photocatalysts and associated protein labeling agents described in Patent Cooperation Treaty Application Serial Number PCT/US2020/036285 can be used with photocatalysts and protein labeling agents described herein. Light having wavelength of 375-450 nm, for example, can be used to effectuate proximity-based labeling with the photocatalysts and protein labeling agents described in PCT/US2020/036285. Moreover, light having wavelength of 650-1100 nm can be used to effectuate proximity-based labeling in some embodiments described herein with tetrapyrrole photocatalysts and conjugates described in Sections I and II above. The differing photocatalysts can have different biomolecular binding agents to target differing cellular environments. Differing protein labeling agents between the photocatalysts may also be used. Under this analytical regime, many local cellular environments may be mapped, thereby elucidating previously unknown biomolecular interactions and relationships.
These and other embodiments are further illustrate by the following non-limiting examples.
Tin (Sn) metalated chlorin e6 photocatalyst was synthesized according to the reaction scheme of FIG. 16. Chlorin e6 trimethylester (7.6 mg, 0.12 mmol) and tin chloride dihydrate (26.8 mg, 0.12 mmol) were added to an 8 ml vial equipped with a magnetic stir bar and dissolved in a 2% NaOAc/glacial acetic solution (0.03 M). This solution was then heated to 60° C. and stirred for 2 hours. The mixture was then let to cool to room temperature, diluted with 10 ml of 1N HCl, and extracted three times with 200 ml DCM. The combined extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield compound S1 as a dark blue solid (3.6 mg, 38.3% yield).
Chlorin e6-PEG3-NHBoc (S2) was synthesized according to the reaction scheme of
Chlorin e6 Sn (OH) DBCO (S3) was synthesized according to the reaction mechanism of
Biotin-PEG3-phenyl azide was synthesized according to the reaction scheme of
Several red light photocatalysts of varying redox properties were tested for the conversion of 4-azidobenzoic acid, as set forth in
From these data, a mechanistic pathway was proposed, the pathway initiating via reductive quenching of the excited-state photocatalyst (4) with NADH to form a highly reducing organic ground state (E1/2=−0.69 V vs Ag/AgCl), as illustrated in
Furthermore, electrochemical reduction of the Sn-chlorin e6 catalyst generated a species with significant spectral overlap with the transient-absorption signal of the photoexcited catalyst in the presence of NADH, supporting the generation of the reduced ground state catalyst.
Tetrapyrrole photocatalyst activity in vitro was established by covalently tagging a recombinant protein in aqueous solution. Carbonic anhydrase was subjected to labeling (10 mol. % tetrapyrrole photocatalyst, 1 mM NADH, 500 μM PhN3-biotin) as set forth in
With a system for red-light activated labeling based on tetrapyrrole photocatalyst established, cellular labeling via the red-light activation was examined. As a model system, epidermal growth factor receptor (EGFR), a cell surface receptor-tyrosine kinase, was selected. Secondary antibodies conjugated to Sn-chlorin photocatalyst were synthesized, which could then be directed with primary antibodies to EGFR, as illustrated in
A549 cells were subjected to immunotargeted photolabeling (1 mM NADH, 500 μM PhN3-biotin, 30 min irradiation) in the presence or absence of anti-EGFR antibodies. Spatially selective biotinylation was assessed via stimulated emission depletion (STED) super-resolution microscopy. As illustrated in
To assess spatial selectivity of labeling, a measurement of the full width at half-maximum (fwhm) of biotinylation clusters estimated the Gaussian distribution oflabeling events to be 87±33 nm (n=50 clusters). This distribution is in agreement with the known longer lifetime of the aminyl radical intermediate (˜50 μs) with respect to carbenes (˜2 ns) generated using blue-light photocatalytic species, but nonetheless affords red-light proximity labeling systems described herein with the ability to profile nanoscale events in individual protein microenvironments.
Membrane lysate fractions from photolabeled cells were then generated and subjected to streptavidin enrichment and quantitative proteomics. Consistent with the STED analysis, EGFR enrichment was observed via Western blot only in samples that had been exposed to anti-EGFR antibody. Here, quantitative tandem mass tag (TMT) proteomics revealed 29 enriched proteins with log2(FC)>1 (
It was next sought to evaluate tetrapyrrole photocatalytic labeling systems described herein in a complex setting where blue light activation would not be feasible. Along these lines, whole blood presents high levels of biochemical complexity, and it was questioned whether tetrapyrrole photocatalytic labeling systems could be used in this setting to achieve selective proximity labeling. TER119, a well-characterized antibody raised against mature erythrocytes, was selected as the targeting modality for cell-surface labeling (
First, we conjugated Sn-chlorin catalysts to both TER119 and a nontargeting isotype conjugated TER119, and minimal signal was observed with isotype conjugates (
In view of the foregoing, a red-light-activated proximity labeling platform has been developed based tetrapyrrole photocatalyst, protein labeling agents, and conjugates described herein. This system exhibits phototonic and spatiotemporal control over labeling and can operate in both simple and complex biological environments.
Various embodiments of the invention have been described in fulfillment of the various objects of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.
The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Ser. No. 63/252,244 filed Oct. 5, 2021, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/045654 | 10/4/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63252244 | Oct 2021 | US |
