The present invention generally relates to nanostructures. In particular embodiments, the present invention relates to the field of nanorobotics. Nanorobotics relates to nanoscale devices that can be programmed and/or designed to perform specific tasks.
The field of nanostructures is becoming more and more relevant in the field of technological applications. The miniaturization of devices is an increasing trend for example in the field of information technology, sensing or medical applications.
Typically, nanostructures are manufactured using technically advanced, highly complex manufacturing processes, that may limit the yield and efficiency and generally is associated with high efforts. However, in recent years new methods for manufacturing nanostructures have been developed. These include methods for manufacturing nucleic acid nanostructures as disclosed in U.S. Pat. No. 7,842,793 B3. The disclosure relates to methods and compositions for generating nanoscale devices, systems, and enzyme factories based upon a nucleic acid nanostructure that can be designed to have a predetermined structure. This technique is widely known as “DNA origami”. While the present invention will mainly be described with regard to DNA origami structures, it should be understood that this is merely exemplary and that the present invention can also be practiced with other nanostructures.
DNA origami is a technology that allows the production of 3D structures on the nanometer scale from DNA molecules. Chemical modifications or single-stranded DNA overhangs can be placed on origami structures with positional control. DNA origami structures can be easily linked to multiple molecules, each of which mediates a specific molecular interaction, thus endowing the DNA origami structure with the interaction capabilities of the linked molecules. In biological systems, molecular complexes with multiple interactions play a role in enabling complex functionalities.
Recently, these techniques have been used for the realization of different nanoscale devices. For example, U.S. Pat. No. 9,863,930 B2 discloses various molecular barcoded bi-stable switches that can be used to detect various analytes, wherein the molecular barcoded bi-stable switch may be manufactured using DNA origami.
Further, WO 2012/061719 A2 discloses DNA origami devices useful in the targeted delivery of biologically active entities to specific cell populations. While this device may enable a targeted delivery of biologically active entities, it relies on specific interaction molecules, i.e. aptamers, that may bind to two different binding targets. This may be disadvantageous since it can restrict the possible applications of such a device.
In light of the above, it is an object of the present invention to overcome or at least alleviate the shortcomings and disadvantages of the prior art. That is, it is an object of the present invention to provide a conditionally switchable nanostructure.
These objects are met by the present invention.
In some embodiment the present invention provides a nanostructure comprising a binding site configured to bind to a binding target, wherein the nanostructure is configured to assume a first configuration and a second configuration, wherein the accessibility of the binding site for the binding target in the second configuration is different to the accessibility of the binding site for the binding target in the first configuration. The nanostructure further comprises a coupling site set comprising at least one coupling site, wherein each coupling site is configured to couple to a respective coupling target. Further, the nanostructure is configured to assume the second configuration when each coupling site of a subset of the coupling site set is coupled to its respective coupling target, wherein the subset comprises at least one coupling site. The nanostructure is further configured to assume the first configuration when none of the coupling sites of the coupling site set is coupled to its respective coupling target.
Generally, it should be understood that the nanostructure comprising a binding site should not be construed to mean that the nanostructure only comprises this binding site. Instead, the nanostructure may also have more than one binding site. In other words (unless explicitly states or unless clear to the skilled person), usage of an article (e.g., “a”, “an”) should not be understood to exclude the plural. For example, also a nanostructure comprising two binding sites is to be construed to be encompassed by “a nanostructure comprising a binding site”.
In other words, the nanostructure may be configured to assume different configurations including a first configuration A and a second configuration B. In some embodiments, these configurations may also be realized as an “open” and “closed” configuration. Depending on the configuration the nanostructure assumes, the binding site may be change its accessibility. For example, the nanostructure may be more accessible in the second configuration than in the first configuration.
Further, the nanostructure may also comprise at least one coupling site (i.e., one coupling site or a plurality of coupling sites). Depending on whether or not these coupling sites are coupled to coupling targets, a likelihood of the nanostructure assuming the different configurations may change. As an example, when all (or a part) of the coupling sites are coupled to their coupling targets, the nanostructure may be more likely to assume the second configuration than would be the case when none of the coupling sites are coupled to their coupling targets.
Thus, depending on the coupling state of the coupling sites, the accessibility of the binding site (or the binding sites) may be changed. Thus, the coupling state of the coupling sites may “activate” and/or “deactivate” the accessibility of the binding site. It will be understood that this may also include that the nanostructure is “further activated”, i.e., that the activity of the nanostructure is further increased.
Thus, depending on the coupling state of the coupling sites, in simplified words, the nanostructure may be switched between different states. This allows many different applications. In particular, such a nanostructure can be used to couple to particular coupling targets and to only be “active” once coupled to such coupling targets. This may, e.g., be used to couple the nanostructures to particular cells and to activate it once coupled to such cells.
It should be understood that the subset of the coupling site set may also coincide with the coupling site set.
Such a nanostructure may thus allow to at least significantly change the probability of being in the first or second configuration depending on the coupling of the coupling sites. Further, a great variety of interactions may be utilized in such a device which may be advantageous compared to the state of the art that relies on specific and limited interactions.
The coupling sites may not be configured to couple to each other. Thus, “self coupling” of the nanostructure to itself may be prohibited.
In some embodiments, each coupling site may be configured to only couple to a single type of coupling target. That is, a coupling site may not couple to distinct coupling targets.
The nanostructure may comprise a plurality of binding sites, wherein each of the binding site may be configured to bind to a respective binding target, and wherein the accessibility of each of the binding sites for the respective binding target in the second configuration may be different to the accessibility in the first configuration.
A different accessibility between the two configurations may generally be advantageous since it allows to condition the accessibility of the binding site on the configuration assumed by the nanostructure, which in turn may be conditioned on the presence or absence of coupling targets. Thus, such a nanostructure may for example enable targeted drug delivery or other forms of treatments of a disease.
The nanostructure may be configured to assume the first configuration with a higher probability than the second configuration when none of the coupling sites of the coupling site set are coupled to its respective coupling target.
That is, also when none of the coupling sites of the coupling site set are coupled to their respective coupling targets, the nanostructure may assume both the first configuration and the second configuration. More particularly, there may be an equilibrium between the first configuration and the second configuration in this binding state. However, in this equilibrium, the first configuration may be “preferred”, i.e., the nanostructure may assume this first configuration with a higher likelihood than the second configuration in this coupling state.
Further, the binding site may be accessible for the binding target in one configuration and may not be accessible for the binding target in the other configuration. That is, the binding site may only be accessible in one of the two configurations. This may be particularly beneficial for applications where the accessibility should be conditioned on the configuration assumed by the nanostructure as it may essentially inhibit accessibility in one configuration.
The nanostructure may be configured to assume first and second equilibrium states between the first configuration and the second configuration, wherein the probability that the nanostructure assumes the second configuration may be different (e.g., higher) in the second equilibrium state than in the first equilibrium state. The nanostructure may be configured to assume the first equilibrium state when all of the respective coupling targets are absent, i.e. none of the coupling sites of the coupling site set can couple to the respective coupling target and to assume the second equilibrium state when all of the respective coupling targets of the subset of coupling sites are present, i.e. all coupling sites of the subset of coupling sites can couple to the respective coupling targets.
That is, the nanostructure may generally be in an equilibrium state between the first configuration and the second configuration, wherein it may assume each configuration with a certain probability. However, in some embodiments an equilibrium state may comprise a probability of essentially zero for being in one of the configurations. In other words, in such an equilibrium state the nanostructure may essentially always remain in one configuration.
The probability of the nanostructure assuming the first configuration in the first equilibrium state may be at least 0.3, preferably at least 0.5, more preferably at least 0.7, such as at least 0.9 and the probability of the nanostructure assuming the second configuration in the first equilibrium state may be at most 0.7, preferably at most 0.5, more preferably at most 0.3, such as at most 0.1.
Further, the probability of the nanostructure assuming the first configuration in the second equilibrium state may be at most 0.7, preferably at most 0.5, more preferably at most 0.3, such as at most 0.1 and the probability of the nanostructure assuming the second configuration in the second equilibrium state may be at least 0.3, preferably at least 0.5, more preferably at least 0.7, such as at least 0.9.
In some embodiments, the nanostructure may comprise a first portion and a second portion, wherein the first portion may be movable with respect to the second portion. Moreover, the first portion and the second portion may each comprise at least one of the coupling sites of the coupling site set and at least one of the first portion and the second portion may comprise the binding site.
Further, the subset of the coupling site set may comprise at least one coupling site comprised by the first portion and at least one coupling site comprised by the second portion. That is, each portion may comprise at least one coupling site.
The first portion and the second portion may be movably attached to each other, e.g. by means of a hinge or a rotational axis.
In some embodiments, the nanostructure may comprise at least one additional portion.
The nanostructure may comprise a maximum length, i.e. a length corresponding to the largest extent of the nanostructure, and the maximum length may be smaller than 1000 nm, preferably smaller than 500 nm, such as 100 nm.
The binding site may bind reversibly to the binding target, i.e. they can bind and detach repeatedly.
Similarly, at least one of the coupling sites of the coupling site set may couple reversibly to the respective coupling target, i.e. they can couple and decouple repeatedly.
In some embodiments, the coupling site set may comprise only identical coupling sites, i.e. coupling sites configured to couple to the same coupling targets. In other embodiments, the coupling site set may comprise at least two distinct coupling sites, configured to couple to distinct coupling targets.
Further, the nanostructure may be configured to couple to coupling targets comprised by a single entity. In other embodiments, the nanostructure may be configured to couple to coupling targets comprised by a plurality of entities. In such cases, the plurality of entities may comprise at least two distinct entities.
The binding target may be comprised by an entity also comprising at least one coupling target.
In some embodiments, the nanostructure may be at least partially formed by a DNA origami structure.
That is, readily available DNA origami techniques that involve comparatively less complex procedures to assemble than standard nanomanufacturing techniques may be used to manufacture the nanostructure.
The DNA origami structure may comprise at least one scaffolding strand, i.e. single-stranded polynucleotide scaffold DNA with a known sequence.
The DNA origami structure may further comprise a plurality of single-stranded oligonucleotide staple strands, wherein each staple strand may be at least partially complementary to at least one scaffolding strand.
Further, each of the staple strands may be configured to bind to the at least one scaffolding strand in two distinct places, wherein the at least one scaffolding strand may be folded and/or arranged such that the desired nanostructure may be formed.
In some embodiments, at least one binding site may be a molecule.
Further, at least one binding site may be configured to bind to a CD28 protein as a binding target. The at least one binding site may also be configured to bind another specific cluster of differentiation (CD) molecule and/or to another disease-associated cell surface molecule.
Similarly, at least one coupling site comprised by the coupling site set may be a molecule.
In some embodiments, at least one coupling site of the coupling site set may be configured to couple to a CD3 antigen as a coupling target.
Further, at least one coupling site of the coupling site set may be configured to couple to an epithelial cell adhesion molecule as a coupling target.
In embodiments, where at least one binding site and/or at least one coupling site is a molecule, said molecule may be bound to the nanostructure by means of linker molecules.
That is, a great number and variety of interactions may be realised for binding sites and/or coupling sites. This may provide an advantage compared to the known state of the art, e.g.
WO 2012/061719 A2, as it may overcome the disadvantage of limited interactions and thus provide a nanostructure for various different applications.
Moreover, in embodiments where the nanostructure is at least partially formed by a DNA origami structure and wherein the DNA origami structure comprises at least one scaffolding strand and a plurality of staple strands, the molecule may be bound to one of the at least one scaffolding strand or a staple strand by means of a linker molecule, wherein the linker molecule may be connected to a DNA strand portion, which is complementary to a portion of the at least one scaffolding strand or to a portion of a staple strand.
In embodiments where the nanostructure comprises a first and second portion, the first portion and the second portion may comprise an identical shape. Further, the shape may be a cuboid, i.e. a rectangular box.
In addition, the two portions may each comprise a cavity, configured to form a chamber when the nanostructure is in the first configuration. Further, at least one binding site may be located in the cavity of the first portion.
Yet further, at least one binding site may be attached to an outer surface of the cavity by means of a rod, wherein the binding site may be configured to leave the cavity when the nanostructure is in the second configuration.
The person skilled in the art will appreciate that a rod may be any type of flexible link between a portion of the cavity and at least one binding site.
In some embodiments in which the nanostructure comprises a first and a second portion, the first portion may be a rod. The rod may comprise a first recess at a first longitudinal end of the rod and a second recess at a second longitudinal end of the rod, each recess comprising a respective bottom surface. The recesses may be oriented perpendicular to a longitudinal axis of the rod and preferably facing in opposite directions, and wherein the bottom surfaces may lie in parallel planes.
In some embodiments, one recess may comprise at least one of the coupling sites and the other recess may comprise at least one binding site.
In some embodiments in which the nanostructure comprises a first and a second portion, the second portion may be a hollow disc. The hollow disc may comprise two outer discs and at least one connection structure connecting the two outer discs to form the hollow disc.
Further, each outer disc may comprise a disc recess. The outer discs may be connected to form the hollow disc such that the two disc recesses may be located at an angle offset of 180° with respect to each other.
In some embodiments wherein the nanostructure comprises a rod, the rod may be located within the disc and the rod may rotate freely around a rotation axis through the centre of the hollow disc, and the disc recesses and the recesses of the rod may be configured to provide access to the coupling site and the at least one binding site in one rotational position of the rod, which may be the second configuration.
The outer disc may comprise a coupling site at an outer rim and adjacent to the disc recess that may be configured to provide access to the coupling site comprised by the recess of the rod.
In some embodiments, the binding site may be more accessible for the binding target in the second configuration than in the first configuration.
However, in other embodiments, the binding site may be more accessible for the binding in the first configuration than in the second configuration.
The coupling site set may be formed by one coupling site. However, in other embodiments, the coupling site set may also be formed by a plurality of coupling sites.
In a further embodiment the present invention relates to a system comprising the nanostructure as described above.
The system may comprise at least one binding target.
The system may compromise a plurality of coupling targets.
The system may comprise at least one first entity. Further, each of the at least one first entity may comprise at least one coupling target. Yet further, each of the at least one first entity may comprise at least one binding target.
In some embodiments, the system may comprise at least one second entity. Further, each of the at least one second entity may comprise at least one coupling target.
The at least one coupling target comprised by the second entity may be distinct to the at least one coupling target comprised by the first entity.
Each of the at least one first entity may be a cell. Further, each of the at least one first entity may be a T-cell.
Similarly, each of the at least one second entity may be a cell. Further, each of the at least one second entity may be a tumor cell.
In some embodiments, the at least one coupling target comprised by each of the at least one first entity may be a CD3 antigen.
Further, the at least one coupling target comprised by each of the at least one second entity may be an epithelial cell adhesion molecule.
Yet further, the at least one binding target may be a CD28 protein.
In a further embodiment, the present invention relates to a method for conditionally binding a binding site to a binding target, wherein the method comprises utilizing a nanostructure or a system according to the present invention.
The method may comprise the nanostructure assuming the first configuration and the second configuration, wherein the accessibility of the binding site for the binding target in the second configuration may be different to the accessibility of the binding site for the binding target in the first configuration, wherein the nanostructure may assume the first configuration when none of the coupling sites of the coupling site set is coupled to its respective coupling target and wherein the nanostructure may assume the second configuration when each of the coupling sites of the subset of the coupling site set is coupled to its respective coupling target.
The method may comprise assuming the first configuration with a higher probability than the second configuration when none of the coupling sites of the coupling site set is coupled to its respective coupling target.
Further, the method may comprise the nanostructure assuming a first and second equilibrium state between a first configuration of the nanostructure and a second configuration of the nanostructure, wherein the probability of assuming the second configuration may be different (e.g., higher) in the second equilibrium state than in the first equilibrium state. Further the method may comprise the nanostructure assuming the first equilibrium state when all of the respective coupling targets are absent, i.e. none of the coupling sites of the coupling site set can couple to the respective coupling target and the nanostructure assuming the second equilibrium state when all of the respective coupling targets of the subset of coupling sites are present, i.e. all coupling sites of the subset of coupling sites can couple to the respective coupling targets.
The method may comprise coupling the coupling sites to identical coupling targets. Alternatively, the method may comprise coupling the coupling sites to distinct coupling targets.
In some embodiments, the method may not comprise coupling the coupling sites to each other. That is, the coupling sites may not be configured to couple to each other or in other words, a coupling site may not also be configured as coupling target.
The method may comprise coupling to coupling targets comprised by a single entity.
Additionally or alternatively, the method may comprise coupling to coupling targets comprised by distinct entities.
In embodiments, wherein the method comprises utilizing a system wherein each of the at least one first entity is a T-cell, the binding target binding to the binding site may at least further activate the T-cell.
In a further embodiment, the present invention relates to a substance comprising a plurality of nanostructures according to the present invention.
The substance may be for use as a medicament. This may be advantageous as the nanostructures may enabled targeted treatment of diseases, such as targeted drug delivery of cell activation.
Further, the substance may be for use in the treatment of cancer.
Additionally or alternatively, the substance may be for use in the treatment of blood clotting disorders.
Further, the substance may be for use in the treatment of immunological disorders.
Additionally or alternatively, the substance may be for use in the treatment of human immunodeficiency virus (HIV) infection.
Further, the substance may be for use in the treatment of macular degeneration.
Additionally or alternatively, the substance may be for use in the treatment of diabetes.
In a further embodiment, the present invention relates to a composition comprising a plurality of nanostructures according to the present invention.
The composition may be for use as a medicament. This may be advantageous as the nanostructures may enabled targeted treatment of diseases, such as targeted drug delivery of cell activation.
Further, the composition may be for use in the treatment of cancer.
Additionally or alternatively, the composition may be for use in the treatment of blood clotting disorders.
Further, the composition may be for use in the treatment of immunological disorders.
Additionally or alternatively, the composition may be for use in the treatment of human immunodeficiency virus (HIV) infection.
Further, the composition may be for use in the treatment of macular degeneration.
Additionally or alternatively, the composition may be for use in the treatment of diabetes.
Thus, in some embodiments the present invention provides a nanostructure that is configured to assume a first and a second configuration, wherein the nanostructure further comprises a binding site for which the accessibility is different between the first and second configuration. Furthermore, the nanostructure comprises a coupling site set comprising at least one coupling site, wherein the nanostructure is configured to assume the first configuration when none of the coupling sites is coupled to its respective coupling target and to assume the second configuration when each coupling site of a subset of the coupling site set is coupled to its respective coupling target.
In other words, the present invention discloses a conditionally switchable nanostructure that may assume different configurations depending on the coupling of at least a subset of the coupling site set comprised by the nanostructure, wherein the accessibility of the binding site may change depending on the configuration the nanostructure assumes. For example, the binding site may be significantly more accessible in one configuration than the other, in some cases it may even be only accessible in one of the two configurations. This may allow to enable access to the binding site only conditioned on the coupling of at least a subset of coupling sites of the coupling site set. This may be advantageous as it may control the access to the binding site based on the presence or absence of at least a subset of the respective coupling targets.
Furthermore, the nanostructure may generally utilize a great variety of interactions, for example the binding and/or coupling sites may be molecules, for example an antibody, an antibody fragment, a DNA strand, a biotin molecule, a streptavidin molecule, a maleimide-thiol chemistry molecule, a click chemistry molecule, etc. This may be advantageous compared to the state of the art such as the devices disclosed in WO 2012/061719 A2, that relies on specific and limited interactions.
Further, the nanostructure may at least partially be manufactured using DNA origami techniques. Owing to the self-assembly of DNA origami structures and the readily available software for designing the corresponding scaffolding and staple strands this may be a comparatively less complex manufacturing process compared to standard nanomanufacturing techniques.
Generally, it will be understood that embodiments of the present invention provide the ability to conditionally activate and deactivate certain molecular interactions based on the coupling state of other interaction sites.
According to some embodiments of the present invention, a DNA origami structure comprising rigid domains that are connected in such a way that the structure can adopt different conformations is provided. At least two interaction sites (e.g., interaction molecules) are attached to the structure, e.g., a coupling site and a binding site. The interaction molecules are attached in such a way, and the structure is designed in such a way, that when a subset A of the interaction molecules is not bound the intended targets, the structure spends the majority of time in a conformation in which a subset B of the interaction molecules cannot bind to their intended targets due to steric hindrance. Once the subset A of the interaction molecules binds to their intended targets, the accessible conformations of the structure are restricted due to the bound targets such that the structure now spends a larger amount of time than in the initial unbound state in conformations in which the subset B of interaction molecules can bind their intended targets. Therefore, the binding rate of the subset B of interaction molecules on the structure is conditionally modified by the binding state of the subset A of interaction molecules. Because the conditional activation is mediated through the shape of the phase space of the structure, and not through the interaction molecules themselves, the function can be applied to any type of interaction, such as antibodies, antibody fragments, DNA strands, biotin-streptavidin, maleimide-thiol chemistry, click chemistry, and many others.
Thus, in embodiments of the present invention, multiple molecular interaction sites are added to a DNA origami structure. Further, the molecular interactions are conditionally activated or deactivated based on the binding state of other molecular interactions present on the origami structure in a generalizable fashion.
In some embodiments of the present invention the nanostructure is comprised in a substance or composition that may be used as a medicament. This may be beneficial, as the nanostructure may enable targeted treatment of diseases. For example, through targeted drug delivery or targeted activation of T-cells. Such diseases may for example be different types of cancer, blood clotting disorders, immunological disorders, HIV infections, macular degeneration or diabetes. That is, embodiments of the present invention may enable treatment of a number of diseases.
Thus, owing to the wide range of interactions available, a more versatile and flexible conditionally switchable nanostructure is provided.
The present invention is also defined by the following numbered embodiments.
Below, reference will be made to nanostructure embodiments. These embodiments are abbreviated by the letter “N” followed by a number. Whenever reference is herein made to “nanostructure embodiments”, these embodiments are meant.
N1. A nanostructure (10) comprising
It should be understood that the subset of the coupling site set may also coincide with the coupling site set.
N2. The nanostructure according to the preceding embodiment, wherein the coupling sites (14,16) are not configured to couple to each other.
N3. The nanostructure according to any of the preceding embodiments, wherein each coupling site (14, 16) is configured to only couple to a single type of coupling target (202, 204).
That is, a coupling site (14, 16) may not couple to distinct coupling targets (202, 204).
N4. The nanostructure (10) according to any of the preceding embodiments, wherein the nanostructure (12) comprises a plurality of binding sites (12),
N5. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) is configured to assume the first configuration (A) with a higher probability than the second configuration (B) when none of the coupling sites (14, 16) of the coupling site set is coupled to its respective coupling target (202, 204).
N6. The nanostructure according to any of the preceding embodiments, wherein the binding site (12) is accessible for the binding target (100) in one configuration (A, B), and is not accessible for the binding target (100) in the other configuration (B, A).
N7. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) is configured to assume first and second equilibrium states between the first configuration (A) and the second configuration (B),
N8. The nanostructure according to the preceding embodiment, wherein the probability of the nanostructure (10) assuming the first configuration (A) in the first equilibrium state is at least 0.3, preferably at least 0.5, more preferably at least 0.7, such as at least 0.9.
N9. The nanostructure according to any of the 2 preceding embodiments, wherein the probability of the nanostructure (10) assuming the second configuration (B) in the first equilibrium state is at most 0.7, preferably at most 0.5, more preferably at most 0.3, such as at most 0.1.
N10. The nanostructure according to any of the 3 preceding embodiments, wherein the probability of the nanostructure (10) assuming the first configuration (A) in the second equilibrium state is at most 0.7, preferably at most 0.5, more preferably at most 0.3, such as at most 0.1.
N11. The nanostructure according to any of the 4 preceding embodiments, wherein the probability of the nanostructure (10) assuming the second configuration (B) in the second equilibrium state is at least 0.3, preferably at least 0.5, more preferably at least 0.7, such as at least 0.9.
N12. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) comprises a first portion (18) and a second portion (20);
N13. The nanostructure according to the preceding embodiment, wherein the subset of the coupling site set comprises at least one coupling site (14, 16) comprised by the first portion (18) and at least one coupling site (14, 16) comprised by the second portion (20).
N14. The nanostructure according to any of the 2 preceding embodiments, wherein the first portion (18) and the second portion (20) are movably attached to each other, e.g. by means of a hinge or a rotational axis.
N15. The nanostructure according to any of the 3 preceding embodiments, wherein the nanostructure (10) comprises at least one additional portion.
N16. The nanostructure according to any of the preceding embodiments, wherein the nanostructure comprises a maximum length, i.e. a length corresponding to the largest extend of the nanostructure, and wherein the maximum length is smaller than 1000 nm, preferably smaller than 500 nm, such as 100 nm.
N17. The nanostructure according to any of the preceding embodiments, wherein the binding site (12) can bind to the binding target (100) reversibly, i.e. they can bind and detach repeatedly.
N18. The nanostructure according to any of the preceding embodiments, wherein at least one of the coupling sites (14, 16) of the coupling site set couples reversibly to the respective coupling target (202, 204), i.e. they can couple and decouple repeatedly.
N19. The nanostructure according to any of the preceding embodiments, wherein the coupling site set comprises only identical coupling sites (14, 16), i.e. configured to couple to the same coupling targets (202, 204).
N20. The nanostructure according to any of the embodiments N1 to N18, wherein the coupling site set comprises at least two distinct coupling sites (14, 16), configured to couple to distinct coupling targets (202, 204).
N21. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) is configured to couple to coupling targets (202, 204) comprised by a single entity (302).
N22. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) is configured to couple to coupling targets (202, 204) comprised by a plurality of entities (302, 304).
N23. The nanostructure according to the preceding embodiment, wherein the plurality of entities (302, 304) comprises at least two distinct entities.
N24. The nanostructure according to any of the preceding embodiments, wherein the binding target (100) is comprised by an entity (302) also comprising at least one coupling target (202, 204).
N25. The nanostructure according to any of the preceding embodiments, wherein the nanostructure (10) is at least partially formed by a DNA origami structure.
N26. The nanostructure according to the preceding embodiment, wherein the DNA origami structure comprises at least one scaffolding strand, i.e. single-stranded polynucleotide scaffold DNA with a known sequence.
N27. The nanostructure according to the preceding embodiment, wherein the DNA origami structure further comprises a plurality of single-stranded oligonucleotide staple strands and wherein each staple strand is at least partially complementary to at least one scaffolding strand.
N28. The nanostructure according to the preceding embodiment, wherein each of the staple strands is configured to bind to the at least one scaffolding strand in two distinct places,
N29. The nanostructure according to any of the preceding embodiments, wherein at least one binding site (12) is a molecule.
N30. The nanostructure according to any of the preceding embodiments, wherein at least one binding site (12) is configured to bind to a cluster differentiation (CD) molecule, e.g., a CD28 protein, and/or to another disease-associated cell surface molecule as a binding target (100).
N31. The nanostructure according to any of the preceding embodiments, wherein at least one coupling site (14, 16) comprised by the coupling site set is a molecule.
N32. The nanostructure according to any the preceding embodiments, wherein at least one coupling site (14, 16) of the coupling site set is configured to couple to a CD3 antigen as a coupling target (202, 204).
N33. The nanostructure according to any of the preceding embodiments, wherein at least one coupling site (14, 16) of the coupling site set is configured to couple to an epithelial cell adhesion molecule as a coupling target (202, 204).
N34. The nanostructure according to any of the preceding embodiments with the features of at least one of N29 and N31, wherein the molecule is bound to the nanostructure (10) by means of linker molecules.
N35. The nanostructure according to the preceding embodiment and with the features of embodiment N26 and N27, wherein the molecule is bound to one of the at least one scaffolding strand or a staple strand by means of a linker molecule;
N36. The nanostructure according to any of the preceding embodiments with the features of embodiment N12, wherein the first portion (18) and the second portion (20) comprise an identical shape.
N37. The nanostructure according to the preceding embodiment, wherein the shape is a cuboid, i.e. a rectangular box.
N38. The nanostructure according to any of the 2 preceding embodiments, wherein the two portions (18, 20) each comprise a cavity (36, 39), configured to form a chamber when the nanostructure (10) is in the first configuration (A).
N39. The nanostructure according to the preceding embodiment, wherein at least one binding site (12) is located in the cavity (36) of the first portion (18).
N40. The nanostructure according to the preceding embodiment, wherein at least one binding site (12) is attached to an outer surface of the cavity (36) by means of a rod (40),
The person skilled in the art will appreciate that a rod (40) may be any type of flexible link between a portion of the cavity (36) and at least one binding site (12).
N41. The nanostructure according to any of the preceding embodiments with the features of embodiment N12, wherein the first portion (18) is a rod (18A).
N42. The nanostructure according to the preceding embodiment, wherein the rod (18A) comprises a first recess (181) at a first longitudinal end of the rod (18A) and a second recess (182) at a second longitudinal end of the rod (18A), each recess (181, 182) comprising a respective bottom surface (1811, 1821).
N43. The nanostructure according to the preceding embodiment, wherein the recesses (181, 182) are oriented perpendicular to a longitudinal axis of the rod and preferably facing in opposite directions, and wherein the bottom surfaces (1811, 1821) may lie in parallel planes.
N44. The nanostructure according to the preceding embodiment, wherein one recess (181) comprises at least one of the coupling sites (16) and the other recess (182) comprises at least one binding site (12).
N45. The nanostructure according to any of the preceding embodiments with the features of embodiment N12, wherein the second portion (20) is a hollow disc (20A).
N46. The nanostructure according to the preceding embodiment, wherein the hollow disc (20A) comprises two outer discs (21, 23) and at least one connection structure (25) connecting the two outer discs (21, 22) to form the hollow disc (20A).
N47. The nanostructure according to the preceding embodiment, wherein each outer disc (21, 23) comprises a disc recess (22, 24).
N48. The nanostructure according to the preceding embodiment, wherein the outer discs (21, 23) are connected to form the hollow disc (20A) such that the two disc recesses (22, 24) are located at an angle offset of 180° with respect to each other.
N49. The nanostructure according to the preceding embodiment with the features of embodiment N44, wherein the rod (18A) is located within the disc (20A) and
wherein the rod (18A) rotates freely around a rotation axis through the centre of the hollow disc (20); and
wherein the disc recesses (22, 24) and the recesses (181, 182) of the rod (18A) are configured to provide access to the coupling site (16) and the at least one binding site (12) in one rotational position of the rod (18A), which is the second configuration (B).
N50. The nanostructure according to the preceding embodiment, wherein the outer disc (23) comprises a coupling site (14) at an outer rim and adjacent to the disc recess (24) that is configured to provide access to the coupling site (16) comprised by the recess (181) of the rod (18A).
N51. The nanostructure (10) according to any of the preceding embodiments, wherein the binding site (12) is more accessible for the binding target (100) in the second configuration (B) than in the first configuration (A).
N52. The nanostructure (10) according to any of the embodiments N1 to N50, wherein the binding site (12) is more accessible for the binding (100) in the first configuration (A) than in the second configuration (B).
N53. The nanostructure (10) according to any of the preceding embodiments, wherein the coupling site set is formed by one coupling site.
N54. The nanostructure (10) according to any of the embodiments N1 to N52, wherein the coupling site set is formed by a plurality of coupling sites (14, 16).
Below, reference will be made to system embodiments. These embodiments are abbreviated by the letter “S” followed by a number. Whenever reference is herein made to “system embodiments”, these embodiments are meant.
S1. A system comprising the nanostructure according to any of the preceding nanostructure embodiments.
S2. The system according to the preceding system embodiment, wherein the system comprises at least one binding target (100).
S3. The system according to any of the preceding system embodiments, wherein the system comprises a plurality of coupling targets (202, 204).
S4. The system according to any of the preceding system embodiments, wherein the system comprises at least one first entity (302).
S5. The system according to the preceding system embodiment and with the features of embodiment S3, wherein each of the at least one first entity (302) comprises at least one coupling target (202, 204).
S6. The system according to any of the 2 preceding embodiments and with the features of S2, wherein each of the at least one first entity (302) comprises at least one binding target (100).
S7. The system according to any of the 3 preceding system embodiments, wherein the system comprises at least one second entity (304).
S8. The system according to the preceding system embodiment and with the features of embodiment S3, wherein each of the at least one second entity (304) comprises at least one coupling target (204).
S9. The system according to the preceding system embodiment and with the features of embodiment S5, wherein the at least one coupling target (204) comprised by the second entity (304) is distinct to the at least one coupling target (202) comprised by the first entity (302).
S10. The system according to any of the preceding system embodiments with the features of S4, wherein each of the at least one first entity (302) is a cell.
S11. The system according to the preceding system embodiment, wherein each of the at least one first entity (302) is a T-cell.
S12. The system according to any of the preceding system embodiments with the features of S7, wherein each of the at least one second entity (304) is a cell.
S13. The system according to the preceding system embodiment, wherein each of the at least one second entity (304) is a tumor cell.
S14. The system according to any of the preceding system embodiments with the features of embodiments S11 and S5, wherein the at least one coupling target (202, 204) comprised by each of the at least one first entity (302) is a CD3 antigen.
S15. The system according to any of the preceding system embodiments with the features of embodiment S12 and S8, wherein the at least one coupling target (202, 204) comprised by each of the at least one second entity (304) is an epithelial cell adhesion molecule.
S16. The system according to any of the preceding system embodiments with the features of embodiments S11 and S6, wherein the at least one binding target (100) is a CD28 protein.
Below, reference will be made to method embodiments. These embodiments are abbreviated by the letter “M” followed by a number. Whenever reference is herein made to “method embodiments”, these embodiments are meant.
M1. A method for conditionally binding a binding site (12) to a binding target (100), wherein the method comprises
M2. The method according to the preceding method embodiment, wherein the method comprises the nanostructure (10) assuming the first configuration (A) and the second configuration (B), wherein the accessibility of the binding site (12) for the binding target (100) in the second configuration (B) is different to the accessibility of the binding site (12) for the binding target (100) in the first configuration (A);
M3. The method according to the preceding method embodiment, wherein the method comprises assuming the first configuration (A) with a higher probability than the second configuration (B) when none of the coupling sites (14, 16) of the coupling site set is coupled to its respective coupling target (202, 204).
M4. The method according to any of the preceding method embodiments, wherein the method comprises
M5. The method according to the preceding method embodiment, wherein the method comprises coupling the coupling sites (14, 16) to identical coupling targets (202, 204).
M6. The method according to any of the penultimate method embodiment, wherein the method comprises coupling the coupling sites (14, 16) to distinct coupling targets (202, 204).
M7. The method according to any of the preceding method embodiments, wherein the method does not comprise coupling the coupling sites (14, 16) to each other.
M8. The method according to any of the preceding method embodiments, wherein the method comprises coupling to coupling targets (202, 204) comprised by a single entity (302).
M9. The method according to any of the preceding method embodiments, wherein the method comprises coupling to coupling targets (202, 204) comprised by distinct entities (302, 304).
M10. The method according to the preceding embodiment, wherein the system comprises the features of embodiment S11,
Below, reference will be made to substance embodiments. These embodiments are abbreviated by the letter “T” followed by a number. Whenever reference is herein made to “substance embodiments”, these embodiments are meant.
T1. A substance comprising a plurality of nanostructures according to any of the preceding nanostructure embodiments.
T2. The substance according to the preceding substance embodiment for use as a medicament.
T3. The substance according to any of the preceding embodiments for use in the treatment of cancer.
T4. The substance according to any of the preceding substance embodiments for use in the treatment of blood clotting disorders.
T5. The substance according to any of the preceding substance embodiments for use in the treatment of immunological disorders.
T6. The substance according to any of the preceding substance embodiments for use in the treatment of human immunodeficiency virus (HIV) infection.
T7. The substance according to any of the preceding substance embodiments for use in the treatment of macular degeneration.
T8. The substance according to any of the preceding substance embodiments for use in the treatment of diabetes.
Below, reference will be made to composition embodiments. These embodiments are abbreviated by the letter “C” followed by a number. Whenever reference is herein made to “composition embodiments”, these embodiments are meant
C1. A composition comprising a plurality of nanostructures according to any of the preceding nanostructure embodiments.
C2. The composition according to the preceding composition embodiment for use as a medicament.
C3. The composition according to any of the preceding composition embodiments for use in the treatment of cancer.
C4. The composition according to any of the preceding composition embodiments for use in the treatment of blood clotting disorders.
C5. The composition according to any of the preceding composition embodiments for use in the treatment of immunological disorders.
C6. The composition according to any of the preceding composition embodiments for use in the treatment of human immunodeficiency virus (HIV) infection.
C7. The composition according to any of the preceding composition embodiments for use in the treatment of macular degeneration.
C8. The composition according to any of the preceding composition embodiments for use in the treatment of diabetes.
Embodiments of the present invention will now be described with reference to the accompanying drawings. These embodiments should only exemplify, but not limit, the present invention.
It is noted that not all the drawings carry all the reference signs. Instead, in some of the drawings, some of the reference signs have been omitted for the sake of brevity and simplicity of the illustration. Embodiments of the present invention will now be described with reference to the accompanying drawings.
In one embodiment, the invention relates to a nanostructure. Very generally, the nanostructure may comprise a first portion, a second portion, a binding site and a coupling site set comprising at least one coupling site. In examples of the present invention, the nanostructure may be a DNA origami structure, i.e., a DNA origami device, as exemplarily disclosed in U.S. Pat. No. 7,842,793 B2. However, it should be understood that the present technology is not limited thereto, and that in fact also other nanostructures may be utilized to exercise the present invention.
It will be appreciated, that the use of the singular article “a” or “the” within this document is not meant to limit the scope of the invention expect if specifically stated. That is, generally “a” may also refer to more than one. In other words, “a” can generally be read as “at least one”. For example, “a binding site” also includes a plurality of binding sites.
Reference will now be made to
The nanostructure 10 further comprises a binding site 12, which may bind reversibly or irreversibly to a binding target 100 (e.g. as illustrated in
As discussed, in one non-limiting example, the nanostructure 10 may be a DNA origami nanostructure 10. When utilizing such a DNA origami nanostructure 10, functional sites, such as biding sites 12, or coupling sites 14 and 16 can be added to the nanostructure 10. A mechanism for this is discussed in conjunction with
Again with reference to
Further, the nanostructure 10 comprises a plurality of coupling sites 14, 16, wherein the first and the second portion 18, 20 may each comprise at least one coupling site 14, 16. The coupling sites 14, 16 may either couple to coupling targets 202, 204 reversibly or irreversibly, i.e. the coupling sites 14, 16 may either bind and detach to the coupling targets 202, 204 repeatedly or generally stay bound to the coupling targets 202, 204 once successfully bound for the first time. The coupling sites 14, 16 and/or the coupling targets 100 may be molecules. For example, reversibly binding molecules may be antibodies, antibody fragments, DNA strands, biotin molecules, streptavidin molecules, whereas irreversibly binding molecules may be maleimide-thiol chemistry molecules, click chemistry molecules, etc.
In some embodiments, the coupling sites 14, 16 may be identical (i.e. configured to couple to the coupling targets 202, 204), as depicted in
The nanostructure 10 may assume the different configurations A and B depending on the binding states of the coupling sites 14, 16. Generally, coupling sites 14, 16 (or, in some embodiments also an individual coupling site) may define a coupling site set, and a coupling site sub set. In the embodiment depicted in
Generally, it will be understood that the nanostructure 10 is configured to assume the configuration A (e.g., the closed configuration) and configuration B (e.g., the open configuration) also when the coupling sites 14, 16 are not coupled to their respective coupling targets 202, 204 (see, e.g.,
In other words, to couple to the coupling targets 202, 204, the nanostructure 10 is in the second configuration. More particularly, the nanostructure 10 may randomly move into the second configuration (usually, this happens rarely). Once this happens and in case the coupling targets 202, 204 are present, the coupling sites 14, 16 can all be bound at the same time. Once this happens, the nanostructure 10 is unlikely to leave the second configuration anymore, thus increasing the time spent in the second configuration.
That is, in this embodiment, the nanostructure 10 may be configured to assume the first configuration A when none of the coupling sites 14, 16 is coupled to its respective coupling target 202, 204. It will be understood that the nanostructure 10 may still be able to assume a different configuration, e.g., the second configuration B. However, when none of the coupling sites 14, 16 is coupled to its respective coupling target 202, 204 (i.e., when coupling site 14 and coupling site 16 is not coupled to its respective coupling target), the nanostructure 10 may be configured to assume the first configuration A. In some embodiments, the nanostructure 10 may be configured to assume the first configuration A with a higher probability than the second configuration B when none of the coupling sites 14, 16 is coupled to its respective coupling target.
Further the nanostructure 10 may be configured to assume the second configuration B when each coupling site 14, 16 of a subset of the coupling site set is coupled to its respective coupling target 202, 204. It will be understood that the nanostructure 10 may also assume the second configuration B in cases where not all coupling sites 14, 16 of the subset are coupled to their respective coupling targets 202, 204 (see
With reference to
Generally, it will be understood that the nanostructure 10 can assume equilibrium states between the first configuration A and the second configuration B (see
Further, the nanostructure 10 may assume a second equilibrium state between the first configuration A and the second configuration B when all of the respective coupling sites 14, 16 of a subset of the set of coupling sites can couple to respective coupling targets 202, 204 (see, e.g.,
It will be understood that the nanostructure may in some embodiments always be in configuration B once the subset of coupling sites 14, 16 is coupled to the respective coupling targets 202, 204. For example, in embodiments where the coupling sites 14, 16 may couple irreversibly to the coupling targets 202, 204.
As discussed, in the first equilibrium state the nanostructure 10 may assume the first configuration A with a probability of 0.9 and the second configuration B with a probability of 0.1. In such a case it may be unlikely that the binding site 12 can bind to its binding target 100. Therefore, the first equilibrium state may in such cases also be referred to as slow binding state. In contrast, in the second equilibrium state the nanostructure 10 may assume the first configuration A with a probability of 0.1 and the second configuration B with a probability of 0.9. Thus, the second equilibrium state may in such cases also be referred to as fast binding state.
It will be understood that the above is merely an example and that the first and second equilibrium state may assume the first and second configuration A, B with different probabilities. In general, the second configuration B may be assumed with a higher probability in the second equilibrium state than in the first equilibrium state.
As described above, in some embodiments the binding site 12 may be more accessible in the second configuration B than in the first configuration A. In some embodiments, the binding site 12 may not be accessible in the first configuration A. However, if the nanostructure 10 assumes the second configuration B, a binding target 100 may bind to the binding site 12 of the nanostructure 10, as depicted in
Again, when the nanostructure 10 is in an environment where its coupling sites 14, 16 can both couple to their respective coupling targets 202, 204 (such as depicted in
Thus, if a binding target 100 is present (see
In the embodiment depicted in
It will be understood that the above is merely an example and that in other embodiments the binding site 12 may be more accessible in configuration A than in configuration B. Such an embodiment is shown in
Again, the nanostructure 10 comprises coupling sites 14, 16, forming a coupling site set and a sub-set (the coupling site set and the sub-set being equal in this embodiment). As in the other embodiments, each coupling site 14, 16 is configured to couple to a coupling target 202, 204.
Again with general reference to
With reference to
It will be understood that the considerations discussed in conjunction with the other nanostructures may also apply accordingly to such a nanostructure 10. For example, the nanostructure depicted in
With reference to
The binding site 12 is more accessible in the second configuration B than in the first configuration A and
Generally, a nanostructure 10 may assume the second configuration when each coupling site 14, 16 of a subset of the coupling site set is coupled to its respective coupling target 202, 204.
With reference to
Throughout the embodiments depicted in
However, as already mentioned above, this is not necessary. In some embodiments of the present invention the coupling sites 14, 16 may bind to distinct coupling targets 202, 204 (i.e., to coupling targets 202, 204 with differing configuration), as schematically depicted in
Generally (i.e., independent of whether or not the coupling sites 14, 16 are of the same or of differing configurations), the respective coupling targets 202, 204 may be comprised by a single entity 302 (see, e.g.,
In some embodiments, a single entity 302, 304 may only comprise the respective coupling targets 202 or 204 for one of the distinct coupling sites 14, 16, wherein in other embodiments a single entity 302, 304 may comprise a plurality of respective coupling sites 202, 204—see
That is, generally, the present technology is applicable in different scenarios. The scenarios include a bivalent nanostructure 10, i.e., a nanostructure 10 having one type of coupling sites 14, 16, and one type of binding site 12, which coupling sites 14, 16 couple to coupling targets 202, 204 of a single entity 302, i.e., to a single object. This case may be referred to as the bivalent (one type of binding site, one type of coupling site) one object case, and is depicted in
In particular, it should be understood that the accessibility of a binding site 12 of the nanostructure 10 may be different when the coupling sites 14, 16 of the subset of the coupling site set are coupled to their respective coupling targets 202, 204 compared to the case that none of the coupling sites 14, 16 are coupled to their respective coupling target.
As already mentioned, the present invention also comprises multivalent nanostructures 10. That is, a nanostructure 10 may comprise a plurality of types of coupling sites 14, 16 and/or binding sites 12. An exemplary embodiment of a multivalent nanostructure 10 is depicted in
The depicted nanostructure 10 comprises a coupling site set comprising four coupling sites 14 A, 14B, 16A, 16B, wherein each coupling site may be of a different configuration. That is, the coupling sites 14 A, 14B, 16A, 16B, may each be distinct from each other. However as previously discussed at least a portion of the coupling sites 14 A, 14B, 16A, 16B, may also be of the same configuration, e.g. the coupling sites attached to the same portion 18, 20 may be identical. Further, the nanostructure 10 may comprise a plurality of binding sites 12A, 12B, wherein the binding sites may be of identical or different configuration. That is, in some embodiments all binding sites may be distinct from each other, whereas in other embodiments at least a portion of the plurality of binding sites may of the same type, i.e. the same configuration.
The nanostructure may be configured assume the first configuration if none of the coupling sites 14 A, 14B, 16A, 16B, is coupled to the respective coupling target (see
It will be understood that albeit being configured to assume the first configuration A when none of the coupling sites 14 A, 14B, 16A, 16B, is coupled to its respective coupling target, the nanostructure 10 may still assume another configuration, such as the second configuration B as depicted on the right hand side of
Furthermore, the nanostructure 10 may be configured to assume a first and second equilibrium state between the first configuration A and the second configuration B. That is, the nanostructure 10 may assume the first configuration A and the second configuration B each with a certain probability as indicated by the two-sided arrow. The probability that the nanostructure 10 assumes the second configuration B may be higher in the second equilibrium state of the nanostructure 10 than in the first equilibrium state. Further, the nanostructure 10 may be configured to assume the second equilibrium state when all coupling sites 14A, 14B, 16A, 16B of a subset of the coupling site set are coupled to the respective coupling targets 202, 204, whereas the nanostructure may be configured to assume the first equilibrium state when none of the coupling sites 14A, 14B, 16A, 16B, of the coupling site set are coupled to their respective coupling targets 202, 204.
As previously discussed, the accessibility of the binding sites 12A, 12B may depend on the configuration assumed by the nanostructure 10. That is, the accessibility of the binding sites 12A, 12B may be different when the nanostructure 10 is in the first configuration A compared to when the nanostructure 10 is in the second configuration B. In the depicted embodiment, the binding sites 12A, 12B may be more accessible when the nanostructure assumes the second configuration B. Moreover, in some embodiments, the binding sites 12A, 12B may not be accessible in the first configuration A.
Whenever the binding sites 12A, 12B are accessible a corresponding binding target 100A, 100B may bind to it as depicted in
Further all previous considerations may apply, particularly the binding of binding sites 12A, 12B and the coupling of coupling sites 14A, 14B, 16A, 16B may be reversible or irreversible depending on the type of interaction site 1000. That is, depending on the configuration (or type) of each coupling or binding site, the corresponding coupling or binding process may be reversible or irreversible. For example, a nanostructure may comprise a coupling site 14A, 14B, 16A, 16B that binds reversibly as well as another coupling site 14A, 14B, 16A, 16B that binds irreversibly.
With reference to
As mentioned before, the nanostructure 10 may be configured to assume the second configuration B when each coupling site 14A, 14B, 16A, 16B of a subset of the coupling site set is coupled to the respective coupling target 202A, 202B, 204A, 204B. A subset may for example comprise one coupling site 14A, 14B, 16A, 16B for each portion 18, 20, wherein the coupling site 14A, 14B, 16A, 16B is attached to said portion 18, 20 of the nanostructure 10. Examples where all coupling sites 14A, 14B, 16A, 16B of such a subset are coupled to the respective coupling targets 202A, 202B, 204A, 204B are shown to the right of the two-sided arrow in
Again, if binding targets 100A, 100B are present and the binding sites 12A, 12B are accessible the binding targets 100A, 100B may bind to the binding sites 12A, 12B as depicted in
In the following an example of a nanostructure 10 comprising two distinct coupling sites 14, 16 is presented, first conceptually and subsequently with reference to an exemplary implementation.
The nanostructure 10 may be in a solution or generally in an environment with the first entity 302 comprising a plurality of binding targets 100 and a plurality of coupling targets 202, wherein the coupling targets 202 may only couple to one of the distinct coupling sites 14, 16.
That is, one coupling site 14 of the nanostructure 10 may couple to a coupling target 202 comprised by the first entity 302. Without other entities being present, the nanostructure 10 may be in a first equilibrium state in which the nanostructure 10 is predominantly, or in some embodiments essentially solely, in the first configuration A and therefore the coupling site 14 only bind solely to the coupling targets comprised by the first entity 302, without the other coupling site 16 coupling to its respective coupling target. In other words, binding of the binding site 12 to the binding target 100 may be (at least mostly) inhibited by the conformation of the nanostructure 10.
However, as depicted in
Such a nanostructure 10 may be beneficial as it may bring two entities into close proximity through coupling of the coupling sites 12, 14 to the respective coupling targets 202, 204 and subsequently for example activate a process within an entity 302, 304 by further binding the binding site 12 to the corresponding binding target 100.
Further still, it will be understood that such a nanostructure 10 may be “inactive” unless activated by the presence of both the first entity 302 comprising the first coupling target 202 and the second entity 304 comprising the second coupling target 204. When such first entities 302 and second entities 304 are present, the nanostructure 10 is “activated” and its binding site 12 becomes accessible. Once accessible, the binding site 12 may bind to a binding target 100. In the example discussed in conjunction with
An exemplary embodiment will now be discussed with reference to
For such an embodiment, the first entity 302 may be a T-cell 302 comprising CD3 antigens 202 as coupling targets 202, and CD28 proteins as binding targets 100, and the second entity 304 may be a tumor cell 304 expressing EpCAM as the coupling target 204 (see
Thus, in a solution or environment such an embodiment may bring a tumor cell 304 and a T-cell 302 in close proximity by coupling to a CD3 antigen 202 on the T-cell 302 and by coupling to an EpCAM 204 on the tumor cell 304. Subsequently the T-cell 302 may be at least further activated by the binding site 12 of the nanostructure 10 binding to the CD28 protein 100. Thus, a solution or composition comprising nanostructures 10 may be used as a medicament.
With reference to
The outer dimensions of each portion 18, 20 may be determined by a length L, a breadth B and a height H, wherein typically L>B>H. The length L may be smaller than 1000 nm, preferably smaller than 500 nm, such as 100 nm.
The two portions 18, 20 may be movably connected along an edge 32, wherein the edge is of dimension B, i.e. it is an edge of a short side of the rectangular portions 18, 20.
Further, each portion 18, 20 comprises a coupling site 14, 16, e.g. a CD3 antigen-coupling site 14 and an EpCAM-coupling site 16. The coupling sites 14, 16 may each be located on a corresponding small end surface 34, 37 of the two portions 18, 20, i.e. a surface with dimensions B×H, wherein the surface is located opposite to the side where the two portions 18, 20 are connected, i.e. opposite to the edge 32. That is, the two coupling sites 14, 16 may be adjacent to each other when the nanostructure 10 assumes a closed configuration, i.e. when the two portions 18, 20 are in contact with the two outer surfaces 35, 38 with dimension L×B and comprising the edge 32 at which the two portions 18, 20 are connected. In this example the closed configuration may correspond to the first configuration A, where the binding site 12 is not accessible.
The two portions 18, 20 may each comprise a cavity 36, 39 configured to form a chamber within the two portions 18, 20 when they assume the closed configuration.
The binding site 12 may be located within the cavity 36 of the portion 18 comprising the coupling target 14. The binding site 12 may be an CD28-binding site 12. The binding site 12 may be attached to the portion 18 by means of a rod 40. That is, once the nanostructure 10 may assume an open configuration, i.e. when the two portions are in a position where the two cavities 36, 39 are exposed to the surrounding of the nanostructure 10, the binding site 12 may leave the cavity 36 and preferably bind to a binding target 100 in the vicinity of the nanostructure 10.
It is noted, that in the context of this document a rod 40 may be any type of flexible link between a portion of the cavity 36 and at least one binding site 12.
Reference will now be made to the illustration in
That is, in the implementation depicted in
Other implementations may for example allow for targeted drug delivery.
Further, the rod 18A may be configured to rotate (with respect to the second portion) around a rotational axis 183 running through the centre of the rod 18A in a direction perpendicular to the longitudinal axis and perpendicular to the bottom surfaces 1811, 1821 of the two recesses 181, 182.
Additionally, one recess 181 may comprise a coupling site 16, whereas the other recess 182 may comprise a binding site 12.
The second portion 20 of the nanostructure 10 may be a hollow disc 20A, comprising two outer discs 21, 23. Each of the two outer discs 21, 23 comprises a disc recess 22, 24 configured to provide access to the end of the rod 18 when assembled. More precisely the disc recesses 22, 24 are configured to provide access to the coupling site 16 and/or the binding site 12 that are attached within the two recesses 181, 182 of the rod 18A.
The two outer discs 21, 23 are connected through at least one connection structure 25 such that a hollow disc is formed, configured to take up the rod 18A. The outer discs 21, 23 may be combined in a way that the two disc recesses 22, 24 are rotated by 180° with respect to each other. In other words, the disc recesses 22, 24 are arranged such that the two recesses 181, 182 of the rod 18A may be accessible at the same time.
The rod 18A may be held in the centre of the hollow disc 20A. That is, the rod 18A may rotate freely within the hollow disc 20A, and the binding site 12 and the coupling site 16 may be accessible only when the rod 18A is in a position where the two recesses 181, 182 line up with the corresponding disc recesses 22, 24 (second configuration B).
Further, the outer disc 23 may comprise a coupling site 14 located at the outer rim and adjacent to the disc recess 24 that is configured to reveal the coupling site 16 comprised by the recess 181 of the rod 18A.
The assembled nanostructure 10 is shown in
With reference to
Further, once the coupling site 16 is bound to its coupling target 204, the binding site 12 is aligned with the other disc recess 22 and therefore accessible for binding to a corresponding binding target 100 (second configuration B). That is, the probability of binding to a binding target 100 is significantly higher compared to a state where the coupling site 16 is not bound to a coupling target (first configuration A).
With exemplary reference to the embodiment depicted in
Such a three-dimensional nanostructure 10 may be realized using DNA origami, i.e. by combining scaffolding strands and staple stands to form the required portions and the overall device. Such designs may for example be performed using software such as caDNAno. That is, a nanostructure 10 comprising multiple portions 18,20 may in some embodiments be made out of one scaffolding strand, whereas in other embodiments portions of a nanostructure 10 may be constructed utilizing a plurality of scaffolding strands.
Again, also with regard to the embodiment depicted in
Whenever a relative term, such as “about”, “substantially” or “approximately” is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., “substantially straight” should be construed to also include “(exactly) straight”.
Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be accidental. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may be accidental. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Yi), . . . , followed by step (Z). Corresponding considerations apply when terms like “after” or “before” are used.
Moreover, reference is made to the following specific examples which are given to further illustrate the present invention, without limiting the invention thereto.
For the subsequent examples 2-4, the following materials and methods were used: All DNA-origami objects were assembled in 20 mM MgCl2 using the folding ramp: 15 min at 65° C., 60° C.-45° C. for 1 h/° C. Staple concentrations were 200 nM and scaffold concentrations were 50 nM. Oligonucleotides were obtained from IDT. DNA scaffolds were produced according to Engelhardt et al. [F. A. S. Engelhardt, F. Praetorius, C. H. Wachauf, G. Bruggenthies, F. Kohler, B. Kick, K. L. Kadletz, P. N. Pham, K. L. Behler, T. Gerling, and H. Dietz, ‘Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds’, ACS Nano (2019), doi 10.1021/acsnano.9b01025]. DNA-origami objects were purified using PEG precipitation [E. Stahl, T. Martin, F. Praetorius, and H. Dietz, ‘Facile and scalable preparation of pure and dense DNA origami solutions’, Angewandte Chemie Intl. Edn., vol 53 (2014), p 12735], before antibody attachment. Purified antibody-DNA conjugates were added to the DNA-origami objects that present the complementary sequences, with a twofold excess per binding site. DNA-origami-antibody objects were purified using PEG precipitation (Stahl et al., ibid.)
All objects were stabilized for flow cytometry and T-cell activation assays using oligolysine-PEG (10 lysine, PEG 5k) as described in [Ponnuswamy, N., Bastings, M., Nathwani, B. et al. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation. Nat Commun 8, 15654 (2017). https://doi.org/10.1038/ncomms15654] with N:P ratio of 1:1 (except for logic gate 2, which was incubated at 1:20 ration overnight). Oligolysine-PEG was removed prior to the experiments using 50k-amicon filtration.
T-cell activation was analyzed using a T-Cell Activation Bioassays (Promega, J1655). The experiment was performed according to the instruction of the supplier. Briefly, CD19 expressing target cells (NALM-6) were added to 96 well microtiter plate at a final concentration of 5*105 cells per ml. Then a serial dilution of the different samples (in RPMI 1640 medium) were added. In the end, the genetic modified TCR/CD3 effector cells were added at a final concentration of 1.3*106 cells per ml. The reaction mixture was incubated for 6 h at 37 degree and 5% C02. The genetic modified effector cells express a luciferase intercellularly, if the interleukin 2 promoter is activated. By addition of Bio-Glo reagent, which includes a substrate for the luciferase, the luminescence signal is a direct proportional signal for activation of the TCR/CD3 effector cells, which was analyzed in a microtiter plate reader (Clariostar Plus, BMG).
In order to demonstrate that a binding site's accessibility can be controlled by the occupancy of another binding site, the present inventors developed a block-like DNA-origami object (logic gate 1), consisting of a moveable piston mounted inside a hollow casing (
Furthermore, the logic gate 1 features binding sites on the piston and on the casing for other objects or molecules (
First, the present inventors wanted to test if the binding of the logic gate 1 to the surface mimic B causes a change in its probability of occupying the compressed or extended state. To this end, the present inventors attached a FRET-dye pair (Cy5 and Cy3) to the piston and case such that the FRET efficiency reports on the state of the logic gate 1. The present inventors prepared logic gates 1 with different DNA springs, mixed these with surface mimics A (excess of surface mimic over logic gate 1), and analysed the mixtures by agarose gel electrophoresis and fluorescence scanning. Mixtures with both, logic gate 1 and surface mimic A, produce a band with slower migration than logic gate 1 only. The present inventors attribute this band to logic-gate-1-surface-mimic-A dimeric objects. Negative-staining TEM imaging of the mixtures also revealed correctly formed dimers (
Next, the present inventors incubated logic-gate-1 variants with surface mimic B or with both surface-mimic A and B and quantified the relative populations at different time points using gel electrophoresis (
The result obtained in the above experiments are shown in
In order to demonstrate a binding-site activation mechanism for cells, the present inventors developed a DNA-origami object with a cylindrical shape (
In order to test this mechanism, the present inventors constructed switchable logic gates 2 that had six anti-CD19 antibodies and non-switchable logic gates 2 that only had two centrally-mounted anti-CD19 antibodies (
One difference between the non-switchable and switchable variant up till now was that the non-switchable variant carried less target-cell binding antibodies. This difference could potentially change the binding rates of the logic gate to the target cell. To rule out that the difference in T-cell activation between the switchable and non-switchable variants was due to a difference in the number of attached antibodies, the present inventors assembled logic-gate variants with the same number of attached antibodies but in different configurations (
Furthermore, the present inventors increased the target-cell affinity for the non-switchable variant by attaching the antibodies in a more distal configuration, thereby increasing their accessibility. To compare the binding affinity of the constructs, the present inventors incubated the switchable and non-switchable variants with CD19-positive NALM6 B cells and performed flow cytometry (
In summary, even though the switchable logic-gate-2 variant has a disadvantage in binding affinity compared to the non-switchable variant, the switchable variants outperformed the non-switchable variant in T-cell activation by presenting the T-cell activating anti-CD3 antibody after switching. The present inventors used anti-CD19 antibodies to recognize B-cell target cells and anti-CD3 antibodies to cause T-cell activation. However, this mechanism can be extended or adapted to other antibodies, proteins or molecules. In particular, using a combination of different target-cell binding moieties, one could specifically target cells that present a combination of antigens (combination of antigen A and antigen B versus antigen A only versus antigen B only).
The results obtained in the above experiments are shown in
Using logic gate 2, the present inventors were able to demonstrate the switching on cell surfaces. However, logic gate 2 has five hinges in parallel that all need to rotate in order to switch, carrying up to eight antibodies. Therefore, the present inventors designed logic gate 3 based on the principles of logic gate 2. Logic gate 3 consists of a base section on which two wing-like plates are flexibly mounted (
Flow cytometry of flexible and always open logic gate variants with CD19-positive NALM6 B cells revealed a comparable binding affinity for the switchable and non-switchable antibody configuration (
In summary, even though the switchable logic-gate-3 variant has a disadvantage in binding affinity compared to the non-switchable variant, the switchable variants outperformed the non-switchable variant in T-cell activation by conditionally presenting the T-cell activating anti-CD3 antibody after switching.
The results obtained in the above experiments are shown in
Right: median fluorescence from flow cytometry experiments after 1.5 h incubation for a flexible and always-open variant with different antibody configurations as depicted in the left schematic. c, T-cell activation of different logic gate variants (Tn indicates number n of thymines in hinge). Both the switchable and the non-switchable antibody configurations had three anti-CD19 antibodies attached but in different configurations (as indicated in b, left).
While in the above, embodiments and examples have been described with reference to the accompanying drawings, the skilled person will understand that these embodiments and examples were provided for illustrative purpose only and should by no means be construed to limit the scope of the present invention, which is defined by the claims.
Number | Date | Country | Kind |
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19181646.1 | Jun 2019 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2020/067329 | 6/22/2020 | WO |