Conditional Cellular Connectors

Abstract

The present invention relates to a nucleic acid nanostructure comprising a first surface and a second surface, the first surface and the second surface being located on opposite sides of the nanostructure, the first surface comprising at least a first targeting agent, and the second surface comprising at least a second targeting agent and at least a third targeting agent. The present invention further relates to compositions comprising the nucleic acid nanostructure. The present invention also relates to nanostructures and compositions for use in medicine, and nanostructures and compositions for use in methods for preventing or treating disease. Furthermore, the present invention relates to methods for preparing the nanostructures, and to the use of the nanostructures for binding a first target and a second target.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a nucleic acid nanostructure comprising a first surface and a second surface, the first surface and the second surface being located on opposite sides of the nanostructure, the first surface comprising at least a first targeting agent, and the second surface comprising at least a second targeting agent and at least a third targeting agent. The present invention further relates to compositions comprising the nucleic acid nanostructure. The present invention also relates to nanostructures and compositions for use in medicine, and nanostructures and compositions for use in methods for preventing or treating disease. Furthermore, the present invention relates to methods for preparing the nanostructures, and to the use of the nanostructures for binding a first target and a second target. [Background technology]

[0002] 2. Background of the Invention Immune cell engaging therapies have great potential for the treatment of cancer. Targeting approaches consider bispecific T-cell engaging antibodies. These compounds target cancer cells on the one hand and T cells on the other. For example, the bispecific antibody blinatumomab (anti-CD3 x anti-CD19 BiTE) was approved by the European Medicines Agency based on its efficacy for the treatment of refractory acute lymphoblastic leukemia (ALL). This antibody recruits CD3-positive cytotoxic T cells (immune cells) to CD19-positive B cells (target cells) and induces the formation of a cytolytic immune synapse between T cells and target cells [1]. The formation of the immune synapse promotes effective target cell killing by cytotoxic T cells through the release of the pore-forming protein perforin and the apoptosis-inducing granzyme B. Importantly, the immune synapse can only be induced if both cells are held in close proximity [2, 3].

[0003] Therefore, T cell-inducing antibodies are designed to have small distances between paratopes. In particular, the 7-nanometer distance between paratopes of blinatumomab contributes to its high target cell killing potency. However, one major challenge facing bispecific antibody therapy is to balance the desired on-tumor T cell-mediated target cell killing with undesired off-tumor activity. Because the target antigen is often not present exclusively on tumor cells but also in healthy tissues, on-target off-tumor activity leads to immune overstimulation that can result in healthy tissue killing and toxicity. For ALL indications where a tumor-specific antigen (CD19) is present, blinatumomab and similar approaches are highly effective with limited side effects. In contrast, for indications where a single tumor-specific antigen cannot be identified, these approaches usually fail due to strong on-target off-tumor side effects [4].

[0004] In recent years, new methods have been developed for fabricating nanostructures, such as the fabrication of nucleic acid nanostructures, such as "DNA origami" as disclosed in U.S. Pat. No. 7,842,793. DNA origami is a nucleic acid nanostructure that can be designed to have a predetermined structure. DNA origami uses DNA molecules for the generation of nanometer-scale three-dimensional structures. WO 2014 / 170898 and WO 2012061719 relate to dynamic bipartite DNA origami devices with a DNA aptamer-latch mechanism for conditionally presenting cargo (e.g., antibodies) on the target cell surface. Such dynamic bipartite DNA origami devices rely on multi-domain objects with flexible portions, whose conformational state depends on the local environment (e.g., ionic strength). Nanostructures that are robust and more inert to the local environment are highly advantageous. Additionally, nanostructures can be protected from digestion from nucleases or degradation from low ionic strength environments using, for example, PEG-oligolysine coatings as described in ["Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation" Ponnuswamy et al., Nat Commun 2017; WO 2015070080]. These coatings and stabilization methods may interfere with the conformational state and configuration of flexible multi-domain objects. Conversely, rigid single-domain nanostructures with one configuration are highly advantageous as they can be stabilized using said stabilization methods without compromising their conditional cell recruitment function. Furthermore, nanostructures that allow for targeting more than one cell type or antigen are highly advantageous. Furthermore, nanostructures that promote cell interactions only when a specific antigen pattern is recognized are highly advantageous. Thus, there is a need to provide enhanced targeting means, such as nanostructures, that can target more than one cell type, and / or more than one cell surface molecule, and / or can target a combination of cell surface molecules. Furthermore, there is a need to provide means for specifically targeting cellular targets, such as immune cells and / or tumor cells. In particular, there is a need for means that allow cellular targets to be brought into close proximity. Furthermore, there is a need for means that allow cellular targets, such as cytotoxic T cells, to be activated, preferably by simultaneously bringing at least one further cellular target into close proximity to the activated cellular target. There is also a need for means that allow immune synapse formation. Furthermore, there is a need for means that allow selective and / or specific targeting of one or more targets. Furthermore, there is a need for means that allow selective and / or specific targeting of cells that present a combination or pattern of cell surface molecules (e.g. antigens). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] “Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation” Ponnuswamy et al., Nat Commun 2017 Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The elements of the present invention are described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments, or combine one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0007] In a first aspect, the present invention relates to a nucleic acid nanostructure comprising a first surface and a second surface, said first surface and said second surface being located on opposite sides of said nanostructure, said first surface comprising at least a first targeting agent and said second surface comprising at least a second targeting agent and at least a third targeting agent.

[0008] In one embodiment, the nanostructure has a longitudinal axis and a lateral axis, and the first surface and the second surface are located on opposite sides along the longitudinal axis.

[0009] In one embodiment, the nanostructures have a maximum longitudinal extension along said longitudinal axis that is greater than the maximum lateral extension along said transverse axis, preferably greater than the synaptic distance, more preferably at least greater than 10 nm, e.g. at least 25 nm; and / or

[0010] The nanostructures have a maximum lateral extension along said horizontal axis that is less than the maximum longitudinal extension along said vertical axis, preferably less than the synaptic distance, more preferably less than 25 nm, for example 10 nm or less.

[0011] In one embodiment, the nanostructures have a maximum longitudinal extension along said longitudinal axis that is greater than the maximum lateral extension along said horizontal axis, preferably greater than the synaptic distance, more preferably at least 10 nm, e.g., at least 25 nm.

[0012] In one embodiment, the nanostructures have a maximum lateral extension along said horizontal axis that is less than the maximum longitudinal extension along said longitudinal axis, preferably less than the synaptic distance, more preferably less than 25 nm, for example 10 nm or less.

[0013] In one embodiment, the first targeting agent is configured to bind to a first target, preferably a first target molecule on a first target cell, more preferably an immune cell, the second targeting agent is configured to bind to a second target, preferably a second target molecule on a second target cell, more preferably a disease cell, and the third targeting agent is configured to bind to a third target molecule on said second target, preferably a second target cell, more preferably a disease cell.

[0014] In one embodiment, the first targeting agent, the second targeting agent, and the third targeting agent are independently selected from an antibody or antigen-binding fragment thereof, an antigen-binding peptide, a Fab fragment, a F(ab')2 fragment, an Fv fragment, a diabody, a single chain Fv fragment, an (scFv)2, a tetrabody, a triabody, a disulfide bond stabilized Fv (dsFv), an Fc domain, an engineered Fc domain, a DNA aptamer, an RNA aptamer, a peptide nucleic acid (PNA), a polypeptide, a peptide, a glycoprotein, a peptidomimetic, anticalins, affilins, affimers, affitins, alphabodies, nanobodies, DARPins, receptor ligands, receptors or fragments thereof, and receptor domains; Preferably, the antibody or antigen-binding fragment thereof, the antigen-binding peptide, the Fab fragment, the F(ab')2 fragment, the Fv fragment, the diabody, the single chain Fv fragment, the (scFv)2, the tetrabody, the triabody, the disulfide bond stabilized Fv (dsFv), the Fc domain, the engineered Fc domain, the polypeptide, the peptide, the glycoprotein, the peptidomimetic, the anticalin, the affilin, the affimer, the affitin, the alphabody, the nanobody, the DARPin, the receptor ligand, the receptor or fragment thereof, and the receptor domain.

[0015] In one embodiment, the second targeting agent and the third targeting agent are separated from each other by a distance, preferably a distance along the longitudinal axis that is greater than the synaptic distance, for example a distance of at least 15 nm, preferably at least 20 nm, more preferably at least 40 nm; and / or the nanostructure is a rigid nanostructure, and / or a nanostructure without a hinge region, and / or a nanostructure with only one configuration, and / or a nanostructure that includes or is a DNA origami, the DNA origami having only one configuration, and / or

[0016] The nanostructures comprise a maximum length which is less than 1000 nm, preferably less than 500 nm, for example 100 nm, and preferably said maximum length is the length of the maximum longitudinal extension.

[0017] In one embodiment, the second targeting agent and the third targeting agent are separated from each other by a distance along the longitudinal axis that is preferably greater than the synaptic distance, for example, a distance of at least 15 nm, preferably at least 20 nm, and more preferably at least 40 nm.

[0018] In one embodiment, the nanostructure is a rigid nanostructure, and / or a nanostructure without a hinge region, and / or a nanostructure having only one configuration, and / or a nanostructure that comprises or is a DNA origami, wherein the DNA origami has only one configuration.

[0019] In one embodiment, the nanostructures comprise a maximum length which is less than 1000 nm, preferably less than 500 nm, for example 100 nm, preferably said maximum length being the length of maximum longitudinal extension.

[0020] In one embodiment, said first target cell is an immune cell, preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells, CAR-T cells, monocytes, macrophages and neutrophils, more preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells and CAR-T cells, and / or Said second target cell is a diseased cell, preferably selected from a cancer cell, a tumor cell, a cell involved in an autoimmune response, and an infected cell, for example a cell infected with a virus, mycoplasma, bacteria or parasite.

[0021] In one embodiment, said first target cell is an immune cell, preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells, CAR-T cells, monocytes, macrophages and neutrophils, more preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells and CAR-T cells.

[0022] In one embodiment, said second target cell is a diseased cell, preferably selected from a cancer cell, a tumor cell, a cell involved in an autoimmune response, and an infected cell, e.g. a cell infected with a virus, mycoplasma, bacteria or parasite.

[0023] In one embodiment, said first target molecule is a surface molecule of an immune cell, preferably selected from CD3, CD3 delta / epsilon, CD3 gamma / epsilon, TCR, TCR alpha, TCR beta, CD2, CD5, CD28, OX40, 4-1BB, CD16, Ly49, NKp30 (CD337), NKp44 (CD336), NKp46 (NCR1), CD3 zeta (CD247), CD27, CD40, CD137, CD64, CD89, toll-like receptors (TLRs), cytokine receptors, Fc domains, engineered Fc domains, GITR and ICOS, and / or

[0024] The second target molecule and the third target molecule are CD2, CD7, CD10, CD13, CD15, CD19, CD24, CD28, CD29, CD33, CD34, CD38, CD44, CD45, CD49f, CD56, CD57, CD60a, CD66 / CEA, CD79a, CD117, CD123, CD138, CD140b, CD227 / MUC1, CD243 / MDR, CD244, CD326 / EpCAM, CD340 / HER2, VEGF-R, EGFR, CSPG4 / MCSP, MAG, CA125, PSMA, HLA-DR, carbonic anhydrase 9, aquaporin, PSMA, TIM3, CLL1 / CLEC12A, EGFR v3 and HLA-A2, preferably said second target molecule and said third target molecule are not identical, and / or

[0025] The second target molecule and the third target molecule are combinations of target molecules, and the combinations include CD33 and CD123, CD326 / EpCAM and CD10, CD326 / EpCAM and CD340 / HER2, CD326 / EpCAM and VEGF-R, CD326 / EpCAM and EGFR, CD326 / EpCAM and CD243 / MDR, CSPG4 / MCSP and CD326 / EpCAM, CSPG4 / M CSP and MAG, CA125 and CD227 / MUC1, CA125 and CD227 / MUC1, CD56 and CD140b, CD56 and CD60a, EGFR and CD340 / HER2, PSMA and CD340 / HER2, CD15 and EGFR, CD44 and CD117, CD44 and CD326 / EpCAM, CD34 and CD19, CD34 and CD79a, CD34 and CD2, CD34 and CD 7, CD34 and HLA-DR, CD34 and CD13, CD34 and CD117, CD34 and CD33, CD34 and CD15, CD33 and CD19, CD33 and CD79a, CD33 and CD2, CD33 and CD7, CD33 and HLA-DR, CD33 and CD13, CD33 and CD117, CD33 and CD15, CD227 / MUC1 and CD10, CD227 / MUC1 and CD66 / and CD45 and HLA-A2.

[0026] In one embodiment, said first target molecule is a surface molecule of an immune cell, preferably selected from CD3, CD3 delta / epsilon, CD3 gamma / epsilon, TCR, TCR alpha, TCR beta, CD2, CD5, CD28, OX40, 4-1BB, CD16, Ly49, NKp30 (CD337), NKp44 (CD336), NKp46 (NCR1), CD3 zeta (CD247), CD27, CD40, CD137, CD64, CD89, toll-like receptor (TLR), cytokine receptor, Fc domain, engineered Fc domain, GITR and ICOS.

[0027] In one embodiment, the second target molecule and the third target molecule are CD2, CD7, CD10, CD13, CD15, CD19, CD24, CD28, CD29, CD33, CD34, CD38, CD44, CD45, CD49f, CD56, CD57, CD60a, CD66 / CEA, CD79a, CD117, CD123, CD138, CD140b, CD227 / MUC1, CD243 / MDR, CD244, CD326 / EpCAM, CD340 / HER2, VEGF-R, EGFR, CSPG4 / MCSP, MAG, CA125, PSMA, HLA-DR, carbonic anhydrase 9, aquaporin, PSMA, TIM3, CLL1 / CLEC12A, EGFR v3 and HLA-A2, and preferably said second target molecule and said third target molecule are not identical.

[0028] In one embodiment, the second target molecule and the third target molecule are a combination of target molecules, the combination being CD33 and CD123, CD326 / EpCAM and CD10, CD326 / EpCAM and CD340 / HER2, CD326 / EpCAM and VEGF-R, CD326 / EpCAM and EGFR, CD326 / EpCAM and CD243 / MDR, CSPG4 / MCSP and CD326 / EpCAM, C SPG4 / MCSP and MAG, CA125 and CD227 / MUC1, CA125 and CD227 / MUC1, CD56 and CD140b, CD56 and CD60a, EGFR and CD340 / HER2, PSMA and CD340 / HER2, CD15 and EGFR, CD44 and CD117, CD44 and CD326 / EpCAM, CD34 and CD19, CD34 and CD79a, CD34 and CD2, CD34 and and CD7, CD34 and HLA-DR, CD34 and CD13, CD34 and CD117, CD34 and CD33, CD34 and CD15, CD33 and CD19, CD33 and CD79a, CD33 and CD2, CD33 and CD7, CD33 and HLA-DR, CD33 and CD13, CD33 and CD117, CD33 and CD15, CD227 / MUC1 and CD10, CD227 / MUC1 and CD6 6 / CEA, CD227 / MUC1 and CD57, CD38 and CD138, CD24 and CD29, CD24 and CD49f, carbonic anhydrase 9 and aquaporin, CD19 and CD33, CD19 and CD22, CD28 and PSMA, CD227 / MUC1 and EGFR, CD33 and TIM3, CD123 and TIM3, CLL1 / CLEC12A and TIM3, CD244 and TIM3, CD33 and EGFR v3, CLL1 / CLEC12A and EGFR v3, CD123 and EGFR v3, and CD45 and HLA-A2.

[0029] In one embodiment, the nucleic acid comprises or consists of DNA, and / or the nanostructure comprises or is a DNA origami structure; The DNA origami structure includes at least one backbone strand, The DNA origami structure further comprises a plurality of single-stranded oligonucleotide staple strands; each staple strand is at least partially complementary to at least one backbone strand; Each of the staple strands is configured to bind to at least one of the at least one backbone chains in at least one location, preferably two or more distinct locations, and the at least one backbone chain is folded and / or arranged to form the desired nanostructure.

[0030] In one embodiment, the nanostructures comprise recesses, preferably oriented perpendicular to the maximum longitudinal extension; Preferably, the first targeting agent is attached to the nanostructure via the recess and / or at least a portion of the first targeting agent is located within the recess.

[0031] In one embodiment, the nanostructure further comprises an active agent, preferably a drug, and / or a marker, e.g., an imaging marker, wherein the active agent and / or marker is optionally attached to the nanostructure via a linker.

[0032] In a further aspect, the present invention relates to a composition, preferably a pharmaceutical composition, comprising a nucleic acid nanostructure as defined above, optionally further comprising a pharma- ceutically acceptable excipient.

[0033] In a further aspect, the present invention relates to a nanostructure as defined above or a composition as defined above for use in medicine.

[0034] In a further aspect, the present invention relates to a method for preventing or treating a disease selected from a proliferative disease, e.g. cancer, an immune disorder, e.g. an autoimmune disease, an infectious disorder, e.g. a viral disease such as human immunodeficiency virus (HIV) infection, a metabolic disorder, and / or diabetes, comprising the steps of: The method preferably comprises binding a first target cell to a second target cell using nanostructures; more preferably, inducing, promoting, stabilizing and / or inhibiting the formation of a synapse, preferably only when the first, second and third targeting agents bind to their respective target molecules; Even more preferably, it relates to a nanostructure as defined above, or a composition as defined above, for use in a method comprising binding a first target cell and a second target cell using the nanostructure such that as a result of said binding, the first target cell and the second target cell have a distance of 25 nm or less, preferably 15 nm or less.

[0035] In a further aspect, the present invention relates to a method for preparing a nanostructure as defined above, comprising the steps of: i) providing a nucleic acid nanostructure, preferably a rigid nucleic acid nanostructure; ii) providing a first targeting agent, a second targeting agent, and a third targeting agent, each of said targeting agents being conjugated to a nucleic acid strand complementary to a nucleic acid strand of said nanostructure, e.g., a backbone strand or a DNA handle strand; and iii) obtaining a nucleic acid nanostructure comprising said first targeting agent, said second targeting agent, and said third targeting agent, preferably by self-assembly of said nanostructure.

[0036] The nucleic acid nanostructure, the rigid nucleic acid nanostructure, the first targeting agent, the second targeting agent, and the third targeting agent are as defined above.

[0037] In a further aspect, the present invention relates to the use of a nanostructure as defined above or a composition as defined above for binding a first target and a second target, comprising: Preferably only when the first, second and third targeting agents bind to their respective target molecules, preferably for inducing, promoting, stabilizing and / or inhibiting the formation of a synapse between the first target cell and the second target cell. More preferably, the use comprises using the nanostructure to bind the first target, e.g. a first target cell, to the second target, e.g. a second target cell, such that as a result of the binding, the first target and the second target have a distance of 25 nm or less, preferably 15 nm or less.

[0038] The nucleic acid nanostructure, the first target, the second target, the first target cell, and the second target cell are as defined above.

[0039] In a further aspect, the present invention relates to the use of a nanostructure as defined above or a composition as defined above for the manufacture of a medicament, e.g. a medicament for preventing or treating a disease selected from a proliferative disease, e.g. cancer, an immune disorder, e.g. an autoimmune disease, an infectious disorder, e.g. a viral disease such as human immunodeficiency virus (HIV) infection, a metabolic disorder, and / or diabetes.

[0040] In a further aspect, the present invention relates to a method for preventing and / or treating a disease, preferably a disease selected from a proliferative disease, e.g. cancer, an immune disorder, e.g. an autoimmune disease, an infectious disorder, e.g. a viral disease such as human immunodeficiency virus (HIV) infection, a metabolic disorder, and / or diabetes, comprising administering to a patient in need thereof a nanostructure as defined above and / or a composition as defined above.

[0041] In one embodiment, said administering comprises administering an effective amount of a nanostructure as defined above and / or a composition as defined above to a patient in need thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description The present invention relies on a rigid DNA origami platform on which targeting agents such as antibodies are placed. Such rigid nucleic acid nanostructures are highly advantageous compared to complex hinge mechanisms or aptamer-latch systems or complex protein engineering, for example, because such rigid nucleic acid nanostructures are more suitable for in vivo use and / or their manufacture is more cost- and / or time-efficient. The nanostructures of the present invention are highly robust, especially in in vivo conditions where the local environment may vary depending on the tissue. Advantageously, compared to nanostructures that include hinge regions, the nanostructures of the present invention do not rely on fine-tuning conformational states. Instead, the nanostructures can be used and / or manufactured efficiently by fine-tuning the size and shape of the DNA origami. The nanostructures of the present invention are also advantageous in that they can target at least two target cells and can even be extended to target additional cells and / or additional targets, for example, by targeting three different antigens. The nanostructures of the present invention are further advantageous in that cells are contacted, recognized and / or eliminated only if at least the first, second and third targeting agents of said nanostructure bind to their respective targets. A further advantage is that only the second target, preferably the second target cell, comprising at least the second target molecule and the third target molecule, is contacted, recognized, and / or eliminated. Thus, only the target cell comprising the respective target molecule is recognized and / or eliminated. Off-binding and / or non-specific binding are thereby reduced. The nanostructure of the present invention is highly advantageous in that it is highly selective and specific, since the first target and the second target bind to each other and / or contact each other, preferably forming a synapse, only when at least the first targeting agent, the second targeting agent, and the third targeting agent are bound to the respective target molecule.

[0043] The modular nature of DNA origami technology, and in particular the nanostructures of the present invention, allows for rapid and efficient tailoring for different indications and environments.

[0044] To make a highly effective method of directing cytotoxic T cells to target cells available for indications where no single tumor-specific antigen can be identified, the inventors have developed a multispecific T cell induction platform, e.g., an antibody platform, using DNA origami techniques. Monospecific target cells and T cell targeting agents, e.g., antibodies, are combined on this platform to form a functional bispecific or multispecific entity that can recruit a first target cell, e.g., an immune cell, e.g., a T cell, to a second target cell, e.g., a cancer cell. However, immune cell, e.g., T cell-mediated killing is initiated only if all the targeting agents of the second target, preferably at least the second targeting agent and the third targeting agent, are bound to their respective target molecules, e.g., antigens on the surface of the target cell. Thus, diseased cells, such as tumor cells, can be targeted based on a complex antigen pattern (i.e., a combination of antigens), rather than based on a single antigen. For example, acute myeloid leukemia (AML) cells can be identified based on a combination of AML markers, such as CD33 and CD123 antigens.

[0045] We achieve this conditional killing of target cells, preferably second target cells, by mounting multiple targeting agents, e.g., antibodies, against target molecules, e.g., target cell antigens, and at least one immune cell-binding targeting agent, preferably a first targeting agent, e.g., an antibody, on a platform (Figure 1). In one embodiment, the term platform relates to nanostructures. The shape of the nanostructure, e.g., DNA origami platform, is designed to have at least one small dimension (e.g., less than 10 nm; this dimension needs to be selected to form the desired immune synapse or cell-cell interaction). In one embodiment, such small dimension is referred to as the maximum lateral extension length and / or maximum lateral length. The second and third targeting agents are attached such that the platform is parallel to the target cell surface, preferably the surface of the second target cell, and the short dimension is perpendicular to the cell surface, only if both the second and third targeting agents are bound to their respective target molecules, e.g., target cell antigens, on the same cell (Figure 1a vs. 1b and 1c). This orientation can improve the accessibility of the first targeting agent, e.g., immune cell-binding antibody, allowing the first target, e.g., cytotoxic T cells, to come into close proximity to the second target. Therefore, the length of the small dimension, the targeting agent and their attachment must be selected so that the cytolytic immune synapse can be formed. If both the second and third targeting agents are not bound, e.g., not all target cell antibodies are bound, the first targeting agent, e.g., immune cell-binding antibody, is inaccessible or has limited access (Figure 1b), or the distance between the first target, e.g., T cells, and the second target, e.g., disease target cells, is too large to initiate the immune synapse (Figure 1c).

[0046] In one embodiment, such a device, preferably a nanostructure, has a rod-like shape (FIG. 2), and optionally a second targeting agent A1 and a third targeting agent A2, such as antibodies A1 and A2, for a second target, such as a disease target cell, are attached to the ends of the device. At one end, a first targeting agent B1, such as an immune cell-binding antibody B1, is attached. Only when both targeting agents A1 and A2 are bound to their respective target molecules, such as antigen targets on the target cell, will the rod be parallel to the cell surface and the immune cells can come into close proximity to form a cytolytic synapse.

[0047] Furthermore, by forming a recess in the DNA origami object where a first targeting agent, such as an immune cell-binding antibody, is placed, the accessibility of the targeting agent, e.g., an immune cell-binding antibody, and thus the conditional properties of the device, e.g., nanostructure, can be tuned (Figure 3). Furthermore, the device can also be used to identify target cells based on their antigen density if the same target cell antigen is attached, and both target cell antibodies can only bind if the antigen density is high enough that the second target cell antibody can bind before the first antibody detaches from the surface.

[0048] Furthermore, which end of the rod-shaped platform the first targeting agent, e.g., immune cell binding antibody, is placed on may be selected according to the distribution of target antigens on target and non-target cells. For example, if antigen RA1 is also common to non-target cells, it may be advantageous to place the immune cell recruiting antibody near A1. This is because binding to RA1 will make B1 inaccessible, so that the immune cell cannot bind, and the antibody-DNA platform will not bridge the two cells, but will leave the cell surface to find the next potential target cell. In one embodiment, when referring to a platform, object, or device, a nanostructure is meant. The approach presented herein can be extended to recognize three or more target molecules, e.g., target antigens, on a second target cell, for example, using a triangular object (Figure 4). Furthermore, two or more immune cell binding moieties may be placed on the platform. This may be advantageous in tumor microenvironments where immune cell activation is inhibited. By placing two or more immune stimulatory molecules, a local immune response may be elicited even in an immune-inhibitory environment. In one embodiment, the first targeting agent, and optionally the fourth or additional targeting agents, are immunostimulatory agents.

[0049] We have previously constructed dynamic DNA origami devices that consist of multiple parts and are linked via flexible hinges to conditionally recruit cells. These nanodevices rely on engineered equilibrium states to fulfill their conditional functions. In particular, the occupancy of conformational states depends on the local environment (e.g., ionic strength, pH, protein corona). The approach presented herein is highly advantageous in that it relies on rigid nanostructures, such as single-body antibody-carrier DNA platforms, that are more inert and robust than flexible multi-domain objects. Thus, the nanostructures of the present invention are highly robust to changes in the local environment (e.g., changes in pH, ionic strength, protein corona, nuclease concentration, etc.). These increased stability and robustness may be particularly important in in vivo environments or when targeting solid tumors, which create tumor microenvironments that differ from healthy tissues.

[0050] In one embodiment, the shape of the DNA origami platform is designed such that certain dimensions are smaller than the distance required for immune synapse formation, e.g., smaller than 20 nm. In one embodiment, the antibodies are arranged such that only multivalent binding, e.g., binding of each targeting agent to its respective target, results in a parallel orientation of the platform, e.g., an orientation parallel to the surface of the target cell. Furthermore, distance-dependent synapse formation is recognized. In one embodiment, the terms "parallel orientation" and "vertical orientation" as used herein refer to the orientation of the cell connector with respect to the target cell surface. In a parallel orientation, the longitudinal axis is almost always approximately parallel to the cell surface, and in a vertical orientation, the longitudinal axis is almost perpendicular to the target cell surface. Those skilled in the art will appreciate that the systems described herein, particularly the nanostructures, are subject to thermal fluctuations and binding and unbinding events may occur. Thus, a parallel orientation is a substantially parallel orientation, and a vertical orientation is a substantially perpendicular orientation. In one embodiment, there are multiple orientations that are substantially parallel or substantially perpendicular to the cell surface.

[0051] In one embodiment, the term "targeting agent" refers to a moiety capable of binding, preferably specifically binding to, a target on a cell surface, such as an antigen-binding peptide. In one embodiment, the targeting agent is either an antibody or an antigen-binding peptide thereof, such as an IgG, a Fab fragment, a single-chain Fab fragment, or a single domain antibody, a DNA and / or RNA aptamer, a peptide, a binding protein, a ligand of a cell surface receptor (e.g., PD-L1), or a lipid, such as an antibody or an antigen-binding fragment thereof, an antigen-binding peptide, a Fab fragment, a F(ab')2 fragment, a Fv fragment, a diabody, a single-chain Fv fragment, (scFv)2, a tetrabody, a triabody, a disulfide bond stabilized Fv (dsFv), a DNA aptamer, a RNA aptamer, a peptide nucleic acid (PNA), a polypeptide, a peptide, a glycoprotein, a peptidomimetic, anticalin, anfilin, anfimer, an affitin, an alphabody, a nanobody, a DARPin, a receptor ligand, a receptor or fragment thereof, and a receptor domain. In one embodiment, the targeting agent is an antigen-binding peptide, such as an antibody, an antibody-like molecule, an antibody mimetic, an antigen-binding derivative or an antigen-binding fragment thereof. In one embodiment, the interaction between the targeting agent and its target is characterized by specific binding of an antibody-antigen interaction. Those skilled in the art can identify similar specific interactions that are within the scope of the present invention. As an example, specific binding of an aptamer to a molecule, or specific binding of a receptor ligand to its receptor, or vice versa, can also be used. However, such nanostructures can utilize a wide variety of interactions, which can be advantageous compared to state-of-the-art technologies that rely on specific and limited interactions, such as receptor-ligand interactions. Thus, due to the wide range of interactions available, a more versatile and tunable DNA nanostructure platform is provided compared to nanostructures disclosed in the prior art.For example, the targeting agent may be an IgG antibody, an IgG antibody fragment, an IgG antibody mimetic, an IgM antibody, an IgM antibody fragment, an IgM antibody mimetic, an IgD antibody, an IgD antibody fragment, an IgD antibody mimetic, an IgA antibody, an IgA antibody fragment, an IgA antibody mimetic, an IgE antibody, an IgE antibody fragment, or an IgE antibody mimetic.

[0052] In one embodiment, the targeting agent binds to its target reversibly or irreversibly. For example, the reversibly binding molecule may be an antibody, an antibody fragment, a DNA strand, a biotin molecule, a streptavidin molecule, and the irreversibly binding molecule may be a maleimide-thiol chemical molecule or a click chemistry molecule. In one embodiment, the first targeting agent, the second targeting agent, and the third targeting agent are the same or different, preferably different from each other. In one embodiment, the second targeting agent and the third targeting agent are the same or different, preferably different from each other. In one embodiment, the second targeting agent and the third targeting agent target the same target molecule and / or different target molecules, preferably different target molecules. In one embodiment, the second targeting molecule and the third targeting molecule are the same or different. In one embodiment, the exemplary first targeting agent is either anti-CD3ε (e.g., Fab), anti-CD28 (e.g., Fab), and IL2, or a combination thereof. In one embodiment, the exemplary second targeting agent is anti-CD19 (e.g., Fab), anti-CD123 (e.g., Fab), and anti-CD33 (e.g., Fab), or any combination thereof. In one embodiment, the exemplary third targeting agent is anti-CD19 (e.g., Fab), anti-CD123 (e.g., Fab), and anti-CD33 (e.g., Fab), or any combination thereof.

[0053] In one embodiment, the immune cell targeting agent is any immune cell binding molecule, protein, moiety, and / or complex, such as an immune cell stimulatory or inhibitory molecule, protein, moiety, and / or complex. In one embodiment, the immune cell targeting agent, such as an immune cell binding antibody, is an agent that can bind to, activate, and / or costimulate T cells or natural killer cells, such as an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-CTLA-4 antibody, a TLR agonist (e.g., TLR-7 / 8, TLR-3, TLR-4, TLR-9), a cytokine (e.g., IL-2), a checkpoint ligand or inhibitor (e.g., anti-PD-1 or PD-L1), or a combination thereof.

[0054] The term "antigen-binding peptide" as used herein relates to a peptide that specifically binds to an antigen. In one embodiment, the antigen-binding molecule is based on an immunoglobulin, such as a polyclonal or monoclonal antibody, or on a protein scaffold structure with antigen-binding capacity, such as anticalin proteins, affilins, affimers, affitins, alphabodies, nanobodies, or DARPins. In one embodiment, the antigen-binding peptide relates to an antibody or an antibody fragment. In one embodiment, the terms "antibody", "antibody fragment", and "antigen-binding peptide" are used interchangeably. There are several classes of antibodies, particularly human antibodies, such as IgA, IgG, IgM, IgD and IgE, and subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. IgG, a hybrid composition of the natural human IgG isotype, may also be useful in the present invention. In one embodiment, the antigen-binding peptide and / or antibody may comprise domains of any antibody class, subclass and / or chain. In one embodiment, the amino acid sequence of the antigen-binding peptide can be modified by protein engineering, e.g., to include constant regions from other immunoglobulin classes that mediate improved binding characteristics, e.g., effector function characteristics, e.g., immune cell stimulatory or immune cell inhibitory functions.

[0055] According to the invention, the antigen-binding peptide may relate, for example, to human antibodies, human chimeric antibodies, humanized antibodies and antibody fragments. The antibody recognizes the antigen via a Fab fragment variable region that contains a paratope. Said paratope specifically targets an epitope on the antigen. According to the invention, the antigen-binding peptide may further relate to a fragment of an antibody, such as a substantially intact antibody, a Fab fragment, a F(ab')2 fragment, a diabody, a single chain Fv fragment, a tetrabody, a triabody, a disulfide bond stabilized Fv (dsFv), or a heavy chain VHH fragment from camelid. However, other forms of antigen-binding peptides are also envisaged by the invention. For example, the antigen-binding peptide may be another (non-antibody) receptor protein derived from a small and robust non-immunoglobulin scaffold, such as one equipped with a binding function, for example by using methods of combinatorial protein design. Such a non-antibody antigen-binding peptide may be, for example, an affibody molecule, an affilin, an affimer, an affitin, an alphabody, an anticalin, a nanobody, or a DARPin. The antigen-binding peptide may also be associated with other antibody mimetic molecules, such as dual affinity retargeting antibodies (DARTs). In one embodiment, the antigen-binding peptide is preferably associated with an antibody or antibody fragment. In one embodiment, the antigen-binding peptide may be associated with any antibody mimetic molecule that mimics the binding properties of an antibody to an antigen, but is structurally different from a naturally occurring antibody.

[0056] The term "human antibody" as used herein refers to an antibody that contains only human sequences or fragments thereof. In one embodiment, human antibodies are produced by display technologies such as phage display or ribosome display. The term "humanized antibody" as used herein refers to an immunoglobulin chain or fragment thereof, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or another antigen-binding fragment of an antibody, that contains sequences from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human antibody in which certain CDR residues have been replaced with CDR residues of non-human origin that have the desired specificity and affinity properties. The term "chimeric human antibody" as used herein refers to an artificial antibody designed by genetic or protein engineering by combining sequences of different species, such as mouse and human. In one embodiment, a human chimeric antibody is a hybrid protein of an antigen-binding domain from a mouse antibody and the constant domains of a human antibody. The term "substantially intact antibody" as used herein refers to an antibody that has not been fragmented, truncated, or otherwise shortened and that retains the ability to specifically bind to a target, such as an antigen.

[0057] The term "targeting" as used herein relates to the ability of a molecular structure, such as an antibody, an antigen-binding peptide, or a surface molecule of a cytotoxic T cell, to bind to a particular structure, such as an antigen, in particular an epitope, by specific interaction. In one embodiment, a molecule on the surface of an immune cell is targeted by a first targeting agent, such as an antigen-binding peptide, and further molecules, such as at least two molecules, on the surface of a target cell, such as a cancer cell, are targeted by a second and a third targeting agent. Such binding of the first targeting agent to the first molecule on the immune cell, and such binding of the second targeting agent to the second molecule on the target cell, such as a cancer cell, and such binding of the third targeting agent to the third molecule on said target cell brings the immune cell and the target cell into close proximity, thereby allowing the formation of an immune synapse. In one embodiment, such proximity of the immune cell and the target cell results in the activation of an effector system, which then leads to the elimination of said target cell, such as a cancer cell, for example, via the caspase cascade. In one embodiment, the formation of the immune synapse leads to cell death of said cancer cell. In one embodiment, the terms "targeting" and "recognition" are used interchangeably. The term "cross-reacting" as used herein relates to off-target binding to structures and / or molecules that are not targets. In one embodiment, the multifunctional nanostructure of the present invention reduces cross-reactivity since immune cells are recruited only when at least two target molecules on said target cells are bound, thus increasing specificity and reducing off-target binding and side effects.

[0058] The term "first targeting agent" as used herein relates to a molecule that binds and / or is configured to bind to a first target, e.g., a first target cell. In one embodiment, the first target cell is an immune cell or other effector cell. In one embodiment, said first targeting agent binds, preferably specifically binds, to a target molecule on an immune cell, such as a lymphocyte, e.g., a T cell or a natural killer cell, or a macrophage or a neutrophil. In one embodiment, said first targeting agent can activate an immune cell and / or stimulate an immune response of the immune cell, the immune response being preferably a cytotoxic immune response, e.g., a cytotoxic immune response against a second target cell. In one embodiment, said first target molecule is preferably a molecule such as CD3, CD3 delta / epsilon, CD3 gamma / epsilon, TCR, TCR alpha, TCR beta, CD2, CD5, CD28, OX40, 4-1BB, CD16, Ly49, NKp30 (CD337), NKp44 (CD336), NKp46 (NCR1), CD3 zeta (CD247), CD27, CD40, CD137, CD64, CD89, toll-like receptors (TLRs), cytokine receptors, Fc domains, engineered Fc domains, GITR and ICOS. In one embodiment, the term "at least one" in the context of a targeting agent, e.g., a first targeting agent, means that there are one or more molecules of one type of said targeting agent, and further, there may be additional targeting agents, such as a fourth, fifth or sixth targeting agent, each of said fourth, fifth or sixth targeting agents being one or more molecules of one type.

[0059] The terms "second targeting agent" and "third targeting agent" as used herein refer to targeting agents that are configured to bind and / or bind to a second target molecule and a third target molecule, respectively. In one embodiment, the second target molecule and the third target molecule are of the same target, e.g., of the same target cell. In one embodiment, the use of a second targeting agent and a third targeting agent increases the specificity of a nanostructure comprising a second and a third targeting agent, etc., over a structure using only one targeting agent. In one embodiment, the specificity and selectivity of the nanostructure of the invention is increased compared to a nanostructure that targets only one target molecule on a target cell, e.g., a second target cell. In one embodiment, when both the second target molecule and the third target molecule are present on said second target cell, only the second target cell, e.g., a diseased cell, e.g., a cancer cell, is targeted and, if necessary, destroyed by cytolytic lysis. Thus, the nanostructure of the invention is advantageous in that it reduces off-binding by increasing the specificity and / or selectivity of cell targeting. Furthermore, the nanostructures of the present invention are advantageous in that they allow for specific and / or selective targeting and / or elimination of diseased cells. An advantage of the present invention is that the nanostructures can specifically and / or selectively induce lysis of diseased cells, such as cancer cells and / or cells involved in an autoimmune response.

[0060] In one embodiment, the nanostructure comprises, in addition to the first, second, and third targeting agents, further targeting agents. In one embodiment, the structure comprises, in addition to the first, second, and third targeting agents, further targeting agents, such as a fourth targeting agent, and / or a fourth and fifth targeting agent, and / or even further targeting agents. In one embodiment, the nanostructure comprises at least the first targeting agent, the second targeting agent, and the third targeting agent, and further comprises a further targeting agent.

[0061] In one embodiment, the term "immune synapse" refers to an interface formed upon recognition between a first cell and a second cell, preferably mediated via the nanostructure of the invention, e.g., upon receptor-mediated and / or nanostructure-mediated recognition between an immune cell and an antigen-presenting cell and / or a cancer cell. In one embodiment, the immune synapse is a cytolytic synapse, meaning that at least one cell is lysed upon formation of the immune synapse. In one embodiment, the nanostructure of the invention induces synapse formation, e.g., a synapse that is or resembles the cytolytic synapse described in

[11] or

[12] . In one embodiment, the immune synapse is a cytolytic T cell synapse, and / or a stimulatory synapse formed between a naive T cell as a first target and a second target cell, e.g., an antigen-presenting cell, and / or a lytic synapse formed between a cytotoxic effector T cell as a first target and a second target cell.

[0062] In one embodiment, the term "synapse formation" relates to forming a synapse and / or synaptic distance between a first target and a second target, e.g., between a first target cell and a second target cell. In one embodiment, such synapse formation is the formation of an immune synapse. In one embodiment, such synapse formation, preferably immune synapse formation, results in dissolution and / or binding of the second target. In one embodiment, the nanostructures of the invention are used to induce and / or promote synapse formation between two cells, e.g., a first target cell and a second target cell, e.g., between an immune cell and a cancer cell. In one embodiment, the nanostructures of the invention are used to bind a cell to a first target, such as a sensor. In one embodiment, the nanostructures of the invention are for use in inducing, promoting, stabilizing, and / or inhibiting synapse formation, preferably only when the first, second, and third targeting agents are bound to their respective target molecules. In a preferred embodiment, the nanostructures of the invention are for use in inducing and / or promoting synapse formation. In one embodiment, the nanostructure of the present invention is for use in promoting a specific distance between two cells and / or between a cell and a target such as a surface, e.g., a sensor surface. In one embodiment, the synapse is an immune synapse. In one embodiment, the nanostructure is for use in inducing and / or promoting the formation of a synapse, preferably an immune synapse. In one embodiment, the nanostructure is used for conditional synapse formation, i.e., such a synapse is formed only under the following conditions: a first targeting agent binds to a first target molecule, a second targeting agent binds to a second target molecule, and a third targeting agent binds to a third target molecule. In an alternative embodiment, the nanostructure of the present invention is for use in inhibiting synapse formation, e.g., for inhibiting an autoimmune response.In one embodiment, such inhibition of synapse formation includes inducing and / or stabilizing synapse formation and inhibiting the biological effect of such synapse formation on a first or second target, for example inhibiting a first target cell from attacking a second target cell, for example inhibiting an immune cell from attacking a cell, for example a healthy cell or a diseased cell, in an autoimmune response. In one embodiment, such inhibition of biological effect can be performed by said first targeting agent, in such a case for example an immune cell inhibitor, and / or by a further agent bound to said first surface, preferably an immune cell inhibitor bound to said first surface. In one embodiment, such inhibition of synapse formation is a specific inhibition of synapse formation, because said first or second target is inhibited only when said first targeting agent binds to said first target molecule, said second targeting agent binds to said second target molecule, and said third targeting agent binds to said third target molecule.

[0063] In alternative embodiments, the nanostructures of the invention inhibit synapse formation, e.g., non-specifically inhibit synapse formation, e.g., by steric hindrance and / or lack of binding specificity, when 1) the second target does not include the second target molecule or the third target molecule, 2) the second target includes only the second target molecule but not the third target molecule, or 3) the second target includes only the third target molecule but not the second target molecule. In one embodiment, the nanostructures of the invention inhibit synapse formation between a target other than the first target including the first target molecule and a second target including the second target molecule and the third target molecule, preferably by steric hindrance and / or binding specificity.

[0064] In one embodiment, the term "synaptic distance" as used herein relates to the distance of a synapse, for example, the distance of the synaptic cleft and / or the distance of the synaptic gap. In one embodiment, the synaptic distance is the distance between a first target and a second target when a synapse is formed. In one embodiment, the synaptic distance is the distance between two cells, such distance being small enough to allow the first cell to affect the second cell and / or to allow biochemical and / or electrochemical communication between the first cell and the second cell. In one embodiment, the synaptic distance is less than 25 nm, for example 20 nm or less, 15 nm or less, or 10 nm or less. In one embodiment, the synaptic distance is 25 nm or less, preferably 15 nm or less, more preferably 10 nm or less. In one embodiment, the synaptic distance is in the range of 1 nm to 25 nm, preferably 5 nm to 15 nm, for example about 13 nm to 15 nm or 5 nm to 13 nm. In one embodiment, a natural synapse between a T cell and an antigen presenting cell has a synaptic distance in the range of 13 to 15 nm. In one embodiment, the artificial synapse, such as the synapse induced by IgG, BiTe, or nanostructure, has a synaptic distance in a similar range to that of natural synapses, or a synaptic distance smaller or larger than that of natural synapses, preferably similar or smaller, more preferably smaller. In one embodiment, the smaller the synaptic distance, the better the effect, for example the cytolytic effect of immune cells on diseased cells. In one embodiment, the first target, for example the first target cell, and the second target, for example the second target cell, are bound using the nanostructure, such that as a result of the binding, the first target and the second target have a distance of 25 nm or less, preferably 15 nm or less, more preferably 10 nm or less.

[0065] The present invention relates to nanostructures that can trigger the formation of synapses, such as immune synapses, between target cells and immune cells upon antigen pattern recognition on the target cell. For example, immune cells that can form immune synapses are T cells, B cells, and natural killer cells. In one embodiment, a synapse is formed between a cytotoxic T cell or natural killer cell and a target cell of interest, such as a cancer cell. In one embodiment, such a synapse allows cytolytic molecules, such as cytolytic molecules released by the cytotoxic T cell or natural killer cell, to be targeted to the target cell, thereby causing target cell death. In one embodiment, the nanostructures are used to form such cytolytic synapses in a method for preventing or treating cancer. In one embodiment, the nanostructures of the present invention allow for the killing of tumor cells by inducing the formation of an immune synapse between an immune cell and a tumor cell. In one embodiment, the nanostructures are also useful in applications where the immune system needs to be locally modulated, such as autoimmune diseases. The nanostructures of the present invention can be used in any application where cells must be in contact and / or in close proximity to each other and / or to a target, such as a sensor surface. In one embodiment, the nanostructures are used to form electrical or electrochemical synapses between neurons and / or cells of the central nervous system. In one embodiment, the nanostructures are used to form electrical or electrochemical synapses between neurons and / or cells of the central nervous system and electrical and / or electrochemical stimulation means, such as electrical and / or electrochemical stimulation devices.

[0066] In one embodiment, the nanostructure is for use in inducing, promoting, stabilizing, and / or inhibiting the formation of a synapse, preferably only when the first, second, and third targeting agents are bound to their respective target molecules. In one embodiment, a synapse is formed only when the first, second, and third targeting agents are bound to their respective target molecules. In one embodiment, a first target cell and a second target cell are bound using the nanostructure only when the first, second, and third targeting agents are bound to their respective target molecules. In one embodiment, a method of preventing or treating a disease comprises binding a first target cell and a second target cell using the nanostructure only when the first, second, and third targeting agents are bound to their respective target molecules. In one embodiment, a method of preventing or treating a disease comprises inducing, promoting, stabilizing, and / or inhibiting the formation of a synapse only when the first, second, and third targeting agents are bound to their respective target molecules. In one embodiment, the use of the nanostructure includes inducing, promoting, stabilizing, and / or inhibiting the formation of a synapse between a first target cell and a second target cell only when the first, second, and third targeting agents are bound to the respective target molecules. In one embodiment, the use of the nanostructure includes binding the first target, e.g., the first target cell, and the second target, e.g., the second target cell, using the nanostructure such that, as a result of said binding, the first target and the second target have a distance of 25 nm or less, preferably 15 nm or less, only when the first, second, and third targeting agents are bound to the respective target molecules. In one embodiment, the "respective target molecule" of the first targeting agent is the first target molecule, the "respective target molecule" of the second targeting agent is the second target molecule, and the "respective target molecule" of the third targeting agent is the third target molecule. In one embodiment, synapse formation is induced, promoted, stabilized, and / or inhibited only when a second target, preferably a second target cell, presents at least one second target molecule and at least one third target molecule.In one embodiment, the use of the nanostructure comprises inducing, promoting, stabilizing and / or inhibiting the formation of a synapse between a first target cell and a second target cell, preferably only if the second target, preferably the second target cell, presents at least the second target molecule and at least the third target molecule. In one embodiment, the method of preventing or treating a disease comprises inducing, promoting, stabilizing and / or inhibiting the formation of a synapse only if the second target, preferably the second target cell, presents at least one second target molecule and at least one third target molecule. In one embodiment, the nanostructure is a conditional cell connector, i.e., a conditional nanostructure, since it induces, promotes, stabilizes and / or inhibits the formation of a synapse only if the first, second and third targeting agents are bound to the respective target molecules. In one embodiment, the "condition" for the binding of the first target to the second target is that the first, second and third targeting agents are bound to the respective target molecules. In one embodiment, the "condition" for binding of the first target is that a second and a third targeting agent are bound to their respective target molecules.

[0067] The term "nanostructure" as used herein relates to nucleic acid nanostructures, preferably DNA origami structures composed of one or more DNA origami subunits (entities). In an embodiment, the nanostructure is a DNA origami nanostructure. Readily available nucleic acid nanostructure technologies, e.g., DNA origami technologies, involve relatively less complicated assembly procedures than standard nanofabrication technologies, and can be used to fabricate nanostructures. In one embodiment, the nanostructure is at least partially fabricated using DNA origami technologies. Due to the self-assembly of DNA origami structures, and the readily available software for designing the corresponding backbone and staple strands, this can be a relatively less complicated manufacturing process compared to standard nanofabrication technologies. In one embodiment, the nanostructure is a DNA origami structure. In one embodiment, the nanostructure comprising a targeting agent is a nanostructure to which the targeting agent is attached. For example, the targeting agent can be attached to the nanostructure via any surface of the nanostructure and / or via the core structure of the nanostructure, e.g., via the core structure and / or an inner portion of the nanostructure. In one embodiment, the nanostructure comprising the targeting agent is a DNA origami to which the targeting agent is attached. In one embodiment, the core structure of the nanostructure comprising the targeting agent is a DNA origami, and the targeting agent is attached to such a core structure, which is a DNA origami. In one embodiment, the targeting agent is not part of the core structure, but is attached to the core structure, e.g., the DNA origami, via a nucleic acid strand optionally conjugated to the targeting agent. In one embodiment, the nanostructure has a maximum length of less than 1000 nm, e.g., in the range of about 25 to about 70 nm, preferably about 30 to about 50 nm. In one embodiment, the maximum length is along the longitudinal axis. In one embodiment, the maximum length is greater than the synaptic distance, e.g., 25 nm or more.

[0068] In one embodiment, the DNA origami structure comprises at least one backbone strand and a plurality of single-stranded oligonucleotide staple strands. In one embodiment, the backbone strand constitutes and / or traverses the major portion of the DNA origami structure and / or the DNA origami subunit ("entity"). In one embodiment, the entity is a DNA origami subunit. The term "staple strand" as used herein refers to a single-stranded oligonucleotide molecule that is at least partially complementary to the backbone strand. In general, the staple strands can be used to introduce, for example, coupling sites into the DNA origami structure and / or the DNA origami subunit ("entity").

[0069] The present invention generally relates to nanostructures. In the present example, 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. However, it should be understood that the present technology is not limited thereto, this is merely exemplary, and it should be understood that other nanostructures may also be utilized to practice the present invention. However, as used herein, the term "nanostructure" preferably refers to a DNA origami structure composed of one or more DNA origami subunits (entities). In one embodiment, the terms "cell connector" and "DNA origami cell connector" as used herein relate to nucleic acid nanostructures, such as DNA origami nanostructures, preferably the nanostructures of the present invention.

[0070] Nanostructures, such as DNA origami nanostructures, may include at least one backbone strand, i.e., a single-stranded polynucleotide backbone DNA having a known sequence. The DNA origami structures may further include a plurality of single-stranded oligonucleotide staple strands, each of which may be at least partially complementary to at least one backbone strand. Furthermore, each of the staple strands may be configured to bind to at least one backbone strand, and the at least one backbone strand may be folded and / or arranged such that a desired nanostructure may be formed. The term "strand" as used herein refers to a nucleic acid strand, e.g., a DNA and / or RNA strand. Such three-dimensional nanostructures may be realized using DNA origami, i.e., by combining backbone strands and staple stands to form the required portions and entire devices. Such designs may be performed, for example, using software such as caDNAno. That is, in some embodiments, nanostructures including multiple portions may be created from one backbone strand, while in other embodiments, portions of the nanostructure may be constructed utilizing multiple backbone strands.

[0071] In an embodiment where the nanostructure is at least partially formed by a DNA origami structure, the DNA origami structure comprises at least one backbone strand and multiple staple strands, the molecule may be linked to at least one of the backbone strands or the staple strands by a linker molecule, and the linker molecule may be linked to a portion of the DNA strand that is complementary to a portion of the backbone strand or a portion of the staple strand. In one embodiment, the first targeting agent is linked to the nanostructure via a linker molecule, and / or the second targeting agent is linked to the nanostructure via a linker molecule, and / or the third targeting agent is linked to the nanostructure via a linker molecule. In one embodiment, the linker molecule is one or more nucleobases, such as a single nucleobase or a nucleic acid sequence. In one embodiment, the linker molecule of the targeting agent is linked to the DNA strand of the nanostructure.

[0072] In one embodiment, the shape of the nanostructure may be any shape, for example, rod, triangle, circle, cube, i.e., rectangle, star, or any other shape. In one embodiment, the nanostructure is not a spherical structure. The nanostructure of the present invention can be any length. In a preferred embodiment, the nanostructure comprises a maximum length, and in a particularly preferred embodiment, the maximum length is less than 1000 nm, preferably less than 500 nm, for example about 100 nm or less.

[0073] In one embodiment, the nanostructure comprises at least one nucleic acid, said nucleic acid may comprise at least one modification, such as a modified internucleoside linkage, a modified nucleobase, or a modified sugar moiety, such as a 2'-O-alkyl modification, such as a 2'-O-methoxy-ethyl (MOE) or 2'-O-methyl (OMe) modification, an ethylene-bridged nucleic acid (ENA), a 2'-fluoro (2'-F) nucleic acid, such as a 2'-fluoro N3-P5'-phosphoramidite, a 1',5'-anhydrohexitol nucleic acid (HNA), or a locked nucleic acid (LNA). In one embodiment, the second targeting agent and the third targeting agent are spaced apart from each other, preferably along the longitudinal axis, such as at least 15 nm, preferably at least 20 nm, more preferably at least 40 nm. In one embodiment, the nanostructure comprises a maximum length of less than 1000 nm, preferably less than 500 nm, such as 100 nm, preferably the maximum length being the length of the maximum longitudinal extension. In one embodiment, the nanostructure is a rigid nanostructure, a nanostructure without a hinge region, a nanostructure having only one configuration, and / or a nanostructure that comprises or is a DNA origami, said DNA origami having only one configuration.

[0074] The term "rigidity" as used herein refers to the characteristics of a rigid body, e.g., a rigid nanostructure or a rigid part of a nanostructure, meaning that such a body, e.g., a nanostructure or a part of a nanostructure, is a solid body with no or negligible deformation. The distance between any two given points on a rigid body, e.g., a nanostructure, remains constant in time regardless of the external force exerted on the rigid body. A rigid body is usually considered as a continuous distribution of mass. In one embodiment, when referring to a nucleic acid that is "rigid", such a term "rigidity" refers to a flexible nanostructure that has only one configuration, i.e., has two or more configurations, e.g., a nanostructure that is not flexible via a hinge region. In one embodiment, such "rigidity" and / or "flexibility" and / or "only one configuration" refers to the core structure of the nanostructure, e.g., the DNA origami, i.e., the nanostructure itself, but not to the targeting agents contained, attached, and / or bound thereto. For example, the bond between the nanostructure, such as a DNA origami, and the targeting agent may be flexible and / or include a hinge region, but the nanostructure itself, such as a DNA origami, is rigid, i.e., inflexible. In one embodiment, the nanostructure comprises a rigid portion, and the second and third targeting agents are attached to the rigid portion of the nanostructure. In one embodiment, the nanostructure comprises a rigid portion and a non-rigid portion, and the second and third targeting agents are attached to the rigid portion, and the first targeting agent is attached to the rigid portion or the non-rigid portion. In one embodiment, the second and third targeting agents are attached to the rigid portion of the nanostructure and / or to the rigid nanostructure. In one embodiment, the first targeting agent is attached to the rigid portion or the non-rigid portion of the nanostructure. In one embodiment, the rigid portion of the nanostructure extends along the maximum longitudinal extension of the nanostructure. In one embodiment, the nanostructure comprises a rigid portion having a maximum longitudinal extension along the longitudinal axis of the nanostructure of at least 10 nm, for example at least 15 nm, 20 nm, 25 nm, or 40 nm.In one embodiment, the second targeting agent and the third targeting agent are spaced apart from each other along the longitudinal axis of the rigid portion of the nanostructure, said distance being at least 15 nm, preferably at least 20 nm, more preferably at least 40 nm. In one embodiment, the nanostructure comprises a rigid portion to which the second and third targeting agents or the first, second and third targeting agents are attached, and the nanostructure optionally further comprises one or more portions flexibly connected to the rigid portion. In one embodiment, the longitudinal extension of the rigid portion is the maximum extension of the nanostructure, preferably the longitudinal extension. In one embodiment, when referring to a nanostructure having only one configuration, such a configuration is only one configuration and relates to the nanostructure itself, e.g., the core structure of the nanostructure, such as DNA origami, but not to the targeting agent and / or the bond between the targeting agent and the nanostructure. For example, the bond between the targeting agent and the nanostructure and / or the targeting agent may have more than one configuration. In one embodiment, such binding between the targeting agent and the nanostructure is via a linker and / or via a nucleic acid attached to said targeting agent. In one embodiment, the nucleic acid nanostructure of the present invention does not have an open and closed configuration. In one embodiment, the nucleic acid nanostructure of the present invention has only one configuration. In one embodiment, the nanostructure does not have a hinge region. In one embodiment, the nanostructure does not have more than one conformational state. In one embodiment, the terms "nanostructure" and "nucleic acid nanostructure" are used interchangeably. In one embodiment, when referring to the "core structure" of a nanostructure, it means a nucleic acid nanostructure that does not include a targeting agent, such as a DNA origami contained in a nucleic acid nanostructure of the present invention. In one embodiment, when referring to a nanostructure "without a hinge region", it means a nanostructure that does not have a hinge region in the core structure of the nanostructure, i.e., the nucleic acid nanostructure does not include a hinge region in the nucleic acid core structure, but may have a hinge region between the targeting agent and the core structure. In one embodiment, when referring to a nanostructure having only one configuration, this means that such nanostructure, preferably the core structure of such nanostructure, e.g., a DNA origami contained in the nanostructure, cannot be switched between states.In one embodiment, the nanostructure has the same configuration and / or state when bound to the first and / or second target and when not bound to the first and / or second target. In one embodiment, the nanostructure does not have an "open" and a "closed" configuration. In one embodiment, the accessibility of the targeting agent does not depend on the configuration of the nanostructure, but on the position, alignment, and / or orientation of the nanostructure with respect to the first and second targets.

[0075] In one embodiment, each of the staple strands is configured to bind to at least one of the at least one backbone strand at at least one, preferably two or more different locations. In one embodiment, the nucleic acid nanostructure and / or the DNA origami contained in the nucleic acid nanostructure has a backbone strand and one or more staple strands. In one embodiment, the nucleic acid nanostructure and / or the DNA origami contained in the nucleic acid nanostructure comprises 100 or less staple strands. In one embodiment, the staple strand has a length of 50-150 nucleobases. In one embodiment, the backbone strand has a length of 100-20000 nucleobases, preferably 120-10000 nucleobases, more preferably 150-8064 nucleobases. In one embodiment, the targeting agent is attached and / or bound to the nucleic acid nanostructure via a nucleic acid sequence conjugated to said targeting agent, preferably a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid nanostructure, e.g., complementary to the backbone strand of said nucleic acid nanostructure, and / or a nucleic acid handle, preferably a DNA handle, attached and / or incorporated into said nucleic acid nanostructure.

[0076] At least three interaction sites, i.e. at least three targeting agents, specifically a first targeting agent, a second targeting agent, and a third targeting agent, are attached to the nanostructure. The targeting agents are designed to be attached such that when at least one of the targeting agents of the subset A of targeting agents, preferably the second and third targeting agents, is not bound to their respective targets, the nanostructure is in an orientation in which the targeting agent subset B, preferably the first targeting agent, cannot bind to their respective targets due to steric hindrance. When the interaction molecule subset A, preferably the second targeting agent and the third targeting agent, bind to their respective targets, the nanostructure becomes oriented in an accessible orientation in which the targeting agent subset B, preferably the first targeting agent, can bind to their respective targets. Thus, the binding rate of the targeting agent subset B on the nanostructure is conditionally modified by the binding state of the targeting agent subset A. Thus, the conditional orientation is mediated by the binding of the targeting agents.

[0077] The terms "first surface" and "second surface" as used herein refer to opposing surfaces of a nanostructure, particularly opposing surfaces along the longitudinal axis of the nanostructure. In one embodiment, a "surface", e.g., the first surface and / or the second surface, refers to a surface that may be in the form of, e.g., a region, a line, an edge, a corner, a DNA helix blunt end, DNA and / or a point. In one embodiment, the surface has any topology. In one embodiment, the surface is a two-dimensional or three-dimensional surface. In one embodiment, the surface, e.g., the first surface or the second surface, is an interrupted or uninterrupted surface. In one embodiment, an interrupted surface has holes, bulges, recesses, and / or depressions in said surface, e.g., holes and / or depressions formed by recesses. In one embodiment, the surface, e.g., the first surface or the second surface, is a region that includes at least one recess, and preferably, said one or more targeting agents are contained within such recess, and preferably, at least a portion of said one or more targeting agents are presented on said surface.

[0078] In one embodiment, the nanostructure comprises a third surface and a fourth surface, the third and fourth surfaces being opposed surfaces along a transverse axis of the nanostructure, and / or the third and fourth surfaces being abutting surfaces of the first and / or second surfaces, and / or the third and fourth surfaces being perpendicular to at least one of the first and second surfaces. In one embodiment, the third surface is separated from at least one of the first and second surfaces via an edge. In one embodiment, the fourth surface is separated from at least one of the first and second surfaces via an edge. In one embodiment, the first targeting agent is attached to the third surface or the fourth surface and / or an edge, and the edge separates the third surface from the first surface or the second surface, or separates the fourth surface from the first surface or the second surface. In one embodiment, the second and / or third targeting agent is attached to a third or fourth surface and / or an edge, said edge separating the third surface from the first or second surface, or separating the fourth surface from the first or second surface. In one embodiment, when a second or third targeting agent is attached to the third or fourth surface and / or the edge, at least a portion of the second or third targeting agent is presented on the second surface. In one embodiment, when a first targeting agent is attached to the third or fourth surface and / or the edge, at least a portion of the first targeting agent is presented on the first surface.

[0079] The terms "surface comprises" and "surface comprises a targeting agent" in the context of a surface, such as a first or second surface comprising a targeting agent, e.g., a first targeting agent, and a second and third targeting agent, respectively, mean that the surface presents at least a portion of the targeting agent, preferably presents at least a portion of the targeting agent on its surface, and / or that at least a portion of the targeting agent is accessible from the surface. In one embodiment, the term "accessible" as used herein means that it can bind to and / or be bound by a target molecule. In one embodiment, the term "accessible from" a targeting agent in the context of a surface means that such a surface does not sterically hinder the accessibility of such a targeting agent.

[0080] In one embodiment, the term "a first surface comprises at least a first targeting agent" means that at least a portion of said at least a first targeting agent is presented on said first surface and / or at least a portion of said first targeting agent is accessible from said first surface. In one embodiment, said first targeting agent is attached on a surface, e.g. on said first surface, and / or subsurface, e.g. subsurface, e.g. within the core structure of said nanostructure. In one embodiment, said first targeting agent is attached to said nanostructure on a surface, e.g. on said first surface, or subsurface, e.g. within a recess or subsurface of said recess. In one embodiment, the first targeting agent can be attached to any part of the nanostructure, e.g. within the core of said nanostructure, and / or in a plane and / or surface perpendicular to the first or second surface, e.g., in a third or fourth surface, in an attachment plane, or in an attachment site. In one embodiment, the first targeting agent can be attached to any portion of the nanostructure, provided that at least a portion of said first targeting agent is displayed on the first surface.

[0081] In one embodiment, the term "the second surface comprises at least a second targeting agent and at least a third targeting agent" means that at least a portion of the at least a second targeting agent and at least a portion of the at least a third targeting agent are presented on the second surface, and / or at least a portion of each of the second and third targeting agents is accessible from the second surface. In one embodiment, the second targeting agent and / or the third targeting agent are attached on a surface, e.g., on the second surface, and / or subsurface, e.g., subsurface, e.g., within a core structure of the nanostructure, e.g., to an inner portion of the nanostructure. In one embodiment, the second targeting agent and / or the third targeting agent are attached to the nanostructure on a surface, e.g., on the second surface, or subsurface, e.g., within a recess or subsurface of the recess. In one embodiment, the second targeting agent and / or the third targeting agent can be attached to any portion of the nanostructure, for example, within the core of the nanostructure and / or attached to a plane and / or surface perpendicular to the first or second surface. In one embodiment, the second targeting agent and / or the third targeting agent can be attached to any portion of the nanostructure, provided that at least a portion of the second targeting agent and at least a portion of the third targeting agent are presented on the second surface.

[0082] In one embodiment, the term "presented" refers to "exposed", e.g., at least a portion of the targeting agent is presented and / or exposed on the surface. In one embodiment, a portion of the targeting agent presented and / or exposed on the surface can bind to its respective target molecule. In one embodiment, a portion of the targeting agent presented and / or exposed on the surface is accessible to its respective target molecule. In one embodiment, the nanostructure of the present invention is a nucleic acid nanostructure comprising a first surface and a second surface, the first surface and the second surface being located on opposite sides of the nanostructure, the first surface comprising at least a first targeting agent, i.e., the first surface presents at least a portion of the at least first targeting agent, and the second surface comprising at least a second targeting agent and at least a third targeting agent, i.e., the second surface presents at least a portion of the at least second targeting agent and at least a portion of the at least third targeting agent.

[0083] In one embodiment, the first targeting agent is attached to the nanostructure in a plane parallel and / or proximate (e.g., 0-5 nm) to the first surface of the nanostructure. In one embodiment, the first targeting agent is attached to the nanostructure at an attachment site and / or attachment plane parallel and / or proximate (e.g., 0-5 nm) to the first surface of the nanostructure. In one embodiment, such attachment site and / or attachment plane intersects with a third or fourth surface, and optionally, said first targeting agent is attached to the nanostructure at such intersection.

[0084] In one embodiment, the second and / or third targeting agents are attached to the nanostructure in a plane parallel and / or proximal (e.g., 0-5 nm) to the second surface of the nanostructure. In one embodiment, the second and / or third targeting agents are attached to the nanostructure at attachment sites and / or attachment planes parallel and / or proximal (e.g., 0-5 nm) to the second surface of the nanostructure. In one embodiment, such attachment sites and / or attachment planes intersect with a third or fourth surface, and optionally, said second and / or third targeting agents are attached to the nanostructure at such intersections.

[0085] In one embodiment, the first targeting agent is attached to a plane and / or site and / or surface that is not the top surface, e.g., not the top first surface. In one embodiment, the second and / or third targeting agent is attached to a plane and / or site and / or surface that is not the top surface, e.g., not the top second surface. In one embodiment, the first and / or second surface may be a surface that is not the top surface of the nanostructure along the longitudinal axis, e.g., a recessed surface. In an alternative embodiment, the first and / or second surface is the top surface of the nanostructure along the longitudinal axis. In a preferred embodiment, at least a portion of the first targeting agent is presented on the top surface, e.g., the first surface, of the nanostructure and / or is accessible on the top surface, e.g., the first surface, of the nanostructure. In a preferred embodiment, at least a portion of the second and / or third targeting agent(s) is presented on the top surface, e.g., the second surface, of the nanostructure and / or is accessible on the top surface, e.g., the second surface, of the nanostructure.

[0086] In one embodiment, the first targeting agent is attached to the nanostructure, e.g., DNA origami object, directly on the first surface, and / or attached to the nanostructure at an edge of the first surface, e.g., an edge separating the first surface from a third or fourth surface, and / or attached to the nanostructure at a third corner of the first surface and an adjacent surface, e.g., a fourth surface. In one embodiment, the second and / or third targeting agent is attached to the nanostructure, e.g., DNA origami object, directly on the second surface, and / or attached to the nanostructure at an edge of the second surface, e.g., an edge separating the second surface from a third or fourth surface, and / or attached to the nanostructure at a third corner of the second surface and an adjacent surface, e.g., a fourth surface. In one embodiment, the terms "adjacent surface" and "abutting surface" are used interchangeably. In one embodiment, the first targeting agent is attached to the nanostructure at a position having a distance of about 0-5 nm from the first surface and / or from the first outermost surface, preferably along the horizontal axis. In one embodiment, the second or third targeting agent is attached to the nanostructure at a distance of about 0-5 nm from the second surface and / or the second top surface, preferably along the transverse axis. The first, second and / or third targeting agent may also be positioned and / or attached in a plane of attachment parallel to the first or second surface to obtain a smaller cell distance. The optimal distance of the plane of attachment from the first or second surface may depend on the size of the targeting agent and the distance between the epitopes of the target molecule from the cell surface. The distance of the parallel plane of attachment to its corresponding surface may be selected from 0-10 nm, preferably less than 5 nm, for example 2 nm.

[0087] The term "longitudinal axis" as used herein refers to a line passing through the center of gravity of a cross-section and / or through the maximum extension and / or length of the nanostructure, e.g., the maximum extension and / or length of a core structure of the nanostructure, and / or a line passing through a principal axis of inertia. In one embodiment, when referring to the first surface and the second surface being located "on opposite sides along the longitudinal axis", such term refers to parallel opposite sides along the longitudinal axis. In one embodiment, the first surface and the second surface lie in a plane parallel to the longitudinal axis.

[0088] The term "transverse axis" as used herein refers to an axis that is, for example, perpendicular to the longitudinal axis, transverse to the longitudinal axis, and / or transverse to the longitudinal axis. In one embodiment, the transverse and longitudinal axes are oriented relative to one another as shown in Figures 1-4.

[0089] The term "maximum longitudinal extension" as used herein preferably relates to the maximum length of the nanostructures along the longitudinal axis. The term "maximum transverse extension" as used herein relates to the length of the nanostructures in the transverse and / or transverse directions, preferably the maximum length of the nanostructures in the transverse and / or transverse directions. In one embodiment, the maximum length of the nanostructures along the transverse axis is smaller than the maximum length of the nanostructures, which is the maximum length along the longitudinal axis. In one embodiment, the maximum transverse extension is smaller than the maximum longitudinal extension. In one embodiment, the maximum length along the transverse extension is smaller than the maximum length along the longitudinal extension.

[0090] The term "target molecule" as used herein relates to any molecule of a target, such as a compound, peptide, antigen, lipid, and / or any other molecule that is bound by a targeting agent. In one embodiment, the target molecule is a molecule that is presented on the surface of the target and / or is accessible on the surface of the target. In one embodiment, the target molecule, such as an antigen, is either a cell surface binding molecule or a sensor surface binding molecule, such as a protein, a cluster of differentiation molecules, a receptor, a lipid, an ion channel, a sugar, or any other cell surface binding molecule or structure. In one embodiment, the target molecule is an antigen.

[0091] The term "first target molecule" as used herein relates to a target molecule on a first target, such as a target molecule displayed on the surface of the first target, such as an immune cell, an effector cell, a sensor surface, or an immobilization surface.

[0092] The term "first target" as used herein relates to any molecule, structure, material, cell, antigen, and / or surface that is bound by a first targeting agent. In one embodiment, said first target is a cell, and / or a molecule, material or surface, such as a sensor surface, immobilization surface or other surface used in an assay. For example, the first target may be a sensor, immobilization surface, bead, column material of a chromatography column, a microtiter plate surface, a biotinylated or streptavidin coated surface, and / or any other surface, material or molecule used in an assay to immobilize an analyte such as a cell. For example, a nanostructure can be targeted to the first target in order to immobilize the nanostructure, and a second target binds to the nanostructure. Such immobilization can be used to perform an assay, a selection and / or sorting of cells, or any other method or assay known to the skilled person. In a preferred embodiment, the first target is a cell, preferably an immune cell. In an alternative embodiment, the first target is a sensor surface or any other material used in an assay to measure, for example, on / off binding kinetics.

[0093] The term "first target cell" as used herein relates to a cell, e.g., a cell that is brought into proximity with a second target cell. In one embodiment, the first target cell is an immune cell, e.g., a lymphocyte, e.g., a T cell, a cytotoxic T cell, a B cell, a natural killer cell, a natural killer T cell, a CAR-T cell, a monocyte, a macrophage or a neutrophil, or other effector cell. The term "effector cell" as used herein refers to a cell, preferably an immune cell, that is involved in an effector system and / or effector mechanism that results in the destruction and / or elimination of the target cell. The term "cytotoxic T cell" as used herein relates to a T lymphocyte that is capable of killing a target cell, such as a cancer cell, an infected cell, or a cell that is otherwise damaged and / or pathological. T cells typically express a T cell receptor (TCR) that specifically recognizes an antigen. Such antigens are typically presented on the surface of the cell by MHC-I molecules. In one embodiment, such antigens are the first targeting agent of the nanostructure. When the T cell receptor of the cytotoxic T cell is specific for an antigen, it binds to the complex of the antigen and the MHC-I molecule and destroys the cell. The interaction of the T cell with the complex of the antigen and the MHC-I molecule of the target cell may also involve a co-receptor, namely CD8. In one embodiment, the nanostructure preferably comprises on said first surface a fourth targeting agent that is or binds to CD8, preferably binds to CD8, more preferably binds to CD8 on said first target cell. In one embodiment, the fourth targeting agent is configured to bind to a fourth target molecule that is CD8. In one embodiment, when the T cell binds to such a complex on the target cell or when an immune synapse is formed using the nanostructure of the invention, the T cell releases any of the cytotoxins perforin, granzymes, and granulysin. In one embodiment, through the action of perforin, granzymes enter the cytoplasm of the second target cell, e.g., the diseased cell, and their serine protease function triggers a caspase cascade that leads to apoptosis of said second target cell.In one embodiment, apoptosis of the second target cell may also be induced by cell surface interaction between the first target cell, e.g., a cytotoxic T cell, and the second target cell, such as by Fas-Fas ligand interaction, and / or by formation of an immune synapse.

[0094] The term "secondary target molecule" as used herein relates to a target molecule on a second target, such as a target molecule displayed on the surface of the second target, such as a disease cell, e.g. a cancer cell or a cell involved in an autoimmune response.

[0095] The term "second target" as used herein relates to any molecule, structure, material, cell, antigen, and / or surface that is bound by a second and / or third targeting agent, e.g., a target cell, such as a diseased cell. In one embodiment, the second and third targeting agents can be used for cell sorting by targeting said second target and said first target, preferably by targeting said second target molecule and a third target molecule on said second target. In one embodiment, by having an immobilized first target, target cells, particularly target cells having a second target molecule and a third target molecule on their surface, can be selectively and / or specifically immobilized via said nanostructure. Furthermore, such second targets, such as diseased cells, can be eliminated and / or destroyed by targeting immune cells and / or effector cells to said second target via the nanostructure.

[0096] The term "second target cell" as used herein relates to a target cell, such as a disease cell or a cell to be fixed. The term "disease cell" as used herein relates to a cell that has at least one pathological characteristic, such as abnormal division or abnormal function, for example, such a cell is involved in and / or causes a disease, such as cancer or an autoimmune disease. For example, when the disease cell is a cancer cell, such a cancer cell is a cell that divides continuously and thereby forms a tumor. For example, when the disease cell is a cell that is involved in an autoimmune response, such a cell may be a cell that induces and / or is targeted by the autoimmune response. In one embodiment, the disease cell is any cell that is involved in a disease and / or the development of a disease, preferably a cell that causes such a disease and / or the development of a disease.

[0097] The term "third target molecule" as used herein refers to a target molecule on a second target, such as a target molecule presented on the surface of the second target, such as a disease cell, for example a cancer cell or a cell involved in an autoimmune response. In one embodiment, the second target molecule and the third target molecule are the same or different target molecules, preferably different target molecules. In one embodiment, by targeting at least two target molecules on the second target, i.e., the second target molecule and the third target molecule, the specificity and / or selectivity of targeting the second target is increased.

[0098] The term "spaced apart from one another" as used herein relates to the spacing between at least two molecules and / or at least two molecules that are positioned at a distance from one another, e.g., a second targeting agent and a third targeting agent along a longitudinal axis.

[0099] The term "surface molecule" as used herein refers to a molecule that is presented on a surface and / or at least partially presented on a surface. The term "non-identical" as used herein in the context of molecules, targets and / or agents refers to molecules or entities that are not the same, e.g., a second target molecule and a third target molecule that are not the same molecule and / or the same type of molecule. The term "combination of target molecules" as used herein refers to a combination of a second target molecule and a third target molecule, preferably a pair of surface molecules, target molecules and / or biomarkers of target cells. For example, such a combination may be a combination of two biomarkers that are typically expressed on a particular disease cell, such as a particular cancer cell.

[0100] The term "DNA origami structure" as used herein refers to nanostructures that include DNA as a building material for creating nanoscale shapes. Preparing DNA origami involves folding one or more "backbone" DNA strands into a specific shape, e.g., by self-assembly, using a plurality of rationally designed "staple" DNA strands. The backbone strands are typically longer than the staple strands. The nucleic acid sequences of the staple strands are designed such that the staple strands hybridize to specific portions of the backbone strands, and hybridization results in a specific shape of the nanostructure. In one embodiment, the term nucleic acid "handle" refers to a staple strand having a nucleic acid sequence extension that can be attached to a moiety, such as a nucleic acid sequence attached to a targeting agent.

[0101] As used herein, the term "oriented perpendicular to the maximum longitudinal extension" refers to being oriented in a transverse and / or perpendicular plane to the longitudinal extension and / or longitudinal axis. In one embodiment, the recesses can have any shape and / or form, such as regular or irregular shapes.

[0102] The term "marker" as used herein relates to any marker known to the person skilled in the art, such as markers and / or dyes for in vitro assays or for in vivo medical imaging, e.g. fluorescent markers, radiographic markers, MRI markers, nuclear medicine markers, tomographic markers, etc.

[0103] The term "specific" or "specifically binds" as used herein means, according to the present invention, that a targeting agent, e.g., an antibody or an antigen-binding peptide, can specifically interact and / or bind to a specific target or a set of specific targets, but does not essentially bind to other molecules, such as antigens. Such binding may be exemplified by the specificity of the lock-and-key principle. In one embodiment, when referring to the term "binding", such term should be construed as including the term "specific binding". In one embodiment, when referring to "targeting", such term should be construed as including the term "specific targeting".

[0104] The term "nucleic acid" as used herein relates to a nucleotide sequence such as a ribonucleic acid or a deoxyribonucleic acid. The term "patient" as used herein may relate to a human or an animal.

[0105] In one embodiment, the term "recess" as used herein relates to a recess in a nanostructure. In one embodiment, the recess has a depth of 0.25 nm to 9 nm, preferably 2 nm to 7 nm, more preferably about 5 nm. In one embodiment, the depth of such recess is oriented along the transverse axis of the nanostructure. In one embodiment, the first targeting agent is bound to the nanostructure via a recess, and / or at least a portion of the first targeting agent is located within the recess, and / or the second targeting agent is bound to the nanostructure via a recess, and / or at least a portion of the second targeting agent is located within the recess, and / or the third targeting agent is bound to the nanostructure via a recess, and / or at least a portion of the third targeting agent is located within the recess. In one embodiment, the first targeting agent, the second targeting agent, and the third targeting agent are bound to the nanostructure via a recess, and / or at least a portion of each of the first targeting agent, the second targeting agent, and the third targeting agent are located within the recess. In one embodiment, the nanostructure has one or more recesses. In one embodiment, incorporating a recess in the nanostructure further enhances the specificity of the nanostructure. In one embodiment, the second targeting agent and the third targeting agent are each incorporated in a recess. In one embodiment, when the second targeting agent and the third targeting agent are each incorporated in a recess, the first targeting agent binds to its target molecule first, and then the second and third targeting agents bind to their target molecules, due to better accessibility of the first targeting agent. In one embodiment, when referring to a targeting agent "incorporated" into a recess, such "incorporation" includes binding to the nanostructure via said recess and / or at least a portion of said targeting agent being located within said recess. In one embodiment, the nanostructure includes a recess that includes said first targeting agent, said second targeting agent, or said third targeting agent. In one embodiment, the recess is oriented perpendicular to the longitudinal axis, which means that the recess is oriented along the transverse axis.

[0106] In one embodiment, when referring to a method, the method is an in vivo, ex vivo, in vitro or in situ method, such as an in vitro method. In one embodiment, when referring to a use, the use is an in vivo, ex vivo, in vitro or in situ use, such as an in vitro use. In one embodiment, the present invention relates to the use of a nanostructure for binding a first target and a second target, preferably for inducing, promoting, stabilizing and / or inhibiting the formation of a synapse between said first target and said second target, more preferably for inducing and / or promoting the formation of a synapse between said first target and said second target, said use being an in vivo, ex vivo, in vitro or in situ use, such as an in vitro use. In one embodiment, the nanostructure of the present invention is for use in vivo or in vitro, preferably in vivo.

[0107] Another aspect of the present invention, which may be combined with any of the other preferred embodiments and / or aspects of the present invention, relates to a vector, in particular an expression vector, comprising a sequence encoding a nanostructure as defined above, a backbone strand and / or a staple strand sequence and / or comprising a nanostructure sequence.

[0108] The composition of the present invention, preferably the pharmaceutical composition, must be formulated to be compatible with its intended route of administration.In a particularly preferred embodiment, examples of routes of administration of the medicament and / or compound of the present invention include intravenous, oral, intranasal, intrathecal, intraarterial, intradermal, subcutaneous, transdermal (topical), intraventricular, intraparenchymal, intratumoral, transmucosal, rectal, vaginal, bronchial, parenteral administration, and any other clinically / medically acceptable method for administering the medicament and / or compound.

[0109] Yet another aspect of the invention, which may be combined with any of the other aspects or specific embodiments of the invention, relates to a method for preventing and / or treating a disease, preferably a proliferative disease such as cancer, an immune disorder such as an autoimmune disease, a viral disease such as human immunodeficiency virus (HIV) infection, a metabolic disorder, an infectious disorder and / or diabetes, comprising the step of administering to a subject a therapeutically effective amount of the nanostructures and / or compositions according to the invention.

[0110] The term "cancer" as used herein refers to a disease characterized by unregulated cell proliferation and / or growth. The term includes benign and malignant cancerous diseases, such as tumors, and may refer to invasive or non-invasive cancers. The term includes all types of cancer, including carcinomas, sarcomas, lymphomas, germ cell tumors, and blastomas. The term "cancer cells" as used herein refers to cells that exhibit abnormal proliferation and divide continuously, thereby forming solid or non-solid tumors.

[0111] Examples of autoimmune diseases include celiac disease, post-infectious IBS, type 1 diabetes, Henoch-Scholein purpura (HSP), sarcoidosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM) and multiple sclerosis (MS).

[0112] In one embodiment, when the nanostructure of the invention is for use in the prevention or treatment of an autoimmune disease, said first target cell is preferably an immune cell, e.g. a macrophage and / or a neutrophil. In one embodiment, the nanostructure is for use in a method of preventing or treating a disease, preferably for use in a method of specifically binding a first target cell, e.g. an immune cell, to a second target cell, e.g. a diseased cell. In one embodiment, the nanostructure of the invention allows at least two cell types to specifically and selectively bind to each other. In one embodiment, the method of preventing or treating a disease comprises a method of forming a synapse between a first target cell and a second target cell. In one embodiment, the method of preventing or treating a disease comprises contacting a first target cell with a second target cell to form a synapse, e.g. an immune synapse. In one embodiment, the method of preventing or treating a disease comprises providing a specific distance between the first target and the second target. In one embodiment, the nanostructure is for use in providing a specific distance between a first target and a second target, for example between a first target cell and a second cell, or between a sensor and a target cell. In one embodiment, the nanostructure is for use in inducing, promoting, stabilizing and / or inhibiting, preferably inducing and / or promoting, the formation of synapses in vivo or in vitro.

[0113] The term "effective amount" as used herein relates to an amount sufficient to elicit a desired effect, such as a therapeutic effect and / or binding of a first target to a second target and / or formation of a synapse. To calculate the amount necessary to be an "effective amount", such as a therapeutically effective amount, a skilled artisan and / or physician can use data obtained from cell culture assays and animal studies to formulate a range of dosages for use in humans. Dosages of such nanostructures and / or compositions are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used, a therapeutically effective dose can be initially estimated from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The pharmaceutical compositions can be included in a container, pack, or dispenser along with instructions for administration.

[0114] The term "drug" as used herein relates to any pharmaceutical agent, such as a chemotherapeutic agent, an immunosuppressant, and / or a checkpoint inhibitor. In one embodiment, the chemotherapeutic agent is a cytotoxic agent typically used in cancer chemotherapy or a checkpoint inhibitor typically used in cancer immunotherapy.

[0115] In one embodiment, in the context of the method of the invention, the term "providing a nucleic acid nanostructure" includes providing an assembled nucleic acid nanostructure and / or providing building materials for the nucleic acid nanostructure, such as a backbone strand and one or more staple strands. In one embodiment, the method of preparing the nanostructure of the invention includes a step of allowing self-assembly of the nanostructure and a step of purifying said self-assembled nanostructure, e.g., step i) of the method, i.e., providing a nucleic acid nanostructure, includes a step of allowing self-assembly and subsequent purification. In one embodiment, allowing self-assembly includes mixing at least one backbone strand and one or more staple strands, and further includes adjusting the ionic strength, as required, e.g., by adding MgCl2 to 20 mM, and / or further includes using a temperature protocol that runs a temperature series. In one embodiment, said purification includes removing remaining excess staple strands, e.g., by precipitation, e.g., PEG precipitation, filtration, and / or liquid chromatography. In one embodiment, the self-assembly and / or allowing self-assembly step includes a denaturation step and a cooling step. In one embodiment, the denaturation step is carried out at a temperature of 50°C to 80°C, preferably 60°C to 70°C, for example about 65°C, for a period of 1 minute to 45 minutes, preferably 10 to 20 minutes, for example about 15 minutes. In one embodiment, the cooling step is carried out at a temperature of 0°C to 70°C, preferably 20°C to 60°C, for example about 50°C to 58°C. In a preferred embodiment, the cooling step is carried out as a slow cooling, preferably at a temperature of about 58°C to about 50°C, decreasing by 1°C per hour. The skilled person will understand that the protocol for self-assembly depends on the design of the nanostructure and / or the nucleic acid sequence, and the protocol can be adjusted according to known protocols for preparing nanostructures. In one embodiment, the nanostructure is configured to have a backbone sequence that does not include a sequence that forms a secondary structure. In one embodiment, the nucleic acid sequence conjugated to the targeting agent, preferably the nucleic acid sequence conjugated to the targeting agent that is complementary to the sequence of the nanostructure, is configured to consist of a sequence that does not form a secondary structure.In one embodiment, the nucleic acid sequences of the nanostructure, e.g., the nucleic acid sequences of the backbone and staple strands, the nucleic acid handle, e.g., the nucleic acid sequences of the DNA handle, and / or the nucleic acid conjugated to the targeting agent, are configured not to form secondary structures. In one embodiment, the self-assembled nanostructure has one or more vacancies, i.e., single-stranded nucleic acid sequences that can be bound by complementary nucleic acid sequences conjugated to a targeting agent.

[0116] In one embodiment, the method of preparing nanostructures comprises coupling a targeting agent, preferably a first targeting agent, a second targeting agent, and / or a third targeting agent, to the nanostructure. In one embodiment, the terms "coupling", "binding", "attached to" and "conjugating" are used interchangeably. In one embodiment, the terms "coupled", "attached", "attached" and "conjugated" are used interchangeably. In one embodiment, the step of coupling the targeting agent to the nanostructure comprises providing a first targeting agent, a second targeting agent, and a third targeting agent, each targeting agent having a unique nucleic acid sequence attached to the targeting agent that is complementary to the nucleic acid strand of the nanostructure, e.g., the backbone strand of the nanostructure or the DNA handle strand attached to the nanostructure. In one embodiment, due to the complementarity of the nucleic acid sequence attached to the targeting agent and the nucleic acid sequence of the nanostructure, such targeting agents bind to the nanostructure via a complementary nucleic acid strand, preferably automatically bind to the nanostructure via a complementary nucleic acid strand. In one embodiment, when referring to a "unique" nucleic acid sequence, such term means that the first targeting agent, the second targeting agent, and the third targeting agent have different nucleic acid sequences attached to the targeting agent. Such "unique" nucleic acid sequences are configured to be complementary to a specific sequence and / or to bind to a specific location of the nanostructure. In one embodiment, the nucleic acid sequence conjugated to the first targeting agent is different from the nucleic acid sequence conjugated to the second targeting agent, which is different from the nucleic acid sequence conjugated to the third targeting agent. In one embodiment, when referring to a "targeting agent", it means the first targeting agent, the second targeting agent, and / or the third targeting agent.

[0117] In one embodiment, the method of preparing nanostructures comprises mixing the nucleic acid nanostructure with a first targeting agent, a second targeting agent, and / or a third targeting agent, preferably the first targeting agent, the second targeting agent, and the third targeting agent, for example in equimolar ratios or in excess of the targeting agents, and optionally further comprising incubating the mixed nucleic acid nanostructure and targeting agent. In one embodiment, the method of the invention comprises, after mixing the nucleic acid nanostructure with the targeting agent, removing the excess targeting agent and / or unbound targeting agent by, for example, precipitation, such as PEG precipitation, filtration, and / or liquid chromatography. In one embodiment, the step of incubating the nanostructure and targeting agent is carried out at a temperature between 20° C. and 50° C., preferably between 30° C. and 40° C., for example about 37° C., for a period of 1 minute to 3 hours, preferably between 30 minutes to 1.5 hours, for example about 1 hour.

[0118] In one embodiment, the method of the invention comprises obtaining a nucleic acid nanostructure comprising said first targeting agent, said second targeting agent, and said third targeting agent, said obtaining comprising: a) mixing one or more building materials for the nanostructures to obtain an assembled nanostructure, and subsequently contacting said assembled nanostructure with one or more targeting agents, thereby obtaining a nanostructure comprising the targeting agents; and / or b) mixing one or more building materials for the nanostructures with one or more targeting agents to obtain nanostructures comprising the targeting agents; and / or c) mixing the assembled nanostructures with one or more targeting agents, thereby obtaining nanostructures containing the targeting agents.

[0119] In one embodiment, a method for preparing nanostructures comprises: iv) preferably by allowing self-assembly of nanostructures, for example by mixing at least one backbone chain and one or more staple chains and purifying said self-assembled nanostructures; Providing a nucleic acid nanostructure, preferably a rigid nucleic acid nanostructure, comprising: Optionally, the self-assembly comprises a step of denaturation and a step of cooling, preferably slow cooling; v) providing a first targeting agent, a second targeting agent, and a third targeting agent, each of which is conjugated to a nucleic acid strand complementary to a nucleic acid strand of the nanostructure, e.g., a backbone strand or a DNA handle strand; vi) preferably mixing the nucleic acid nanostructure with a first targeting agent, a second targeting agent, and / or a third targeting agent, and optionally incubating; vii) optionally removing unbound first targeting agent, second targeting agent, and / or third targeting agent; and viii) obtaining a nucleic acid nanostructure comprising the first targeting agent, the second targeting agent, and the third targeting agent.

[0120] In one embodiment, a method of preparing nanostructures comprising a targeting agent comprises attaching a targeting agent-nucleic acid conjugate to a nanostructure, said method comprising: 1. Allowing nanostructures to self-assemble and purifying said assembled nanostructures, e.g. DNA origami objects, preferably by mixing one or more backbone strands, preferably one backbone strand with one or more staple strands; adjusting the ionic strength, if necessary (e.g., by adding MgCl to 20 mM) and subjecting the mixture to a series of specific temperatures; Optionally, removing excess unbound staple strands (e.g., using PEG precipitation, filtration, liquid chromatography); 2. coupling a nucleic acid strand to a targeting agent, wherein each of the first targeting agent, the second targeting agent, and the third targeting agent is coupled to a different nucleic acid sequence, and the nucleic acid sequence is preferably complementary to a DNA sequence protruding from a nanostructure, e.g., a DNA origami object, e.g., a nucleic acid handle, or a backbone DNA strand; 3. Preferably in equimolar ratios or with an excess of targeting agent-nucleic acid conjugate, e.g. DNA-antibody-conjugate, mixing the nanostructures, e.g., DNA origami objects, with a targeting agent-nucleic acid conjugate, e.g., a DNA-antibody conjugate; Preferably, the method comprises incubating the nanostructures and the targeting agent at about 37° C. for about 1 hour; 4. Removing excess targeting agent-nucleic acid conjugate, e.g., DNA-antibody-conjugate, e.g., using PEG precipitation, filtration, liquid chromatography.

[0121] In one embodiment, the nanostructures may be stabilized against nuclease degradation, against degradation by low ionic strength environments, and / or against in vivo conditions using coatings described in ["Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation", Ponnuswamy et al., Nat Commun 2017; WO 2015070080] and / or stabilization methods described in ["Sequence-programmable covalent bonding of designed DNA assemblies", T. Gerlin, Science Advances, 2018; WO 2019234122].

[0122] In one embodiment, the nanostructures of the present invention, e.g., rigid single-domain nanostructures having one configuration, are highly advantageous as they can be stabilized using the stabilization methods described above without compromising their conditional cell recruitment function.

[0123] As used herein, the term "comprising" should be interpreted as embracing both "including" and "consisting of," both meanings being specifically intended and thus being individually disclosed embodiments according to the present invention. As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that the skilled artisan will understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, and for example ±5% from the indicated numerical value. As will be understood by those skilled in the art, such a specific deviation for the numerical value of a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater bias than man-made or engineered technical effects. It is understood that the use of the singular article "a" or "the" in this document is not meant to limit the scope of the invention unless specifically stated. That is, generally, "a" may refer to more than one. In other words, "a" can generally be read as "at least one". For example, "a targeting agent" also includes multiple targeting agents. In one embodiment, the term "as defined above" relates to "as defined in any of the above embodiments".

[0124] As used herein, the terms "of the [present] invention," "in accordance with the invention," "according to the invention," and the like are intended to refer to all aspects and embodiments of the present invention that are described and / or claimed herein.

[0125] Whenever method steps are listed in this specification and in the appended claims, it should be noted that the order in which the steps are listed in this document may be incidental. That is, unless otherwise specified or obvious to one skilled in the art, the order in which the steps are listed may also be different. For example, when this specification states that, for example, a method includes steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), and it is also possible that step (A) is performed (at least partially) simultaneously with step (B), or that step (B) precedes step (A). Furthermore, when it is said that step (X) precedes another step (Z), this does not mean that there is no step between step (X) and (Z). That is, step (X) preceding step (Z) can encompass not only the situation in which step (X) is performed immediately before step (Z), but also the situation in which step (X) is performed before one or more steps, e.g., (Y1), (Y2), (Y3), followed by step (Z). Where terms such as "after" or "before" are used, corresponding considerations apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The invention will now be further described with reference to the following figures.

[0127] All methods referred to in the following figure descriptions were performed as detailed in the Examples. [Brief description of the drawings]

[0128] [Figure 1] FIG. 1 shows an exemplary scheme of the mechanism of conditional T cell-mediated killing of target cells by the DNA origami antibody platform.

[0129] Exemplary nanostructures of the present invention, in particular an exemplary DNA origami carrier platform with two targeting agents A1 (exemplary second targeting agent) and A2 (exemplary third targeting agent) for target cells (e.g., antibodies against target cell antigens) and one targeting agent B1 (exemplary first targeting agent) for immune cells (e.g., antibodies against immune cell antigens). The vertical axis is indicated by the letter l and the horizontal axis is indicated by the letter t. A. The antibody platform can bind with both target cell antibodies to cells with both antigens RA1 and RA2 (exemplary second and third target molecules). In order for both antibodies A1 and A2 to bind to their respective targets RA1 and RA2, the platform needs to assume an orientation where the vertical axis is parallel to the cell surface of the target cell. This presents an immune cell antibody B1 that can bind to a first target molecule such as an antigen of the immune cell (e.g., CD3 on a cytotoxic T cell). The dimensions of the platform, the targeting agent, e.g., antibody, and antibody attachment are such that the distance d between the cell surfaces is the minimum distance d required for immune synapse formation. min (e.g., 10 nm). B. If the cell carries only antigen RA1, the antibody platform may bind with antibody A1 but may not adopt a stable parallel orientation to the cell surface. The platform will fluctuate and antibody B1 will not be accessible to the immune cell. Furthermore, the antibody platform may dissociate. To promote rapid dissociation, a low affinity antibody can be used at position A1. C. If the cell carries only antigen RA2, the antibody platform may bind with antibody A2 but may not adopt a stable parallel orientation to the cell surface. Antibody B2 will be accessible to recruit immune cells. However, the distance d between the cell surfaces is smaller than the distance d allowed for immune synapse formation. min Thus, cells bearing only RA2 are not killed by recruited immune cells.

[0130] [Figure 2-1] Figure 2 shows the schemes of exemplary nanostructures of the present invention. A. Schemes of rod-shaped DNA origami carrier platforms with attached targeting agents (2D schemes at the top, 3D schemes in the middle) for target cells (A1, A2) and immune cells (B1). l indicates the direction of the vertical axis, t1 and t2 indicate the direction of the horizontal axis. As in BA, but the cylinders represent the double-stranded interconnected DNA helices and Fab fragments as targeting agents. C. caDNAno design of rod-shaped antibody-DNA origami carrier platforms. Exemplary targeting agent attachment positions are indicated by black circles.

[0131] [Figure 2-2] Same as above.

[0132] [Diagram 3] FIG. 3 shows a scheme of an exemplary nanostructure of the present invention having a recess.

[0133] Schemes of rod-shaped antibody-DNA origami platform with recess and targeting agent attached for ABC target cells (A1, A2) and immune cells (B1). l indicates the direction of the vertical axis, t1 and t2 indicate the direction of the horizontal axis. A. 2D schematic. BC cylinder represents double-stranded DNA helix and Fab fragment as targeting agent. D. The recessed targeting agent B1 will reduce the accessibility of antibody B1 in both cases where only one antigen RA1 or RA2 is present.

[0134] [Figure 4]Figure 4 shows schematic diagrams of exemplary DNA origami antibody platforms (gray cylinders represent DNA double helices) with Fab fragments (cyan, blue, purple, orange) attached as targeting agents. Rod-shaped (A, B, C, D), plate-shaped (E), and L-shaped (F) platforms for conditional synapse formation. The DNA origami platform may carry different numbers and valencies of first targeting agents (B1 and B2) for a first cell (e.g., immune cells). The DNA origami platform may carry different numbers and valencies of second targeting agents (A1, A2, and A3) for a second cell (e.g., cancer cells) at different locations. l indicates the direction of the vertical axis, and t1 and t2 indicate the direction of the horizontal axis. The second targeting agents (A1, A2, and A3) are positioned on the DNA origami platform to promote parallel orientation of the vertical axis to the cell surface of the second cell when all second targeting agents are bound to their target molecules.

[0135] [Diagram 5]FIG. 5 shows an exemplary attachment strategy for an exemplary nanostructure, e.g., a DNA-antibody conjugate. A. Part of a caDNAno diagram of a DNA origami object with a protruding single-stranded DNA attachment handle (left). The DNA sequence of the DNA-antibody conjugate (x is the position of the antibody) is complementary to the sequence of the attachment handle. By mixing the DNA origami object with the DNA-antibody conjugate (equimolar ratio, 1 hour incubation at 37° C.), the DNA handle on the antibody hybridizes to the attachment handle of the DNA origami (right). The sequence can be selected such that 0-5 bases (e.g., thymine) remain unpaired to improve or limit the range of spatial angles of the antibody (dotted lines in caDNAno diagram). B. Part of a caDNAno diagram of a DNA origami object with a binding site (i.e., a vacancy) for a single-stranded DNA oligonucleotide (left). The DNA sequence of the DNA-antibody conjugate (x is the position of the antibody) is complementary to the sequence of the backbone at the binding site. By mixing the DNA origami object with the DNA-antibody conjugate (equimolar ratio, 1 h incubation at 37 °C), the DNA handles on the antibody hybridize to the vacant holes of the DNA origami (right). The sequence can be chosen such that 0-5 bases (e.g. thymine) remain unpaired to improve or limit the range of spatial angles of the antibody (dotted line in caDNAno diagram).

[0136] [Figure 6]FIG. 6 shows two exemplary attachment positions of targeting agents (TA1, TA2, and TA3). A. The first targeting agent (TA1) is attached directly to the first surface of the nanostructure, e.g., the DNA origami platform, and the second and third targeting agents (TA2 and TA3) are attached directly to the second surface. l indicates the direction of the vertical axis, and t indicates the direction of the horizontal axis. B. The first targeting agent (TA1) is attached to the DNA origami platform at a surface adjacent to the first surface, e.g., the third surface or the fourth surface. For example, the attachment position may be close to the edge where the first surface and its adjacent surface, e.g., the third surface or the fourth surface, meet. This attachment strategy allows the first targeting agent to bind to the first target, e.g., immune cells, whose surface, e.g., the cell surface, is parallel to the first surface while providing a smaller target-target distance, e.g., a smaller cell-cell distance. The second and third targeting agents (TA2 and TA3) are attached to a surface adjacent to the second surface, e.g., the third or fourth surface, at positions close to the edge. The attachment positions shown in B may reduce the distance between the binding sites of the targeting agents and provide for close attachment of the DNA origami to the cell surface. Thus, providing a small distance between the first target and the second target, e.g., the first and second cells, while maintaining the targeting agents within the plane of the first or second surface, respectively. l indicates the direction of the vertical axis and t indicates the direction of the horizontal axis.

[0137] [Figure 7]FIG. 7 shows an exemplary nanostructure of the invention, e.g., a DNA origami platform with attached targeting agents (TA1, TA2, TA3) that are bound to epitopes of target molecules of a first target, e.g., a first target cell (top, immune cell) and a second target, e.g., a second target cell (bottom, target cell). The targeting agents are attached in a plane parallel and adjacent to the first and second surfaces of the DNA origami platform, but are not directly attached to the outer surface. This attachment strategy allows for improved orientation of the targeting agent when it is bound to an epitope of the target molecule. Thus, this attachment strategy allows for small inter-target distances, e.g., small inter-cell distances, the distance being limited only by the maximum lateral extension of the DNA origami platform. l indicates the direction of the vertical axis, and t indicates the direction of the horizontal axis.

[0138] [Figure 8-1]Figure 8 shows the scheme of three DNA origami cell connectors and their experimental validation. A. Side and top views of three rod-shaped DNA origami cell connector variant models (DNA helices in white, average predicted structure using oxDNA

[13] ) with attached targeting agents (Fab fragments) for immune cells (TA1) and for target cells (TA2 and TA3). l indicates the direction of the vertical axis, t1 and t2 indicate the direction of the horizontal axis. The three cell connector variants (short, medium, long) realize different TA2 to TA3 distances along the vertical axis. The immune cell-binding antibody (TA1) is shown to be centered along the vertical axis. The bars give the distance between the Fab attachment sites along the vertical axis. B. Average negative stain electron micrographs of the side views of the variants (scale bars represent 20 nm, left: short, center: medium, right: long). C. Images of 3.5% agarose gels electrophoresed with cy5 modified cell connector variants (left: short, center: medium, right: long) with different target cell antibody configurations (anti-human CD19 Fab fragments) and without immune cell antibodies. 1-1: 1 aCD19 Fab on the left and 1 aCD19 Fab on the right; 2-0: 2 aCD19 Fabs on the left; 1-0: 1 aCD19 Fab on the left; 0-0: no aCD19 Fab. D. Images of 3.5% agarose gels electrophoresed with cell connector variants (left: short, center: medium, right: long) with different target cell antibody configurations and with or without immune cell antibodies. + indicates the presence of anti-CD3 Fab fragment DNA conjugates in the assembly reaction, - indicates the absence of anti-CD3 Fab fragment DNA conjugates in the assembly reaction. [Figure 8-2] Same as above.

[0139] [Figure 9-1]Figure 9 shows how target cell binding configuration and cell connector length affect the binding affinity and lysis efficiency of target cells in a cytotoxic T cell assay. A. Schematic of the cell binding assay (cell connector variants on the left and NALM6 target cells on the right). The cell connector carried one fluorescent Cy5 dye (black star) attached in the center and anti-human CD19 Fab fragments of various configurations. Top (circle): one aCD19 Fab on the left and one aCD19 Fab on the right (variant 1-1); center (triangle): two aCD19 Fabs on the left (variant 2-0); bottom (grey square): one aCD19 Fab on the left (variant 1-0). B. Median normalized Cy5 fluorescence of NALM6 cells incubated for 1 h at 37 °C with Cy5-modified cell connector variants (short -26 nm, medium -55 nm, long -69 nm, control without aCD19 Fab fragment) in different concentrations of complete cell culture medium. Black circles indicate cell connector variant 1-1, grey triangles indicate cell connector variant 2-0, and light grey squares indicate cell connector variant 1-0. Error bars represent standard deviation from three biological replicates. C. Normalized median fluorescence signal obtained from flow cytometer experiments of incubation with 2500 pM cell connector plotted against cell connector length. Cell connector variant with a different aCD19 antibody configuration than in the DA case, but with anti-human CD3 Fab attached in the center (left). Schematic of the cytotoxic T cell assay (right) with cytotoxic T cells (peripheral blood mononuclear cells, PBMC) and CD19+ target NALM6 cells. E. Percentage of killed NALM6 target cells from cytotoxic T cell assays with different concentrations of cell connector variants. NALM6 target cells (1×105 cells / ml) were incubated with PBMCs (5×105 cells / ml) in complete cell culture medium with different cell connector concentrations and variants for 24 h at 37° C. The cell connectors carried anti-human CD19 Fab fragment(s) and one anti-human CD3 Fab fragment. Markers indicate cell connector variants as in D. The grey line represents the NALM6 and PBMC control without cell connector.The solid line is a Hill equation model fit to the data. Controls (right) are cell connector variants with two aCD19 Fab fragments (black diamonds), or one aCD3 Fab fragment (gray downward triangles), or no antibody (light gray star). F. The percentage of dead NALM6 target cells from incubation with 2500 pM cell connector was plotted against the length of the cell connector. GHI is as in D, E, F, but with anti-human EpCAM Fab fragments and with EpCAM+MCF7 target cells. The percentage of viable MCF7 target cells was determined by quantifying the amount of ATP after 48 hours with the Bio-Glo Cell Titer System (Promega). MCF7 target cells were cultured to form a confluent monolayer. MCF7 target cells were then co-cultured with unstimulated PBMCs at an effector to target ratio of 5:1 in complete cell culture medium with different amounts of cell connectors carrying anti-EpCAM Fab fragment(s) and one anti-human CD3 Fab fragment for 48 hours at 37° C. The grey line represents the control of MCF7 and PBMCs without cell connectors. As shown, the nucleic acid nanostructures of the present invention promote efficient lysis of target cells. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 9-7] Same as above. [Figure 9-8] Same as above. [Figure 9-9] Same as above.

[0140] [Figure 10-1]FIG. 10 shows a comparison of target and bystander cell lysis between trispecific DNA origami cell connectors and trispecific antibody control constructs. A. Schematic of T cell-mediated lysis of a double positive target cell (RA2-first target antigen, RA3-second target antigen) by trispecific antibody (center) and trispecific cell connector (right). B. Schematic of T cell-mediated lysis of a single positive bystander cell by trispecific antibody (center) and trispecific cell connector (right). The cell connector adopts a vertical orientation, thus inhibiting bystander lysis (indicated by cross arrow). C. Schematic of a small molecule trispecific control antibody and average predicted structure using oxDNA with Fab fragments. A control antibody construct was assembled from equimolar ratios of two ssDNA strands and three Fab fragment conjugates (anti-EpCAM, anti-PSMA, anti-CD3) in PBS according to FIG. 12. D. Schematic of a trispecific cell connector and average predicted structure using oxDNA with Fab fragments. The aCD3 Fab fragment was located on the left. The trispecific cell connector had an intermediate length (55 nm variant) and carried an anti-PSMA Fab fragment and an anti-CD3 Fab fragment on the left and an anti-EpCAM Fab fragment on the right. Target cell antibodies TA2 and TA3 were attached in a 1-1 configuration. E. Percentage of dead target (left) or bystander (center and right) cells from a cytotoxic T cell assay with a trispecific control antibody (grey triangles and lines) or a trispecific cell connector (black circles and lines) or a bispecific aCD3-aEpCAM cell connector (light grey diamonds and dashed lines) or a monospecific aCD3 cell connector (light grey squares and dotted lines) or a cell connector without antibody (light grey stars and dotted lines). Insets show the target cell model and the bystander cell model. Target cells: LNCaP (EpCAM+, PSMA+); Bystander cells 1: MCF7 (EpCAM+, PSMA-); Bystander cells 2: BT549 (EpCAM+, PSMA-); Horizontal dashed lines: target cells or bystander cells with PBMC only. EC50 fit parameters obtained from fitting a Hill equation model to the data shown in FE (solid lines).As demonstrated, the nucleic acid nanostructures of the present invention have reduced off-target effects compared to controls. In particular, the nucleic acid nanostructures promote efficient lysis of target cells and avoid off-target effects on non-target cells by promoting the formation of immune synapses only if the target cell presents both the second and third target molecules. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0141] [Figure 11-1] FIG. 11 shows the blueprints and antibody attachment sites of three cell connector variants (A, short variant; B, medium variant; C, long variant) and a trispecific antibody control construct (D). Blueprints and cross sections are exported from cadnano. Antibody attachment sites are shown as solid circles (TA2), dotted circles (TA1) and solid squares (TA3). The cell connector variants are assembled with a 1033 base long scaffold. The control antibody is assembled from an 86 base long scaffold strand, three antibody-DNA conjugates and one 26 base long ssDNA strand. The 3D model of the control antibody is the average predicted structure from oxDNA. Exemplary sequences used to provide the nanostructures of the invention are defined in SEQ ID NOs: 1-5. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0142] Reference will now be made to the following examples, which are given to illustrate but not to limit the invention. EXAMPLES

[0143] Working Example Example 1: Design and construction of multivalent and multispecific DNA origami antibody objects. DNA objects were designed and assembled according to DNA origami methods [5-7]. Antibodies were attached to the DNA objects by conjugating a DNA strand (e.g., 25 bases) to the antibody. Ion-exchange chromatography purified antibody-DNA conjugates were mixed with PEG-purified [8] DNA origami objects in equimolar ratios or with an excess of antibody-DNA-conjugate. Single-stranded DNA handles were placed at different positions on the surface of the DNA origami objects. These protruding DNA handles serve as antibody attachment sites and can be included in the folding reaction by the designer. Alternatively, antibodies were modified with sequences that are complementary to the backbone and thus directly bind to the DNA origami object. Finally, the DNA origami antibody constructs self-assemble by relying on DNA-DNA hybridization of the protruding or backbone strands with complementary antibody conjugate strands. The DNA origami antibody objects can be further purified or stabilized against nucleases and low ionic strength conditions according to established methods [9, 10].

[0144] Example 2: Protocol for attachment of antibody-DNA conjugates to DNA origami objects 1. Self-assembly and purification of DNA origami objects: Mix the backbone and staple strands (at least one staple strand per backbone strand), adjust the ionic strength (e.g., by adding MgCl2 to 20 mM), expose the mixture to a series of specific temperatures, and finally remove the remaining excess staple strands (e.g., using PEG precipitation, filtration, liquid chromatography). 2. Coupling a DNA strand to a targeting agent, such as an antibody. Each targeting agent, such as an antibody type, carries a DNA strand with a unique sequence. The DNA sequence is complementary to the DNA sequence protruding from the DNA origami object or to the sequence of the backbone DNA strand. 3. Mix the DNA origami object with DNA-antibody conjugate (equimolar ratio or excess of DNA-antibody-conjugate) and incubate at 37° C. for 1 hour. 4. Remove excess DNA-antibody-conjugate using, for example, PEG precipitation, filtration, liquid chromatography.

[0145] Example 3: Length of cell connector and composition of tumor-specific antibody determine lysis efficiency As described above (see, e.g., FIG. 1), the DNA origami cell connector assumes a parallel orientation to the surface of the target cell when both target cell-binding antibodies are bound to their corresponding antigens on the target cell. This parallel orientation shortens the distance between the immune-cell engaging paratope (e.g., anti-CD3 paratope) and the surface of the target cell, so that a functional cytolytic synapse can be formed between the immune cell and the target cell. Conversely, when only one of the target cell-binding antibodies is bound to their corresponding antigen, the DNA origami cell connector assumes a perpendicular orientation to the surface of the target cell. The accessibility of the immune cell-binding antibodies is reduced in the perpendicular orientation compared to the parallel orientation. Furthermore, the distance between the surface of the immune cell and the surface of the target cell is longer in the perpendicular orientation than in the parallel orientation. For these two reasons, separately or in combination, the perpendicular orientation prevents the initiation and / or formation of a functional cytolytic synapse between the immune cell and the target cell.

[0146] Without wishing to be bound by any theory, the inventors believe that in this context, the aspect ratio, i.e., the length ratio of the stretching of the DNA origami cell connector along the vertical and horizontal axes, determines its melting efficiency in parallel or perpendicular orientation. For example, a cell connector variant with a low aspect ratio, i.e., a short variant, has similar melting efficiency in parallel or perpendicular orientation. In contrast, a cell connector variant with a high aspect ratio, i.e., a long variant, has a high melting efficiency difference between parallel and perpendicular orientations.

[0147] To study this relationship, we created three cell connector variants with short, medium and long distances between the target cell binding antibodies. All variants have similar masses but different aspect ratios (Figure 8A). The short variant is a compact 18-helix bundle with a length (distance between Fab attachment sites) of 26 nm. The medium variant is an 8-helix bundle with a length of 55 nm. The long variant is a 6-helix bundle with a length of 69 nm. We designed, assembled and imaged these variants using negative staining electron microscopy (Figure 8B). The variants have designed shapes, sizes and aspect ratios. The left side of each cell connector variant can have no target cell binding antibodies attached, one or two target cell binding antibodies attached. The right side can have no target cell binding antibodies attached or one. Finally, an immune cell recruiting anti-CD3 antibody is placed in the middle.

[0148] To test whether the length of the cell connector affects the lysis efficiency in a parallel or perpendicular orientation, we constructed variants with three target cell-antibody configurations. In all three of these target cell-antibody configurations, the immune cell-binding antibody (anti-CD3 Fab fragment, clone UCHT1) was attached in the center of the plane parallel to the first surface and close to the first surface (Figure 8A). In the first target cell-antibody configuration, one target cell-binding antibody is attached on each side (one on the left and one on the right) of the plane parallel to the second surface and close to the second surface. This "1-1" variant mimics the case when the cell connector is bound to a double-positive target cell. In this 1-1 configuration, the cell connector can adopt a parallel orientation if both antibodies bind to their respective cell surface antigens. The second target cell antibody configuration is parallel to the second surface and has one target cell antibody on the left and no antibody on the right in the plane close to the second surface. This "1-0" variant mimics the case where the cell connector is bound to a single positive bystander cell (i.e., a cell that is not a target cell). In this 1-0 configuration, the cell connector can only assume a vertical orientation. The configuration of the third target cell antibody is parallel to the second surface, with two target cell antibodies on the left and no antibodies on the right, in a plane close to the second surface. This "2-0" variant serves as an artificial control that can only exist in a vertical orientation, but has similar binding affinity to the 1-1 variant. 2-0 and 1-1 have similar target cell binding affinity, since they carry the same number of target cell binding antibodies.

[0149] We used two different cell culture models to investigate these variants (CD19-positive NALM6 cells and EpCAM-positive MCF7 cells). First, we quantified the target cell binding affinity of each variant by attaching an anti-human CD19 Fab fragment (clone HIB19) to each construct (Figure 8C) and performed cell binding assays with CD19-positive NALM6 cells (Figure 9A-C). NALM6 cells were cultured with different cell connector variants carrying one fluorescent Cy5 dye at different cell connector concentrations and at 105 NALM6 cells / ml were incubated for 1 h at 37 °C in complete cell culture medium (Figure 9A). NALM6 cells were then washed, fixed, and their fluorescence was analyzed using flow cytometry. For all cell connector variants, the 2-0 configuration had the strongest fluorescent signal, followed by the 1-1 configuration (Figure 9B-C). Thus, both variants 1-1 and 2-0 have similar cell binding affinity.

[0150] To investigate how the length of the cell connector affects the lysis efficacy in each orientation, we performed a T cell-mediated cytotoxic lysis assay with cell connector variants bearing anti-CD3 Fab fragments and anti-CD19 Fab fragments (Figure 9D). Briefly, NALM6 target cells were co-cultured with unstimulated PBMCs at 37°C for 24 hours with different amounts of cell connectors carrying anti-CD19 Fab fragments and one anti-CD3 Fab fragment at an effector-to-target ratio of 5:1 in complete cell culture medium (Figure 9E). For all cell connector lengths, the 1-1 variant had higher lysis efficiency than the 2-0 or 1-0 variants. Since the 1-1 variant can adopt a parallel orientation and the 2-0 variant can only adopt a perpendicular orientation, we speculate that the parallel orientation results in improved target cell lysis compared to the 2-0 configuration. Indeed, even though the 2-0 variant has a higher cell binding affinity than the 1-1 variant, the 2-0 variant has a lower cell lysis efficiency. Moreover, the difference in target cell lysis efficiency between the 1-1 and 2-0 configurations increased with the length of the DNA origami cell connector (Figure 9F), thus further supporting the proposed mechanism that lysis efficiency depends on the orientation of the cell connector on the cell surface.

[0151] The 2-0 configuration served as a control to examine only the orientation effect on cell lysis efficiency, since it has a similar affinity as the 1-1 variant. On the other hand, the 1-0 variant more closely mimics the off-target reaction on single positive bystander cells. Here, the lysis efficiency is significantly reduced compared to the 1-1 variant, due to the smaller affinity and the orientation being perpendicular to the cell surface. Thus, single positive bystander cells are lysed with much lower efficiency, opening the therapeutic window.

[0152] To further confirm the mechanism of the cell connector, we constructed cell connector variants with different configurations of anti-human EpCAM Fab fragments (clone VU-1D9) and performed cytotoxic T cell assays with EpCAM-positive MCF7 cells. Briefly, MCF7 target cells were cultured to form confluent monolayers. MCF7 target cells were then co-cultured with unstimulated PBMCs for 48 h at 37 °C with different amounts of cell connectors carrying anti-EpCAM Fab fragments (Figure 9G) at an effector-to-target ratio of 5:1 in complete cell culture medium. Again, for all cell connector lengths, the 1-1 variant had similar or higher lysis efficiency than the 2-0 or 1-0 variants (Figure 9H). The lysis efficiency decreased in a length-dependent manner for the cell connectors 2-0 and 1-0, which can only adopt a perpendicular orientation (Figure 9I). In contrast, the lysis potency of the 1-1 variant, which can adopt a parallel orientation, was similar for all length variants. These results are consistent with the NALM6 data and further support the proposed mechanism in which lytic efficacy depends on the orientation of cellular connectors on the cell surface. Moreover, the strength of this effect may be antigen-, cell type-, and / or antibody-dependent.

[0153] As demonstrated, the nucleic acid nanostructure of the present invention allows for efficient lysis of target cells. Furthermore, the targeting agent configuration of the nanostructure, i.e., a first targeting agent present on a first surface, and a second targeting agent and a third targeting agent present on a second surface of the nanostructure opposite the first surface, allows for specific and selective targeting of target cells.

[0154] Example 4: Comparison of trispecific DNA origami cell connectors with trispecific antibodies As demonstrated (Figure 9), the DNA origami cell connector triggers T cell-mediated target cell lysis with high efficiency only when it is able to adopt a parallel orientation relative to the cell surface. Thus, the nucleic acid nanostructure of the present invention, comprising a first surface with at least a first targeting agent and a second surface with at least a second targeting agent and at least a third targeting agent, is highly advantageous in promoting highly efficient lysis of target cells and preventing off-target lysis. By attaching different target cell binding antibodies on each side, the cell connector can be programmed to adopt a parallel orientation only when both antigens are present on the target cell (Figure 10A). Thus, only cells expressing both corresponding antigens, i.e., double-positive target cells, are lysed with high efficiency. Cells expressing only one of the antigens, i.e., single-positive bystander cells, are not lysed or are lysed with reduced efficiency due to the inability of the cell connector to adopt a stable parallel orientation (Figure 10B). The nucleic acid nanostructure of the present invention is thereby highly selective and specific in promoting lysis of target cells. To demonstrate this antigen pattern-dependent lysis mechanism, we constructed a cell connector carrying both anti-human PSMA Fab fragment (clone GCP-05) and anti-human EpCAM Fab fragment in a 1-1 configuration (Figure 10D). The cell connector carried anti-human PSMA on the left and anti-human EpCAM Fab fragment on the right, both in the plane parallel to the second surface and close to the second surface. In addition, the cell connector carried anti-human CD3 Fab fragment on the left. In addition, we assembled a trispecific control antibody from anti-EpCAM, anti-PSMA, and anti-CD3 Fab-DNA conjugates (Figure 10C and Figure 12). This control antibody mimics the function of a conventional trispecific antibody without a rigid DNA origami carrier object. The three Fab fragments are connected via a single DNA helix and a three-thymine linker, which is more flexibly connected than in the DNA origami cell connector and also has a shorter distance from each other (EpCAM-PSMA distance is approximately 9 nm for the control antibody versus 55 nm for the DNA origami cell connector).EpCAM-positive LNCaP cells and PSMA-positive LNCaP cells serve as double-positive target cells, while EpCAM-positive MCF7 cells and PSMA-negative MCF7 cells or EpCAM-positive BT549 cells and PSMA-negative BT549 cells serve as single-positive bystander cells. Both the trispecific control construct and the DNA origami cell connectors cause efficient T cell-mediated cell lysis with similar EC50 values ​​against double-positive target cells (Figure 10E). Furthermore, the trispecific control construct also causes high bystander cell lysis (EC50 of 235 pM for MCF7 and 800 pM for BT549). In contrast, the trispecific DNA origami cell connectors cause bystander cell lysis only at significantly higher concentrations (EC50 of 2325 pM for MCF7 and 13 nM for BT549). Thus, the placement of target cell-binding Fab fragments on DNA origami cell connectors reduces bystander lysis while maintaining high on-target lysis efficiency. Thus, the nucleic acid nanostructures of the present invention promote lysis of target cells with high specificity and selectivity.

[0155] Example 5: Methods Assembly of cell connectors: The reaction mixture contained the 1033 base long backbone DNA at a concentration of 50 nM and the oligonucleotide strands at 200 nM each. The reaction buffer contained 5 mM TRIS, 1 mM EDTA, 5 mM NaCl (pH 8) and 20 mM MgCl2. The reaction mixture was subjected to a thermal annealing ramp using a TETRAD (MJ Research, now Biorad) thermal cycling device. All variants were first subjected to 65 °C for 15 min. The short variants were annealed from 54 °C to 51 °C at a rate of 2 h / °C. The medium and long variants were annealed from 58 °C to 55 °C at a rate of 1 h / °C. The cell connectors were purified using size exclusion filtration through 50k Amicon filters (see [7]). The purified cell connectors were incubated with equimolar or binding site-to-conjugate ratios of Fab fragment-DNA conjugates in PBS with 15 mM MgCl2 for 1 h at 37° C. Finally, the cell connector-Fab fragment assemblies were stabilized using 5k-PEG-lysine 10 as described in [9].

[0156] Assembly of trispecific control antibody: The trispecific control antibody was assembled from the 86-mer backbone at 100 nM in PBS, staples and Fab-DNA conjugates at 150 nM for 1 h at 37° C. The construct was then stabilized with 5k-PEG-lysine 10 according to [9].

[0157] Cytotoxic T cell assay with NALM6 target cells: Target cells (NALM6) were fluorescently stained using the CellTrace CSFE Cell Proliferation Kit (Thermo Fisher, C34554) according to the manufacturer's protocol. For the cytotoxic T cell killing assay, frozen human peripheral blood mononuclear cells (PBMC, Stemcell, 70025.1) were used. PBMC cells were handled according to the supplier's instructions. 2 × 10 per ml 5 Culture CSFE-stained target cells at 1 × 10 in cell culture medium at 37 °C (5% CO2). 6PBMC / ml and various concentrations of cell connectors were incubated with or without additional cell connectors. After 24 h, the cell mixture was stained with anti-CD8-APC and anti-CD69-PerCP-eFluor710 for 20 min at 4 °C. Cell fluorescence and scatter intensity were then determined using an Attune Nxt flow cytometer (ThermoFisher) equipped with a Cytkick Max autosampler. NALM6 target cells were gated for CSFE signal. Dead and live NALM6 cells were identified based on their distinct populations in the FSC-SSC scatter plot. The percentage of dead target cells was calculated as dead NALM6 cells / (dead NALM6 cells + live NALM6 cells).

[0158] Cytotoxic T cell assays using MCF7, BT549 or LNCaP target cells: MCF7, BT549 or LNCaP target cells were cultured to form a confluent monolayer. Cells were then co-cultured with unstimulated PBMCs at a 5:1 effector-to-target ratio in complete cell culture medium with different amounts of cell connectors for 48 h at 37 °C (5% CO2). After 48 h, cell culture supernatant was removed and adherent cells were washed with calcium- and magnesium-containing PBS. Luciferase assays were performed on the remaining cells using the Bio-Glo Cell Titer System (Promega), which contains luciferase and luciferin in the lysis buffer, according to the manufacturer's protocol. This allowed the amount of remaining ATP, and therefore the number of viable cells, to be quantified. Luminescence signals were measured using a Clariostar plate reader. The buffer-only signal was subtracted from each sample. The percentage of viable cells was calculated by normalizing the luminescence intensity to the luminescence intensity of samples containing only target cells and PBMCs.

[0159] References [ka] [ka]

[0160] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. 1. A nucleic acid nanostructure comprising a first surface and a second surface, the first surface and the second surface being located on opposite sides of the nanostructure, the first surface comprising at least a first targeting agent, and the second surface comprising at least a second targeting agent and at least a third targeting agent.

2. 2. The nucleic acid nanostructure of claim 1, wherein the nanostructure has a longitudinal axis and a transverse axis, and the first surface and the second surface are located on opposite sides along the longitudinal axis.

3. the nanostructures have a maximum longitudinal extension along the longitudinal axis that is greater than a maximum lateral extension along the transverse axis, preferably greater than the synaptic distance, more preferably at least greater than 10 nm, e.g. at least 25 nm; and / or 3. The nucleic acid nanostructure of claim 1 or 2, wherein the nanostructure has a maximum lateral extension along the horizontal axis that is less than a maximum longitudinal extension along the vertical axis, preferably less than the synaptic distance, more preferably less than 25 nm, for example 10 nm or less.

4. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the first targeting agent is configured to bind to a first target, preferably a first target molecule on a first target cell, more preferably an immune cell; the second targeting agent is configured to bind to a second target, preferably a second target cell, more preferably a second target molecule on a diseased cell; and the third targeting agent is configured to bind to a third target molecule on the second target, preferably a second target cell, more preferably a diseased cell.

5. The first targeting agent, the second targeting agent and the third targeting agent are selected from the group consisting of an antibody or an antigen-binding fragment thereof, an antigen-binding peptide, a Fab fragment, an F(ab') ２ Fragments, Fv fragments, diabodies, single chain Fv fragments, (scFv) ２, a tetrabody, a triabody, a disulfide bond stabilized Fv (dsFv), an Fc domain, an engineered Fc domain, a DNA aptamer, an RNA aptamer, a peptide nucleic acid (PNA), a polypeptide, a peptide, a glycoprotein, a peptidomimetic, anticalins, affilins, affimers, affitins, alphabodies, nanobodies, DARPins, receptor ligands, receptors or fragments thereof, and receptor domains; Preferably, the antibody or antigen-binding fragment thereof, antigen-binding peptide, Fab fragment, F(ab') ２ Fragments, Fv fragments, diabodies, single chain Fv fragments, (scFv) ２ , a tetrabody, a triabody, a disulfide bond stabilized Fv (dsFv), an Fc domain, an engineered Fc domain, a polypeptide, a peptide, a glycoprotein, a peptidomimetic, anticalins, affilins, affimers, affitins, alphabodies, nanobodies, DARPins, receptor ligands, receptors or fragments thereof, and receptor domains.

6. the second targeting agent and the third targeting agent are spaced apart from each other by a distance along the longitudinal axis that is preferably greater than the synaptic distance, for example by a distance of at least 15 nm, preferably at least 20 nm, more preferably at least 40 nm; and / or the nanostructure is a rigid nanostructure, and / or a nanostructure without a hinge region, and / or a nanostructure having only one configuration, and / or a nanostructure that comprises or is a DNA origami, the DNA origami having only one configuration, and / or 10. A nucleic acid nanostructure according to any one of the preceding claims, wherein the nanostructure comprises a maximum length, the maximum length being shorter than 1000 nm, preferably shorter than 500 nm, e.g. 100 nm, preferably the maximum length being the length of the maximum longitudinal extension.

7. said first target cell is an immune cell, preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells, CAR-T cells, monocytes, macrophages and neutrophils, more preferably selected from the group consisting of lymphocytes, such as T cells, cytotoxic T cells, B cells, natural killer cells, natural killer T cells and CAR-T cells; and / or said second target cell is a diseased cell, preferably selected from a cancer cell, a tumor cell, a cell involved in an autoimmune response, and an infected cell, e.g. a cell infected with a virus, mycoplasma, bacteria or parasite; 2. A nucleic acid nanostructure according to any one of the preceding claims.

8. said first target molecule is a surface molecule of an immune cell, preferably selected from CD3, CD3 delta / epsilon, CD3 gamma / epsilon, TCR, TCR alpha, TCR beta, CD2, CD5, CD28, OX40, 4-1BB, CD16, Ly49, NKp30 (CD337), NKp44 (CD336), NKp46 (NCR1), CD3 zeta (CD247), CD27, CD40, CD137, CD64, CD89, toll-like receptors (TLRs), cytokine receptors, Fc domains, engineered Fc domains, GITR and ICOS; and / or The second target molecule and the third target molecule are CD2, CD7, CD10, CD13, CD15, CD19, CD24, CD28, CD29, CD33, CD34, CD38, CD44, CD45, CD49f, CD56, CD57, CD60a, CD66 / CEA, CD79a, CD117, CD123, CD138, CD140b, CD227 / MUC1, CD243 / MDR, CD244, CD326 / EpCAM, CD340 / HER2, VEGF-R, EGFR, CSPG4 / MCSP, MAG, CA125, PSMA, HLA-DR, carbonic anhydrase 9, aquaporin, PSMA, TIM3, CLL1 / CLEC12A, EGFR v3 and HLA-A2, preferably said second target molecule and said third target molecule are not identical, and / or The second target molecule and the third target molecule are combinations of target molecules, and the combinations are selected from the group consisting of CD33 and CD123, CD326 / EpCAM and CD10, CD326 / EpCAM and CD340 / HER2, CD326 / EpCAM and VEGF-R, CD326 / EpCAM and EGFR, CD326 / EpCAM and CD243 / MDR, CSPG4 / MCSP and CD326 / EpCAM, CSPG4 / M CSP and MAG, CA125 and CD227 / MUC1, CA125 and CD227 / MUC1, CD56 and CD140b, CD56 and CD60a, EGFR and CD340 / HER2, PSMA and CD340 / HER2, CD15 and EGFR, CD44 and CD117, CD44 and CD326 / EpCAM, CD34 and CD19, CD34 and CD79a, CD34 and CD2, CD34 and CD 7, CD34 and HLA-DR, CD34 and CD13, CD34 and CD117, CD34 and CD33, CD34 and CD15, CD33 and CD19, CD33 and CD79a, CD33 and CD2, CD33 and CD7, CD33 and HLA-DR, CD33 and CD13, CD33 and CD117, CD33 and CD15, CD227 / MUC1 and CD10, CD227 / MUC1 and CD66 / 2. The nucleic acid nanostructure of any one of the preceding claims, selected from CEA, CD227 / MUC1 and CD57, CD38 and CD138, CD24 and CD29, CD24 and CD49f, carbonic anhydrase 9 and aquaporin, CD19 and CD33, CD19 and CD22, CD28 and PSMA, CD227 / MUC1 and EGFR, CD33 and TIM3, CD123 and TIM3, CLL1 / CLEC12A and TIM3, CD244 and TIM3, CD33 and EGFR v3, CLL1 / CLEC12A and EGFR v3, CD123 and EGFR v3, and CD45 and HLA-A2.

9. the nucleic acid comprises or consists of DNA, and / or the nanostructure comprises or is a DNA origami structure; The DNA origami structure includes at least one backbone strand, the DNA origami structure further comprises a plurality of single-stranded oligonucleotide staple strands; each staple strand is at least partially complementary to at least one backbone strand; 2. The nucleic acid nanostructure of any one of the preceding claims, wherein each of the staple strands is configured to bind to at least one of the at least one backbone strands in at least one location, preferably two or more distinct locations, and wherein the at least one backbone strand is folded and / or arranged to form the desired nanostructure.

10. the nanostructures comprise recesses, preferably recesses oriented perpendicular to the maximum longitudinal extension; 2. The nucleic acid nanostructure of any one of the preceding claims, preferably wherein the first targeting agent is bound to the nanostructure via the recess and / or at least a portion of the first targeting agent is located within the recess.

11. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the nanostructure further comprises an active agent, preferably a drug, and / or a marker, such as an imaging marker, wherein the active agent and / or marker is optionally attached to the nanostructure via a linker.

12. A composition, preferably a pharmaceutical composition, comprising the nucleic acid nanostructure of any one of claims 1 to 11, optionally further comprising a pharma- ceutically acceptable excipient.

13. A composition comprising a nanostructure according to any one of claims 1 to 11 or a composition according to claim 12 for use in medicine.

14. 1. A method for preventing or treating a disease selected from a proliferative disease, e.g., cancer, an immune disorder, e.g., an autoimmune disease, an infectious disorder, e.g., a viral disease such as human immunodeficiency virus (HIV) infection, a metabolic disorder, and / or diabetes, comprising: The method preferably comprises using the nanostructure to bind a first target cell and a second target cell; preferably only when said first, second and third targeting agents bind to their respective target molecules, more preferably inducing, promoting, stabilizing and / or inhibiting the formation of a synapse; Even more preferably, a composition comprising the nanostructure of any one of claims 1 to 11 or the composition of claim 12 for use in a method comprising binding a first target cell and a second target cell using the nanostructure such that as a result of the binding, the first target cell and the second target cell have a distance of 25 nm or less, preferably 15 nm or less.

15. A method for preparing a nanostructure according to any one of claims 1 to 11, comprising the steps of: i) providing a nucleic acid nanostructure, preferably a rigid nucleic acid nanostructure; ii) providing a first targeting agent, a second targeting agent, and a third targeting agent, each of said targeting agents being conjugated to a nucleic acid strand complementary to a nucleic acid strand of said nanostructure, e.g., a backbone strand or a DNA handle strand; and iii) obtaining, preferably by self-assembly of said nanostructure, a nucleic acid nanostructure comprising said first targeting agent, said second targeting agent, and said third targeting agent.

16. A composition comprising the nanostructure of any one of claims 1 to 11 or the composition of claim 12 for use in a method for binding a first target and a second target, comprising: preferably for inducing, promoting, stabilising and / or inhibiting the formation of a synapse between a first target cell and a second target cell, preferably only when said first, second and third targeting agents are bound to their respective target molecules; More preferably, the method comprises using the nanostructure to bind the first target, e.g., a first target cell, to the second target, e.g., a second target cell, such that as a result of the binding, the first target and the second target have a distance of 25 nm or less, preferably 15 nm or less.

17. Use of the nanostructure of any one of claims 1 to 11 or the composition of claim 12 for binding a first target and a second target, comprising: use for inducing, promoting, stabilising and / or inhibiting the formation of a synapse between a first target cell and a second target cell, preferably only when said first, second and third targeting agents are bound to their respective target molecules, More preferably, the method comprises binding said first target, e.g. a first target cell, to said second target, e.g. a second target cell, using said nanostructure such that as a result of said binding, said first target and said second target have a distance of 25 nm or less, preferably 15 nm or less; The use is an in vitro or ex vivo use; use.

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