Aptamer-based CAR T-cell switch

Abstract

Aptamer-based switch technology provides enhanced control over the use of chimeric antigen receptor (CAR)-associated immunotherapies. Aptamer-based switches utilize synthetic bridge molecules containing a target-binding aptamer linked to a CAR-binding aptamer via a linker. Systems containing a CAR and a corresponding aptamer bridge can (i) target any desired antigen by selecting the target-binding aptamer of the bridge; (ii) redirect from one target to another by changing the target-binding aptamer; (iii) be administered according to the changing needs of an individual patient over time by changing the administration protocol of the bridge; (iv) be switched on or off rapidly or gradually; (v) be used as a companion diagnostic for a specific CAR therapy; (vi) be integrated with in vitro or in vivo CAR expression; (vii) be non-immunogenic; and (viii) provide an immunotherapy platform with low production costs.

Description

[Technical field]

[0001] The present invention relates to aptamer-based switch technology that enhances control over the use of chimeric antigen receptor (CAR)-associated immunotherapies. [Background technology]

[0002] Chimeric antigen receptors (CARs) utilize single-chain variable domain (scFv) antibodies to endow a patient's T cells with the ability to recognize and kill cancer cells. However, despite early successful applications, the use of CAR T cells is complicated by the difficulty of controlling immune responses in vivo, which can result in harmful excess release of cytokines and unwanted toxicity. Furthermore, off-target reactions can occur and antigen escape by tumors may necessitate a next round of CAR T-cell therapy with altered antigen specificity. Thus, strategies must be developed to enhance the interaction to increase specificity and redirect the CAR to new antigens or develop an "off" or "kill" switch to inhibit or terminate CAR T-cell therapy.

[0003] Switchable CAR T cell approaches have been developed that allow for the administration to patients of an adaptable molecular switch that mediates the interaction between CAR T-cells and target cells. In the absence of the switch, no CAR-mediated immune response occurs. For example, a kill switch can be used to eliminate CAR-T cells and prevent toxicity (Straathof et al., Di Stasi et al.). Another approach utilizes split chimeric receptors that bind in the presence of a small molecule switch (Wu et al.). Antibody-based switches have also been developed to mediate the interaction between CAR and target (Tamada et al., Urbanska et al. (2012a), Urbanska et al. (2012b)). In a variant of the antibody switch technology, a target-specific antibody can be integrated with a peptide neoepitope (PNE) that binds to the scFv portion of the CAR (Rodgers et al.). The PNE is distinct from the endogenous epitope, allowing for a modular switch approach in which new switches can be designed by combining a single PNE and anti-PNE CAR with different target-specific antibodies. Summary of the Invention [Problem to be solved by the invention]

[0004] However, this approach still requires complex engineering of the antibody molecule and raises the possibility of immune reactions against the switch, especially if the scFv is not fully humanized. [Means for solving the problem]

[0005] The present technology provides an aptamer-based switch technology that creates a high degree of control in implementing chimeric antigen receptor (CAR)-related immunotherapy. At its most basic level, the present technology's aptamer-based switch, or aptamer bridge, utilizes a synthetic bridge molecule that contains a target-binding aptamer attached to a CAR-binding aptamer via a linker. The system containing the CAR and the corresponding aptamer bridge provides an immunotherapy platform that (i) can be targeted to any desired antigen by selecting the target-binding aptamer of the bridge, (ii) can be redirected from one target to another by changing the target-binding aptamer, (iii) can be dosed according to the changing needs of an individual patient over time by changing the administration protocol of the bridge, (iv) can be switched on or off quickly or gradually, (v) can be used as a companion diagnostic for a particular CAR therapy, (vi) can be integrated with CAR expression either in vivo or ex vivo, and (vii) is non-immunogenic. (viii) It has low manufacturing costs.

[0006] The present technology provides cell redirecting aptamers (e.g., bispecific or multivalent aptamers) that can be used as aptamer bridges in aptamer-based CAR immunotherapy systems, as well as for in vivo or ex vivo genetic modification of cells. The aptamer bridges, cells, kits, and methods of the present technology can be used in a wide variety of applications, including as immunotherapies for the treatment of cancer (e.g., hematological or non-hematological, individual cell or solid tumors), autoimmune diseases (e.g., arthritis, myasthenia gravis, pemphigus), neuroinflammatory diseases, eye diseases, neurodegenerative diseases (e.g., ALS, Huntington's disease, Alzheimer's disease), neuromuscular diseases (including Duchenne muscular dystrophy, SMA), infectious diseases (e.g., HIV, HSV, HPV, HBV, Ebola, tuberculosis, cryptococcus), and metabolic diseases (e.g., type 1 diabetes). They can also be used to provide diagnostics, kits, and methods for use in such immunotherapy, including imaging, analysis of cell trafficking, and research and development of new immunotherapies, as well as to provide prophylaxis when combined with stem cell therapy (e.g., HSCT).

[0007] As used herein, a "chimeric antigen receptor cell" or "CAR cell" is a cell (e.g., T cell, NK cell, monocyte, or other) that has been genetically engineered ex vivo or in vivo to express a single-chain variable domain (scFv) antibody fused via a stalk or transmembrane domain to an intracellular domain of a receptor (such as CD3-TCR), endowing the cell with the ability to recognize and bind to one or more specific antigens and activate a cellular immune response (e.g., the ability to kill cancer cells or destroy cells infected with a virus).

[0008] As used herein, "antigen loss" or "antigen escape" can refer to any of several mechanisms of resistance or adaptation to immunotherapy, such as downregulation of tumor antigens or upregulation of inhibitory ligands (e.g., PD-L1, TIM3, LAG3), which may contribute to CAR-T cell failure, inability of the CAR cell to reach its target (e.g., tumor site), immunity to the antibody portion of the CAR (e.g., T cell response to scFv, especially if it is not fully humanized), fitness of the CAR-T cell (i.e., reduced memory self-renewal potential and increased tendency for attrition), or splicing or mutation of the antigen.

[0009] Candidate aptamers are a set of nucleic acids with different sequences from which the desired aptamer is selected. The source of candidate aptamers can be naturally occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids, or nucleic acids produced by a combination of the aforementioned techniques. Nucleic acids for use in the present technology can be, for example, DNA, RNA, or XNA (foreign nucleic acid) molecules, including chemical modifications thereof, and can be either single-stranded, double-stranded, or a mixture of single- and double-stranded.

[0010] The target is a structural unit, usually a component of a biological entity, that binds to the aptamer. The target can be a molecule, such as a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, drug, nutrient, growth factor, etc. The target can be a cell or part of a cell, such as a T cell, NK cell, monocyte, B cell, tumor cell, or a pathologically altered cell (e.g., altered by a virus or other microorganism, or due to genetic rearrangement). The target can also be an organ or organelle.

[0011] Binding affinity refers to the strength of interaction between two or more structures that bind reversibly to each other, such as a ligand and a receptor. Binding affinity is measured by the on-rate (k on ) and off-rate (k off) and is a measure of the concentration dependence of binding, or the Gibbs free energy of dissociation (ΔG D ) from the determination of the dissociation constant (K d ) or the association constant (K A ) can be measured or estimated by any known method, including the determination of 。 Increased binding affinity results from stronger intermolecular forces between interacting structures (e.g., ligand and receptor), resulting in increased residence time at the binding site. Ligands exhibiting higher binding affinity may have higher "on" rates and / or lower "off" rates. Increased binding affinity may be advantageous in therapeutic products, since full receptor activity is achieved at lower concentrations of ligand. Binding affinity may be determined by the collective strength of the multiple affinities of the individual non-covalent interactions in a multivalent structure. The overall binding affinity of a multivalent ligand depends on its individual binding affinities, the valency of both the ligand and receptor, and the configuration of the interacting structures. However, the overall binding affinity of a multimeric construct is not simply the average of the individual binding affinities of the individual aptamers. Factors that affect the affinity of a multimeric structure include the stereochemical fit between the ligand and receptor, the size of the contact area between them, and the distribution of charged and hydrophobic groups.

[0012] A safety profile refers to a collection of side effects associated with a drug, treatment, or intervention and the likelihood of such side effects. A better safety profile may result from less frequent and / or less severe side effects and / or adverse events associated with the same level of therapeutic efficacy. A better safety profile may be associated with an improved therapeutic index (TI), i.e., a larger safety window between the minimum effective dose and the maximum tolerated dose.

[0013] An immune response refers to the induction of humoral and / or cell-mediated responses specific to an antigen. The humoral component may include the production of antibodies specific to the antigen, while the cell-mediated component may include delayed-type hypersensitivity to the antigen and the generation of cytotoxic effector cells. Immune cells participate in the immune response in a variety of ways, by direct interaction with the antigen, interaction with other cells of the immune system, and release and / or react with cytokines.

[0014] Activation or stimulation of the immune system is mediated by activation of immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), and cytotoxic T lymphocytes (CTLs). It can be mediated by activation and maturation of antigen-presenting cells, such as dendritic cells. It can also be mediated by blocking inhibitory pathways, such as by suppressing immune checkpoint molecules. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a schematic diagram of some embodiments of the aptamer bridges of the present technology. [Diagram 2] Figure 2 shows a schematic diagram of the system and method for CAR T cell therapy using in vivo expression of the CAR platform together with aptamer bridge-mediated targeting. [Diagram 3] FIG. 3 is a schematic diagram of a method of target redirection according to the present technology. [Figure 4] FIG. 4 is a sequence listing of peptide neoantigens and monomeric aptamers. [Diagram 5] FIG. 5 shows some predicted secondary structures of monomeric aptamers. [Figure 6] FIG. 6 shows a click chemistry reaction scheme for linking aptamers. [Figure 7A] FIG. 7A shows binding of anti-PSMA aptamers to cells that do and do not express the respective antigens. [Figure 7B] FIG. 7B shows binding of anti-CD3 aptamers to cells that do and do not express the respective antigens. [Figure 8A] FIG. 8A shows an agarose gel of monomeric and dimeric (bispecific) aptamers. [Figure 8B] FIG. 8B also shows an agarose gel of monomeric and dimeric (bispecific) aptamers. [Figure 9] FIG. 9 shows the time course of RNA aptamers in serum. [Figure 10] Figures 10A-B show the binding affinity of the bispecific aptamers to PSMA positive and negative cells. [Figure 11] Figures 11A-B show the binding affinity of the bispecific aptamers to CD3 positive and negative cells. [Figure 12] FIG. 12 shows the cytotoxicity of bispecific aptamers against PSMA-positive cells. [Figure 13] Figure 13A shows a sequence alignment of several anti-PNE RNA aptamers, and Figure 13B shows the predicted secondary structures of two RNA aptamers from 13A. [Figure 14] Figures 14A-C show binding of the anti-CAR PNE aptamer to cells that do not express the receptor, the CD19 CAR, or the PNE CAR. [Figure 15] FIG. 15 shows the stability of the anti-CAR PNE RNA aptamer in serum. [Figure 16] Figure 16 shows the binding of anti-CAR PNE aptamers to peripheral blood mononuclear cells (PBMCs) transduced with CAR-PNE. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description The present technology provides aptamer-based switches for use in immunotherapy, such as CAR T-cell therapy. Aptamer switches act as a physical bridge between CAR-expressing immune cells and target cells, such as cancer cells or cells of a pathogen. However, aptamer switches or bridges also serve as agile and rapidly adaptable tools to modulate the specificity and strength of immune responses. Because both their CAR-binding and target-binding functions are performed by aptamers, aptamer bridges can be rapidly selected and adapted to the evolving needs of patients in vitro at a rapid pace and at a low cost that antibody-based approaches cannot accommodate. Furthermore, the present technology's aptamer bridges can be combined with direct injection of CAR-encoding vectors and in vivo transduction of selected immune cells and expression of the CAR (i.e., transduction within the body of the immunotherapy subject), for example, from polymer-coated viral vector particles, resulting in advanced immunotherapy with additional time and cost savings compared to traditional ex vivo transduction and expansion of CAR T cells.

[0017] The cell redirecting aptamer technology (e.g., bispecific or multivalent binding) and the aptamer bridge of the present technology, as well as the systems and kits containing them, can be used in a variety of applications, including autologous or heterologous cancer therapy and immunotherapy. The cell redirecting aptamer technology, aptamer bridge, aptamer-based CAR immunotherapy system, kit, and method of the present technology can be employed in a wide variety of immunotherapies, including the treatment of cancer (e.g., hematological or non-hematological), individual cells or solid tumors), autoimmune diseases (e.g., arthritis, myasthenia gravis, pemphigus), neuroinflammatory diseases, eye diseases, neurodegenerative diseases (e.g., ALS, Huntington's disease, Alzheimer's disease), neuromuscular diseases (Duchenne's disease, SMA), infectious diseases (e.g., HIV, HSV, HPV, HBV, Ebola, tuberculosis, cryptococcus), and metabolic diseases (e.g., type 1 diabetes). They can also be used to provide diagnostics, kits, and methods for use in such immunotherapy, including imaging, analysis of cell trafficking, and research and development of new immunotherapies, as well as to provide prophylaxis when combined with stem cell therapy (e.g., HSCT).

[0018] Aptamer bridges can enhance immunotherapy by improving the quality and strength of immune responses, such as cell-mediated immune responses to cancer and infectious diseases. In addition, aptamer bridges can be used to target a universal CAR to many different targets or to redirect an immune response to a different target. Aptamer bridges can also be used to tune the strength of an immune response, to turn a response on or off simply by administering aptamer bridges for different targets or by adjusting the dose or administration schedule of the aptamer bridge over time. A further use of aptamer bridges is to convert inhibitory ligands, such as PDL-1, into immune stimulatory effects, i.e., converting an inhibitory ligand into a CAR-binding ligand by replacing the target-binding aptamer of the aptamer bridge with an aptamer that binds to the inhibitory ligand.

[0019] CAR therapy by this technology typically begins with the introduction of CAR-expressing cells into a subject in need of immunotherapy. CAR cells can be obtained ex vivo, for example by transducing cells removed from the subject with a viral vector or by transducing them with a plasmid or transposon, so that the CAR is expressed on the cells, followed by growing the cells in culture and introducing the grown cells into the subject, for example by intravenous injection. Alternatively, a viral vector, such as a lentiviral vector, encoding the CAR can be administered to the subject, for example by intravenous injection, so that the CAR is expressed in the desired immune cells (such as autologous or allogeneic T cells, NK cells, B cells, monocytes, macrophages, or dendritic cells). Either way, once a baseline of CAR-expressing cells is established in the subject's body, the CAR cells will expand when activated by binding a target to the scFv portion of the CAR.

[0020] Administration of a suitable aptamer bridge to a subject containing a sufficient number of CAR cells will cause an immune response against the target specified by the target-binding aptamer of the bridge when the CAR-binding aptamer of the bridge binds to the CAR. The strength of the resulting immune response can vary over time and is a function of the number of CAR cells in the subject, the access of the CAR cells to the target, and the concentration of the aptamer bridge at the target. The strength of the immune response can be adjusted up or down by adjusting the number of CAR cells and / or by increasing or decreasing the amount of bridge administered, or by adjusting the concentration of the bridge by shortening or increasing the time interval between doses of the bridge administered.

[0021] In the treatment of the present technology, prior to administering the aptamer bridge to initiate immunotherapy, the total amount of CAR cells in the subject is estimated, and then an administration protocol (e.g., dosage, number of doses, and / or interval between doses) is designed (e.g., using an algorithm) to generate the desired strength and / or duration of the immune response. The strength of the immune response can be monitored using known methods (e.g., fluorescence-activated cell sorting (FACS) analysis) to quantify the number and type of specific activated immune cells in a blood sample from the patient, or by determining the levels of specific cytokines in such a sample. If the immune response is too strong and may pose a risk to the patient, the response can be reduced by reducing the dosage or frequency of administration of the aptamer bridge, stopping it altogether, or administering a "kill" switch, including: That is, a single CAR-binding aptamer (that interferes with binding of the aptamer bridge to the CAR), a peptide or antibody that binds to the CAR and interferes with binding of the aptamer bridge, a single target-binding aptamer (that interferes with binding of the aptamer bridge to the target), or a soluble domain or epitope from the CAR or the target antigen.

[0022] The aptamer bridge of the present technology can be used to redirect CAR cells to another target. This can be useful in cases of antigen escape by the first target, or a weak response to the first target, requiring the pursuit of a different or additional target. In one embodiment of this method, a different (e.g., second or subsequent) aptamer bridge is administered to the subject, which has the same or similar CAR-binding aptamer, but has a different target-binding aptamer than the first bridge. In another embodiment, a conventional CAR that is directly targeted to the first target (i.e., without using a CAR switch) is expressed on the subject's cells, and an aptamer bridge is administered to the subject to redirect the CAR response to a different (second or more) target. The aptamer bridge includes a CAR-binding aptamer specific to the target-binding CAR and a target-binding aptamer, where the two aptamers are linked by a linker. This approach can be used several times (e.g., 2, 3, 4, 5, or more) in succession or simultaneously with different targets in the same subject to tailor immunotherapy or find the optimal target or combination of targets. One embodiment of the aptamer bridge of the present technology is coupled to a detectable label for use in imaging or quantifying either the CAR cells or the target in a subject. For example, the bridge may be attached to a linker or to either the CAR-binding aptamer or the target-binding aptamer for use in PET scanning. 18 The F moiety can be included. The same method can be performed using the individual aptamers (either CAR-binding or target-binding) used in the aptamer bridge for treatment. This method can be used, for example, to quantify a subject's CAR cells before or during treatment.

[0023] The aptamer bridge of the present technology contains two or more aptamers covalently or non-covalently linked by a linking moiety. The two or more aptamers form a CAR-binding moiety and a target-binding moiety, each of which contains one or more aptamers. The CAR-binding aptamer binds to a CAR expressed in an immune cell, such as a T cell, and in some embodiments activates the immune cell, but in other embodiments (e.g., when acting as a "kill" switch), does not activate the immune cell. The target is the intended target of the immunotherapy, i.e., the cell intended for elimination. Thus, the CAR-expressing cell and the aptamer bridge are intended to be used together as a system in immunotherapy, such as CAR-T cell therapy. The binding of the aptamer bridge to the CAR and the target is preferably a high affinity binding. The target can be a protein (such as a cell surface receptor protein), a cell, a small molecule, or a nucleic acid. The target is preferably located on the surface of the target cell, such as a cancer cell, and may or may not be found on other cells (normal cells) of the subject.

[0024] In some embodiments, the target is a tumor antigen such as CD19, CD20, CD22, CD30, CD123, BCMA, NY-ESO-1, mesothelin, PSA, PSMA, MART-1, MART-2, Gp100, tyrosinase, p53, ras, Ftt3, NKG2D Ligang, Lewis-Y, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, Her3, EGFRvIII, BRCA1 / 2, CD70, CD73, CD16A, CD40, VEGF-α, VEGF, TGF-β, CD32B, CD79B, cMet, PCSK9, IL-4RA, IL-17, IL-23, 4-1BB, LAG-3, CTLA-4, PD-L1, PD-1, OX-40, or mutant SOD. The component aptamers of the aptamer bridge can also specifically bind to such combinations of targets.In some embodiments, the targets are antigens of infectious pathogens, such as gag, reverse transcriptase, tat, HIV-1 envelope protein, circumsporozoite protein, HCV nonstructural protein, hemagglutinin, etc. The aptamer bridge can also specifically bind to such combinations of targets.

[0025] In a preferred embodiment, one or more CAR-binding aptamers are selected for specific binding to peptide neo-epitope (PNE), i.e., the extracellular domain of the CAR that has affinity for anti-PNE CAR. Because PNE is an epitope that does not exist in the subject's body, immune cells expressing anti-PNE CAR are not activated by endogenous biomolecules, but await the administration of an aptamer bridge to the subject, which acts as an "on" switch for immune cells, targeting CAR-expressing cells to the desired antigen or cell type that bears the antigen. The immune activation and in vivo proliferation of CAR-expressing immune cells can be turned off by administering to the subject either a peptide containing PNE, or a CAR-binding aptamer or a target-binding aptamer of the bridge in monomeric form, one of which will terminate the activation of CAR-expressing immune cells by the target.

[0026] PNE can be any peptide epitope that is not found in the host proteome (e.g., not found in the human proteome) and for which an anti-PNE CAR can be obtained. A preferred example of PNE is the peptide fragment of the GCN4 transcription factor from Saccharomyces cerevisiae (NYHLENEVARLKKL, SEQ ID NO: 1). High affinity (K d = 5.2 pM) has been described by Rodgers et al. Additional PNEs suitable for use with CARs and corresponding aptamer bridges include:

[0027] (i) the N-terminal 15-mer peptide ESQPDPKPDELHKSS (SEQ ID NO:2) of Staphylococcus aureus enterotoxin B, paired with an antibody that binds to it, and described in Clin. Vaccine Immunol. 17(11):1708-1717; (ii) the E. coli mycotoxin deoxynivalenol has been paired with a binding scFv and described in Protein Expr. Purif. 35(1): 84-92; (iii) HPV-16 protein E5 paired with the antibody described in BiomedInt. Res. Int. 2018;2018:5809028; (iv) rabies virus proteins and scFvs that bind to them, as described in Protein Expression and Purification 86 (2012) 75-81; (v) Influenza A matrix protein paired with an scFv that binds to it, as described in Bioconjugate Chem. 2010, 21, 1134-1141;

[0028] (vi) amino acids 134 to 145 of the pre-S2 protein of HBV (PRVRGLYFPAGG, SEQ ID NO: 3), paired with an scFv that binds to it, as described in ViralImmunol. 2018 May 30; (vii) Duck hepatitis virus type 1 VP3 peptide paired with scFv described in J. of Virological Methods 257(2018)73-78; (viii) a peptide from glycoprotein D of bovine herpesvirus type 1 (MEESKGYEPP, SEQ ID NO: 4) paired with a scFv described in Appl Microbiol Biotechnol. 2017 Dec; 101(23-24):8331-8344; (ix) a peptide comprising amino acid 159 of the VP1 protein of South African Territorial 2 (SAT2) foot-and-mouth disease virus, paired with an scFv that binds to it, and described in Virus Research 167 (2012) 370-379; (x) A peptide of OmpD from Salmonella typhimurium (DRTNNQVKA, SEQ ID NO: 5) paired with a scFv that binds to it is described in Veterinary Microbiology 147 (2011) 162-169;

[0029] (xi) a peptide of isoferritin from Escherichia coli paired with an scFv that binds to it, as described in Journal of Biotechnology 102 (2003) 177 / 189; (xii) a peptide located at the N-terminus of the grapevine leafroll virus 3 coat protein (AQEPPRQ, SEQ ID NO: 6) paired with an scFv that binds to it, as described in Arch. Virol. (2008) 153:1075-1084; (xiii) a peptide of the N protein of SARS-CoV (PTDSTDNNQNGGRNGARPKQRRPQ, SEQ ID NO: 7) paired with an scFv that binds to it, and described in Acta Biochimica et Biophysica Sinica 2004, 36(8)541-547; (xiv) a peptide comprising amino acids 1-15 of the HIV Tat protein and paired with an scFv that binds thereto, as described in J.Virol. 2004 Apr; 78(7):3792-3796; and (xv) a peptide from amino acids 1363-1454 of the helicase domain of HCV NS3, paired with an scFv that binds thereto, as described in J. Hepatology 37(2002)660-668, Virol 1994; 68:4829-4836, and ArchVirol 1997; 142:601-610. Other examples of universal CARs that can be combined with the aptamer bridges of the present technology are described in J. Autoimmun. 2013 May.42 :105-16; Blood Cancer J. 2016 Aug, 6(8): e458; Oncotarget. 2017 Dec 12, 8(65):108584-108603; Oncotarget 2017 May 9, 8(19): 31368-31385; Oncotarget 2018 Jan 26, 9(7): 7487-7500; and WO2016-030414.

[0030] The linking moieties of the aptamer bridge can be simple covalent bonds or bonds between the individual aptamers, or can be synthetic or naturally occurring polymers such as hydrocarbons, polyethers, polyamines, polyamides, nucleic acids, peptides, carbohydrates, or lipids. In certain embodiments, the linking moieties are not peptides. In certain embodiments, the aptamer bridges are devoid of peptides, and are devoid of polypeptides and proteins. The linking moieties can also take the form of nanoscale structures (such as polymers, proteins, nanoparticles, nanotubes, nanocrystals, nanowires, nanoribbons, nanocrystals, micelles, or liposomes), or microscale structures (such as microbeads or cells), or larger structures (such as solid supports). Preferably, the linking moieties are biodegradable polymers. The linking moieties can be polymers that are linear, branched, cyclic, or a combination of these structures. The linking moieties can also function as the backbone of dendrimer structures, or hub or star structures (such as a core structure to which two or more aptamers are attached). In the case of non-covalent association, two or more individual aptamers can be linked through non-covalent interaction, either directly between aptamers or through interaction with linking moieties.Non-covalent interaction can be, for example, one or more hydrogen bonds, ionic bonds, hydrophobic bonds, van der Waals interactions, or combinations thereof.A high affinity binding pair such as streptavidin-biotin can be used to non-covalently link the aptamers in the aptamer bridge.

[0031] The linker or linking moiety can be any chemical moiety that covalently or non-covalently links the monomeric aptamer units. The linker can contain or consist of, for example, an oligonucleotide, a polynucleotide, a peptide, a polypeptide, or a carbohydrate. The linker can contain or consist of a cell receptor, a ligand, or a lipid. The linker can contain or consist of a hydrocarbon chain or polymer, such as a substituted or unsubstituted alkyl chain or ring structure, a polyethylene glycol polymer, or a modified or unmodified oligonucleotide or polynucleotide. The linker can be a single covalent bond or can include one or more ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals interactions. The linker can include disulfide bridges, oligosaccharides (glycosaminoglycans) derived from heparin or heparan sulfate, chemical crosslinkers, hydrazones, thioethers, esters, or triazoles. The linker can be enzymatically cleavable, allowing for the release of individual aptamers and / or termination of the CAR-target interaction by the aptamer bridge.

[0032] The linker can have a net positive, negative, or neutral charge. The linker can be flexible or rigid as needed to ensure preservation of the functional properties of the individual monomeric aptamer units in the multimeric construct and to facilitate binding to or interaction between the CAR and the target. The linker can include a flexible portion, such as a polymer of 5-20 glycine and / or serine residues. The linker can also contain a rigid, defined structure, such as a polymer of glutamate, alanine, lysine, and / or leucine. The linker can include a hinge portion or a spacer portion. The linker can include a substituted or unsubstituted C2-C 50The linker may comprise a chain or ring structure, a polyethylene glycol polymer (e.g., hexaethylene glycol), or a modified or unmodified oligonucleotide or polynucleotide. The linker may comprise an oligosaccharide (glycosaminoglycan) derived from heparin or heparan sulfate, a chemical cross-linker, a peptide, a polypeptide, a hydrazone, a thioether, or an ester.

[0033] C2-C 50 The linker can comprise a backbone of 2-50 carbon atoms (saturated or unsaturated, linear, branched, or cyclic), 0-10 aryl groups, 0-10 heteroaryl groups, and 0-10 heterocyclic groups, which optionally contain ether linkages (e.g., one or more alkylene glycol units, including but not limited to, one or more ethylene glycol units -O-(CH2CHO)-); one or more 1,3-propanediol units; amines, amides, or thioethers. Each backbone carbon atom (i.e., containing only -H substituents) can be independently unsubstituted or can be substituted with C1-C3 alkyl, -OH, -NH 2、 In some embodiments, the linker is optionally substituted with one or more groups selected from -SH, -O-(C1-C6 alkyl), -S-(C1-C6 alkyl), halogen, -OC(O)(C1-C6 alkyl), and -NH-(C1-C6 alkyl). 20 Linker, C2-C 10 linker, a C2-C8 linker, a C2-C6 linker, a C2-C5 linker, a C2-C4 linker, or a C3 linker, where each carbon can be independently substituted as described above.

[0034] In certain embodiments, there are non-covalent bonds between aptamers, mediated, for example, through ionic bonds, hydrogen bonds, hydrophobic bonds, van der Waals interactions, or mixtures thereof, and there are no intervening linking moieties linking the individual aptamers. A single multimeric aptamer construct can also use a mixture of covalent bonds via intervening linker moieties connecting certain aptamers, and non-covalent bonds at other binding sites between aptamers, without intervening linker moieties.

[0035] A linker can optionally have one or more functions, for example, in some embodiments, the linker is temperature and / or pH sensitive, meaning that the linker changes conformation or is cleaved at a predesigned temperature and / or pH range.

[0036] Each of the CAR-binding portion and the target-binding portion of the aptamer bridge can comprise a single aptamer, or can comprise two or more identical or non-identical aptamer units. The use of two or more identical aptamers, or aptamers that are not identical but bind to the same target, can greatly increase binding affinity through binding of the same target presented differently or cooperativity of binding, especially for targets such as cells that have multiple copies of a given target molecule. Identical monomeric aptamers can have completely identical nucleotide sequences (100% identical), or have sequences that are about 99% identical, about 98% identical, about 97% identical, about 96% identical, about 95% identical, about 94% identical, about 93% identical, about 92% identical, about 91% identical, or about 90% identical, or at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical.

[0037] Each of the CAR-binding portion and the target-binding portion of the aptamer bridge is not identical and can include two or more individual aptamers that bind to the same or different targets. Non-identical aptamers can differ in structure, nucleotide sequence, and / or binding specificity or binding affinity. Such bridges can specifically bind to two or more different sites on the target, such as different epitopes of a protein or different surface proteins of a target cell, or different CARs, thereby increasing the affinity and / or specificity for the target, or binding to different target cells and / or different CAR-expressing cells. Different targets or different CARS can be selected to maximize the desired therapeutic effect through a combination of actions involving different target cells and / or different CAR-expressing cells.

[0038] The function of CAR cells can be improved by directing them to two or more antigens on target cells, for example, two or more antigens on a given tumor cell, such as CD19 and CD22, CD19 and CD123, or CD19 and BCMA. Such an approach can help overcome tumor antigen loss and achieve improved persistence of responses. Aptamer bridges can be engineered to recognize a CAR and two or more antigens on the same target cell by linking two or more aptamers together with target-binding moieties. This approach is similar to engineering T cells to simultaneously express two tumor-associated antigen receptors, but extends beyond the dual-targeting limitations of antibodies, offering lower costs and greater flexibility than antibody-based CAR switches.

[0039] The aptamer bridge constructs of the present technology can be used in immunotherapy compositions. An embodiment of such a composition includes an aptamer bridge and an excipient. The aptamer bridge can be associated with other molecules, or structures such as liposomes, micro- or nanoparticles, receptor or cell-targeting molecules, or oral, topical or other formulations to aid in uptake, distribution, and / or absorption. In some embodiments, the aptamer bridge is encapsulated, conjugated (complexed), or otherwise associated with a therapeutic delivery vehicle. In some embodiments, the therapeutic delivery vehicle is a gene delivery vehicle, such as a viral vector, or includes a viral vector. The viral vector can be any type of suitable vector, such as an expression vector or a plasmid. In a preferred embodiment, the vector is a lentiviral vector. In a preferred embodiment, a CAR-encoding vector is first administered to a subject, and an initial baseline of CAR expression, and in some embodiments, CAR cell proliferation, is established in the subject before administering the aptamer bridge to the subject to activate an immune response against the target. CAR expression, and optionally proliferation of CAR-expressing cells, can be detected and / or quantified in a subject, for example, by administering a CAR-binding aptamer (such as the CAR-binding portion of an aptamer bridge) in a labeled form, followed by determining the bound label of peripheral blood cells or determining the distribution of the label in the body.

[0040] The constituent aptamers of the aptamer bridge may include any desired modification to the aptamer structure or sequence. Modifications may include substitutions introduced into one or more ribose or deoxyribose moieties of the candidate aptamer. Modifications may include substitutions in one or more phosphate moieties of the candidate sequence. Modifications may include substitutions in one or more purine or pyrimidine moieties of the candidate aptamer, or the use of non-natural or rare natural nucleotides in place of natural nucleotides of the candidate aptamer. Chemical modifications may be introduced into the internucleotide phosphate linkages, such as replacement of the internucleotide phosphate with phosphorothioate or boranophosphate, addition of biotin, azide or alkyne groups. Chemical modifications may be introduced into the C2' position of the ribose ring, such as introduction of fluorine, LNA (locked-in nucleic acid) units, or 2'-O-alkyl modifications. One or more nucleosides of the aptamer may include a modification selected from a sugar modification at the 2' position, such as (2'-amino (2'-NH2), 2'-fluoro (2'-F), or 2'-O-methyl (2'-OMe)), a modification at the cytosine exocyclic amine, an internucleoside linkage modification, or a 5-methylcytosine. The aptamer may include a 3' cap, a 5' cap, and / or an inverted deoxythymidine at the 3' end. Rare nucleotides such as 2-thiouridine (s2U), pseudouridine (ψ), dihydrouridine (D), or non-natural nucleotides such as peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), or threose nucleic acid (TNA) can be used in place of commonly occurring nucleotides. Non-natural nucleic acids (xeno nucleic acids (XNA)) can also be included.

[0041] Any suitable method for generating or selecting aptamers for a target can be used to obtain the constituent aptamers of the aptamer bridge. For example, aptamers can be identified by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). SELEX is described, for example, in U.S. Patent No. 5,270,163, which is incorporated herein by reference. Briefly, SELEX begins with a plurality of nucleic acids (i.e., candidate aptamer sequences) that contain various nucleotide sequences that contact the target. Unbound nucleic acids are separated from the nucleic acids that form aptamer-target complexes. The aptamer-target complexes are then dissociated, the nucleic acids are amplified, and the steps of binding, separation, dissociation, and amplification are repeated through as many cycles as desired to result in a population of aptamers with increasingly higher affinity for the target. Cycles of selection and amplification can be repeated until no significant improvement in binding affinity is achieved with further repetitions of the cycles.

[0042] Cycles of selection and amplification can be interrupted before a single aptamer is identified. In such cases, a population of aptamers is identified, which can provide important information about the sequences, structures, or motifs that allow the aptamer to bind to the target. Such a population of candidate aptamers can also inform which parts of the aptamer are not important for target binding. This information can guide the generation of other aptamers to the same target. Aptamers thus generated can be used as input for a new round of SELEX, potentially generating aptamers with better binding affinity or other properties of interest.

[0043] In some embodiments, the candidate aptamer sequence containing the multimeric aptamer construct, such as the candidate aptamer bridge, is formed, and then it is subjected to further rounds of selection as a multimeric construct.The multimeric candidate aptamer construct can be made by linking each candidate aptamer portion with a linking moiety, and optionally using such a construct as the input for one or more rounds of SELEX.In some embodiments, each aptamer is independently selected through one or more rounds of SELEX, and finally linked together with a linking moiety.Thus, the multimerization of the monomeric aptamer and the multimeric aptamer construct can be carried out before, during, or after the SELEX procedure.

[0044] To select the component aptamers of the aptamer bridge, the selection can be performed differently for the aptamers that bind to the CAR compared to the aptamers that bind to the target on the target cell. The selection of the aptamers that bind to the target cell can be performed by associating the candidate aptamers with intact target cells, recombinant cells, pathogen cells, virus-like particles, or purified target proteins or peptide epitopes, or with membrane preparations from the target cells, or with recombinant cells that contain antigenic surface proteins. However, the selection of the aptamers that bind to the anti-PNE CAR can be performed by contacting the candidate aptamers with the anti-PNE CAR expressed on cells in the presence and absence of PNE. Thus, according to this method, the aptamers that have the desired binding affinity to the anti-PNE CAR are those that can replace the binding to the CAR in the absence of PNE by adding a peptide that contains PNE. However, aptamers can also be selected using peptides from different regions of the CAR, such as adjacent to the PNE binding site, and aptamers selected in this manner may not be displaced by PNE but may provide high affinity binding to the anti-PNE CAR. Furthermore, to promote successful immunotherapy, activation of CAR-expressing cells, such as T cells expressing CAR, should be monitored during the aptamer selection process. To this end, selection of candidate CAR-binding aptamers can be performed using candidate CAR-binding aptamers placed in an aptamer switch along with candidate target-binding aptamers.

[0045] The component aptamers of the aptamer bridge can have any desired length. In some embodiments, the component aptamers comprise at least about 15 nucleotides. In some embodiments, the component aptamers comprise about 100 nucleotides. The length of the aptamer bridge can determine the distance between cells interacting through the bridge. Thus, the length of the bridge is desirably in the range of about 70 to about 200 angstroms, or about 70 to about 150 angstroms, or about 100 to about 170 angstroms, or about 120 to about 150 angstroms.

[0046] This technique can be used to identify or generate aptamers with any desired binding affinity. In a preferred embodiment, one or more selected aptamers have a K for binding to a target ranging from about 1 pM to about 10 nM for high affinity (specific) binding. d and for low affinity binding, on the order of about 100 nM to 10 μM. In some embodiments, the aptamer has a K of about 1 pM to about 10 μM, about 1 pM to about 1 μM; about 1 pM to about 100 nM; about 100 pM to about 10 μM; about 100 pM to about 1 μM; about 100 pM to about 100 nM; or about 1 nM to about 10 μM; about 1 nM to about 1 μM; about 1 nM to about 200 nM; about 1 nM to about 100 nM; about 500 nM to about 10 μM; or about 500 nM to about 1 μM. dIn some embodiments, the binding affinity of the component aptamers in the aptamer bridge is 4-50 times higher than that of the monomeric candidate aptamer. In some embodiments, the affinity of the component aptamers of the aptamer bridge to one or more targets can be fine-tuned by varying the degree of multimerization in the bridge, the component aptamers included in the bridge, or the linker. In certain embodiments, the CAR-binding portion and / or the target-binding portion of the bridge are multivalent, meaning that they contain two or more individual aptamers that bind to the same or different sites on the CAR or target. Multivalent aptamer structures offer the advantage of high binding specificity and / or high binding affinity to the CAR or target. The binding affinity of a multivalent aptamer can be much higher than that of a single aptamer because the binding of the multimeric complex can exhibit positive cooperativity or positive allosterism between the individual binding sites. The ability to carefully select and attach binding sites with multimeric aptamer bridge moieties enhances the safety profile by limiting adverse or toxic effects and increasing the efficacy of effective treatment.

[0047] In some embodiments, the aptamer of the aptamer bridge is stabilized and / or polymerized by conjugation with another compound, such as biotin avidin or polyethylene glycol. In some embodiments, the aptamer is synthesized using a solid support.

[0048] The aptamer bridges disclosed herein are useful for preventing or treating proliferative diseases or infectious diseases. One aspect of the invention is a method of preventing or treating cancer or infection by administering an aptamer bridge or an immunotherapeutic formulation or a therapeutic delivery vehicle that includes the aptamer bridge.

[0049] The invention also provides methods of inducing or enhancing an immune response against cancer or an infectious disease in a subject by administering an aptamer bridge or an immunotherapeutic formulation or a therapeutic delivery vehicle comprising the aptamer bridge.

[0050] The invention further provides a method for specifically associating immune cells expressing a CAR with target cells against which an immune response or enhanced immune response is desired: administration of an aptamer bridge with binding specificity for both the CAR and a molecular target on the target cell leads to activation of the CAR-expressing immune cell, initiating or enhancing an immune response against the target cell. EXAMPLES

[0051] Example 1: Preparation of bispecific aptamers specific for PSMA and CD3 The A10 RNA aptamer (SEQ ID NO: 8) is a 39 nucleotide long sequence selected against human prostate specific membrane antigen (PSMA) and used as a prostate-specific delivery agent for siRNA (McNamara et al. 2006-Dassie et al. 2009). CELTIC_1S, CELTIC_19s, CELTIC-core are DNA aptamers (SEQ ID NOs: 9, 10, 11), ARACD3-3700006 and ARACD3-0010209 are RNA aptamers (SEQ ID NOs: 12 and 13), all of which were previously selected against human CD3. These DNA or 2'-deoxy-2'-fluorothymidine modified RNA (2'F-RNA) aptamers were purchased from BaseClick (Neuried, Germany) as HPLC-RP purified single standard oligos synthesized via standard solid-phase phosphoramidite chemistry. The anti-CD3 aptamer did not activate cytokine secretion or surface marker expression when used in combination with a costimulatory anti-CD28 antibody, unlike the anti-CD3 monoclonal antibody (data not shown).

[0052] The A10 aptamer was modified with an azide group at its 3' end for subsequent triazole-internucleotide dimerization. Biotin was added to the 5'-end of the A10 aptamer as biotin-TEG, which introduces a 16-atom mixed polarity spacer between the aptamer sequence and the biotin flag. A Cy5-labeled version of A10 was also synthesized. Subsequent triazole-nucleoside-nucleotides ERTIC_1s, CELTIC_19s, CELTIC_core, ARACD3-3700006, and ARACD3-0010209 were modified with an alkyne group at the 5' end. The molecular weight, purity, and integrity were verified by HPLC-MS. The affinity and specificity of the A10 anti-PSMA RNA aptamer were evaluated for PSMA-positive and PSMA-negative cells (Figure 7A).

[0053] Anti-PSMA A10 and anti-CD3 aptamers were heterodimerized by copper-catalyzed click reaction performed at 45 °C for 60 min with Oligo2-Click Kit L (BaseClick, Neuried, Germany) according to the manufacturer's instructions. Reaction products on a 3% agarose gel migrated in 1XTBE buffer (Invitrogen) at 100 V for 30 min were separated by gel electrophoresis. The gel was visualized using a Bio-Rad imaging system and the results are shown in Figure 8A and 8B. Gel slices corresponding to dimeric aptamers were excised from the gel and nucleic acids were extracted by passive elution in 25 mM NaCl-TE buffer for 72 h at 8 °C. Bispecific aptamer dimers were recovered by standard sodium acetate precipitation method, resuspended in sterile water, and stored at -20 °C until use.

[0054] [Example 2] Functional stability of PSMA-specific aptamer A10 The stability of the A10 RNA aptamer was measured in Dulbecco's phosphate buffered saline (DPBS) containing 5% FBS or FBS alone. The biotinylated aptamer was denatured at 85 °C for 5 min and then immediately cooled to 4 °C on an ice block for 5 min. The aptamer was then diluted to a final concentration of 2 μM in DPBS supplemented with 5% of FBS or in pure FBS. Samples were incubated at 37 °C for 10 min, 30 min, 1 h, 2 h, 4 h, or 24 h. Control samples contained freshly prepared aptamer without incubation at 37 °C. Then, 100 nM streptavidin PE was added to each solution and the aptamer was incubated with PSMA-positive LNCaP cells (human prostate cancer-ATCC CRL-1740). The half-life of aptamer A10 in DPBS buffer containing 5% FBS or in pure FBS was then measured using flow cytometry on the YL-1 channel based on the change in the number of fluorescence-positive cells as a function of incubation time at 37 °C. The results are shown in Figure 9. Aptamer A10 incubated in DPBS buffer containing 5% serum was stable over 24 hours. When tested with pure serum, half of the binding activity was lost within the first 2 hours of incubation.

[0055] [Example 3] Determination of affinity and specificity of anti-PSMA x anti-CD3 bispecific aptamers for targets expressed on cells. The affinity and specificity of the anti-PSMAxanti-CD3 bispecific aptamers for the target proteins expressed on cells was assessed by flow cytometry. These studies were performed by incubation of biotinylated RNA / DNA aptamers in SELEX buffer or RNA / RNA aptamers in DPBS buffer supplemented with 5% of FBS on CD3 positive Jurkat (acute T cell leukemia human cell line-ATCC TIB-152), CD3 negative Ramos (Burkitt lymphoma human cell line-ATCC CRL-1596), PSMA positive LNCaP (human prostate cancer-ATCC CRL-1740) and PSMA negative PC-3 (human prostate cancer-ATCC CRL-1435) cells.

[0056] Cells were cultured in RPMI-1640 medium (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen) and 1% penicillin / stephomycin (Gibco Invitrogen) before use. Jurkat, Ramos, LnCaP, and PC-3 cells (2.5 × 10 5 Cells / well ) were seeded in 96-well plates and centrifuged at 2500 rpm for 2 min. The supernatant was discarded and the pelleted cells were washed twice with 200 μL of SELEX or DPBS-5% FBS buffer pre-warmed at 37 °C. Each washing step was followed by centrifugation at 2500 rpm for 2 min. The aptamers were denatured at 85 °C for 5 min and immediately placed on an ice block at 4 °C for 5 min. Test samples were then diluted in two different concentration ranges: 3, 10, 30, 100 and 300 nM (CD3 binding assay) and 30, 100 and 300 nM (PSMA binding assay), followed by the addition of 100 nM phycoerythrin-labeled streptavidin (Streptavidin-PE, eBioscience) to each solution. Jurkat, Ramos, LnCap, and PC-3 cells were resuspended in aptamer dilutions (100 μL / well) and incubated for 30 min at 37 °C in a humidified 5% CO2 atmosphere.

[0057] As controls, cells were incubated with CD3 monoclonal antibody (PE-labeled, OKT3 human anti-CD3, Invitrogen), PSMA monoclonal antibody (Alexa Fluor 488-labeled, GCP-05 human anti-PSMA, Invitrogen), PE-streptavidin or the respective buffers without additional reagents. After incubation, cells were centrifuged at 2500 rpm for 2 min and the supernatant with unbound sequences was discarded. Pelleted cells were washed with SELEX or DPBS-5% FBS buffer (200 μL / well), centrifuged, and centrifuged twice to remove all weakly and nonspecifically attached sequences. Afterwards, cells were washed with 1 mg / mL salmon sperm DNA solution (100 μL / well) at 37 °C in a 5% CO2 humidified atmosphere. After 30 min, salmon sperm was removed by centrifugation at 2500 rpm for 2 min, and cells were washed twice with SELEX or DPBS-5% buffer (200 μL / well) followed by centrifugation. Jurkat, Ramos, LnCap, and PC-3 cells with DNA or RNA sequences attached were then fixed (BD CellFix solution #340181), and fluorescent positive cells were counted by flow cytometry (AttuneNXt; Invitrogen, Inc.) on the YL-1 channel.

[0058] The results of the binding study to PSMA-positive cells are shown in Figures 10A and 10B. Three RNA / DNA aptamers (A10 x Celtic_1S, A10 x celtic_19S, A10 x Celtic_Core) and two RNA / RNA aptamers (A10 x ARACD3-3700006, A10 x ARACD3-0010209) were analyzed together with the A10 monomeric aptamer. For comparison, the binding of the tested reagents to PSMA-negative PC-3 cells was also measured. For A10, a dose-dependent binding to PSMA-positive LNCaP cells was observed without reaching signal saturation at the highest concentration tested. The intensity of the signal was as strong as the antibody control. Residual binding of A10 monomer to PC-3 cells was observed only at the highest concentration tested. All bispecific PSMAxCD3 aptamers showed similar binding properties to the A10 monomer, but with reduced residual binding to PSMA-negative cells, resulting in improved specificity for target-positive cells. At each concentration tested, the signal intensity of the bispecific aptamers was superior to that measured with the A10 monomer, suggesting improved affinity through heterodimerization.

[0059] In a separate experiment, the binding of the same aptamers to CD3 positive Jurkat and CD-3 negative Ramos cells was investigated. See Figures 11A and 11B. As expected, the A10 aptamer did not bind to these two cell lines. Residual binding of anti-CD3 monomers to Ramos cells was observed only at the highest concentration tested. All bispecific PSMAxCD3 aptamers showed similar dose-dependent binding, but with a strong reduction in residual binding to CD3 negative cells, resulting in excellent specificity for target positive cells. At each concentration tested, the signal intensity of the bispecific aptamers was inferior to that measured for anti-CD3 monomers, suggesting reduced affinity due to heterodimerization. These results suggest that after heterodimerization, the binding properties of selected aptamers against different targets are not destroyed due to steric hindrance when assessed separately, and depending on the selected partners, the specificity and affinity for each target may even be improved upon dimerization.

[0060] Example 4: Binding of bispecific aptamers targeting PSMA and CD3 measured by surface plasmon resonance Binding affinity measurements were performed using a BiAcore T200 instrument (GE Healthcare). To analyze the interaction between the aptamers and CD3 and PSMA proteins, 300 resonance units of biotinylated aptamers were immobilized on a Series S Sensor Chip SA (GE Healthcare) according to the manufacturer's instructions (GE Healthcare). DPBS buffer was used as the running buffer. The interactions were measured by injecting different concentrations of human CD3 ε / γ, CD3 ε / δ, lgG1 Fc, PSMA (Acro Biosystems) in the "single kinetic cycle" mode at a flow rate of 30 μl / min. The highest aptamer concentration used was 300 nM. Other concentrations were obtained by 3-fold dilution. All kinetic data of the interactions were evaluated using the BIAcore T200 evaluation software.

[0061] K of monomeric and bispecific aptamers D Comparison of values ​​indicates that dimerization does not interfere with the binding properties of each subunit for its specific target. Simultaneous binding of PSMA and CD3 ε / γ can be recorded in manual injection mode at a flow rate of 10 μl / min by injecting a solution of the first target at saturating concentration, then a solution of the second target at saturating concentration. A second injection with the reverse sequence is performed. In both sequences, each injection resulting in a response of the same intensity indicates that both arms of the bispecific aptamer are able to bind the second target while the binding site for the first antigen is occupied. The failure of the monomer to respond to both injections of target solution indicates that the bispecific aptamer is able to bind both targets simultaneously.

[0062] [Example 5] Biological activity of bispecific aptamers specific for PSMA and CD3 Cytotoxicity assays were performed on unstimulated peripheral blood mononuclear cells (PBMCs). Freshly prepared PBMCs were isolated from buffy coats obtained from healthy donors (Etablissement Francais du Sang, Division Rhones-Alpes). After dilution of blood with DPBS, PBMCs were separated on a FICOLL density gradient (FICOLL-PAQUE PREMIUM 1.077 GE Healthcare), washed twice with DPBS, resuspended in RPMI-1640 medium (Gibco Invitrogen) and diluted at 5x10 6 The cell density in cells / ml was obtained. These PBMCs were used as effector cells.

[0063] LNCaP target cells were labeled with 2 μM calcein AM (Trevigen Inc, Gaithersburg, MD, USA) for 30 min at 37 °C in cell culture medium. Calcein AM fluorochrome is a dye that is trapped inside living LNCaP cells and released only upon redirected lysis. After washing twice with cell culture medium, 5 × 10 5 A cell density of 100 μl of 50,000 cells was adjusted to 100 cells / ml and a 100 μl aliquot of 50,000 cells was used per assay reaction. Standard reactions at 37°C / 5% CO2 lasted 4 hours and contained 5x10 4 Cell Calcein AM labeled target cells, 5x10 5 PBMCS (E / T ratio 1:10), 20 μl of bispecific aptamer solution was used at 1 μM in a total volume of 200 μl. After the cytotoxicity reaction, the dye released in the culture medium was quantified with a fluorescence reader (VariosKan Lux, ThermFisher, Waltham, MA, USA) and compared with the fluorescence signal from a control reaction in which no cytotoxic compound was present and a reaction in which the fluorescence signal was determined for completely lysed cells (aptamer was replaced by A100 reagent purchased from Chemometec, Allerod, Denmark). Based on these measurements, the specific cytotoxicity was calculated according to the formula: [fluorescence (sample)-fluorescence (control)] / [fluorescence (total lysis)-fluorescence (control)] x100.

[0064] The results of the cytotoxicity assay obtained after 4 h of incubation in the presence of 100 nM of aptamer with a single E:T ratio of 10:1 are shown in Figure 12. Zero to weak specific cell killing activity (<10%) was observed with the PSMAXCD3 bispecific RNA / DNA aptamers. Excellent specific cytotoxicity was measured with the RNA / RNA aptamers A10 x ARACD3-3700006 and A10 x ARACD3-0010209, which induced 40-50% killing of LNCaP cells. The control monomeric A10, lacking the CD3 binding moiety, did not induce cytotoxicity. These results suggest that the engineered aptamer switch can attract effector T lymphocytes to target cells, redirect the cytolytic machinery, and eliminate specific cell populations.

[0065] [Example 6] Anti-CAR PNE apatamer Libraries and primers The initial RNA library template and primers were prepared by IDT (Coralville, IA, USA) using single-stranded DNA: :5'-CCTCTCTATGGGCAGTCGGTGAT-(N20)-TTTCTGCAGCGATTCTTGTT-(N10)-GGAGAATGAGGAACCCAGTGCAG-3' (Template, SEQ ID NO: 14) ,5'-TAATACGACTCACTATAGGGCCTCTCTATGGGCAGTCGGTGA T-3' (forward primer SEQ ID NO:15 ), 5'-CTGCACTGGGTTCCTCATTCTCC-3' (reverse primer SEQ ID NO:16 ) was synthesized. To minimize the effect of the primer on secondary structure, two short "blocking" sequences (purchased from IDT) were synthesized that complemented the 5' and 3' invariant primer regions. 5'-ATCACCGACTGCCCATAGAGAGG-3' (forward blocking sequence SEQ ID NO:17 ), 5'-CTGCACTGGGTTCCTCATTCTCC-3' (reverse blocking sequence SEQ ID NO:18An additional biotinylation "capture" sequence that complements the invariant central region of the library was also synthesized by IDT: 5'-biotin-GTC-PEG-6 spacer-CAAGAATCGCTGCAG-3' (SEQ ID NO:19) All materials were ordered at the 250 nmol scale and underwent desalting purification.

[0066] RNA libraries for RNA aptamer selection were modified with 2'fluoro-(2'F-) pyrimidines to enhance stability in the final application. T7 primers were combined with Titanium Taq DNA polymerase (Clontech; Mountain View, CA, USA) and library template sequences for primer extension. Primer extension materials were provided by Durascribe (R) Libraries were transcribed using a T7 transcription kit (Epicentre; Madison, WI, USA) and purified with denaturing polyacrylamide in 8 M urea (Sequence NE Reagent, Part A and Part B) and purchased from American Bioanalytical (Natick, MA, USA). During selection, libraries were reverse transcribed using Supercript IV reverse transcriptase (Invitrogen; Carlsbad, CA, USA) according to the manufacturer's protocol and amplified using Clontech Titanium Taq DNA polymerase. During selection, libraries were amplified using the following PCR protocol (95°C for 10 s, 60°C for 30 s, followed by an initial HotStart activation at 95°C for 60 s). Libraries were then amplified using Durascribe (R) The RNA library was transcribed using a T7 transcription kit and purified on a polyacrylamide gel (PAGE). The gel elution buffer for library recovery after overnight purification at 4°C was 0.5M NH4OAc, 1mM EDTA (both purchased from Teknova), 0.2% SDS (purchased from Amresco), pH 7.4.

[0067] RNA aptamer selection Screening of the RNA library was performed in six rounds of selection using a melting-off approach. Rounds 1-6 of selection were performed on a stable HEK293T cell line (human embryonic kidney - ATCC CRL-1573), expressing the GCN4 (S25R14) CAR (chimeric antigen receptor) directed against a peptide neoepitope (PNE) on the cell surface, and encapsulating an anti-CD19 CAR for a negative selection step in stably transduced HEK293T cells (cell-SELEX). The HEK293T cell line was obtained from the American Type Cell Collection and was cultured in DMEM medium (Gibco Invitrogen), supplemented with 10% FBS (Gibco Invitrogen) and 1% penicillin / steptomycin (Gibco Invitrogen). All selections were performed in DMEM medium supplemented with 10% serum matrix, and each of the SELEX rounds included the following steps: immobilization of the RNA library on streptavidin-coated magnetic beads, counter selection, incubation with the target, reverse transcription of the target-recognized sequences, PCR amplification, and transcription into RNA. Before each round, an aliquot of streptavidin-coated magnetic beads (MyOne Streptavidin T1 Dynabeads) was (TM) Typically, 1 pmole of biotinylated material is added to the dynamite. (TM)The RNA libraries were refolded in serum-free DMEM medium (denatured at 90 °C for 1 min, annealed at 60 °C for 5 min, and annealed at 23 °C for 5 min) with twice the library molar amount of primer blocking sequences and capture sequences. This was done to minimize the effect of constant primer regions on the secondary structure of the aptamer, capture the library by magnetic beads via streptavidin-biotin binding interactions, and protect the ends of the aptamers from exogenous nuclear cells.

[0068] After refolding and primer blocking, the library was captured on the magnetic beads by incubation at room temperature for 15 min. The magnetic beads were then separated from the solution and washed three times with 200 μL of selection buffer at 37 °C to remove PBS-T and non-specifically bound library species. The magnetic beads with immobilized RNA libraries were incubated for 10 min at 37 °C. 6A counter selection incubation was performed with 200 μL of each paired cell preparation, resulting in the release of nonspecific sequences from the magnetic beads. The nonspecific library members were then discarded and the magnetic beads were washed 6 times for 7 min with 200 μL of selection buffer and incubated for 1 h (7 min at 37 °C with DPBS, magnetically separated for 3 min, discarded the supernatant, and repeated the process). The nonspecific library members are less likely to survive to respond during the positive selection step. Positive selection consisted of incubation of the magnetic bead RNA library with 200 μL of the positive cell preparation for 60 min at 37 °C during rounds 1-4, and 30 min during rounds 5-6, with parallel evaluation (cell counting in Table 1). Once the positive selection was completed, the supernatant containing the sequences that recognized the target was separated from the magnetic beads and collected. The supernatant was then subjected to a second magnetic separation to ensure that the magnetic beads were completely removed. Targeted HEK293T CAR PNE cells were pelleted by centrifugation at 5,000xg and washed once with 200μL of DPBS buffer to remove weakly associated aptamer species. Libraries were recovered from cells by heat denaturation at 70°C. Libraries recovered from all rounds underwent protein precipitation with MPC reagent (Lucigen Corp, Middleton, WI, USA), ethanol precipitation, sample concentration, and purified by denaturing PAGE with 8M urea 10%. Libraries were reverse transcribed using superscript IV reverse transcriptase according to the manufacturer's instructions and titrated on a 100% column. (R)The RNA libraries were amplified using Taq DNA polymerase and transcribed using the Durascrib® T7 Transcription Kit according to the manufacturer's instructions. The transcription products were purified by 10% denaturing polyacrylamide gel electrophoresis (PAGE) in 8 M urea. Gel slices were excised and eluted overnight at 4 °C in gel elution buffer, and the concentration of the RNA library was calculated by measuring the absorbance at 260 nm on a Nanodrop-1000. During successive rounds of the SELEX process, the RNA library concentration and target cell number were gradually decreased. Additional parallel evaluations and "crossover fitness tests" were performed to facilitate the identification of good aptamer candidates during post-selection bioinformatics analysis.

[0069] [Table 1]

[0070] Aptamer candidates were selected by next-generation sequencing using a MiniSeq Mid Output (150 cycles) system (Illumina). Several aptamers were selected for further testing. For this purpose, 2'-deoxy-2'-fluorothymidine modified RNA aptamers were purchased from Eurogentec-Kaneka (Liege, Belgium) as HPLC-RP purified single-stranded oligos synthesized by standard solid-phase phosphoramidite chemistry. Biotin was added to the 5' end of the aptamer as biotin-TEG, which introduces a 16 atom mixed polarity spacer between the aptamer sequence and the biotin flag. Molecular weight, purity and integrity were verified by HPLC-MS. The nucleic acid sequences of these aptamers are shown in Figure 13A.

[0071] Example 7: Determination of affinity and specificity of anti-CAR PNE aptamers for targets expressed on human cells The affinity and specificity of the ARAA-00100001, ARAA-05200001, ARAA-0060095, ARAA-01300011, and ARAA-01700001 aptamers to target proteins expressed in cells were assessed by flow cytometry. These studies were performed on HEK293T, HEK293T-CAR CD19 (a HEK293T cell line transduced with a lentiviral vector encoding a CAR directed against the CD19 antigen) and HEK293T-CAR PNE (a HEK293T cell line transduced with a lentiviral vector encoding a CAR directed against the PNE peptide) cells by culturing them with biotinylated aptamers in DPBS buffer, supplemented with 5% of FBS.

[0072] Cells were cultured in DMEM medium (Gibco Invitrogen) and supplemented with 10% FBS (Gibco Invitrogen) and 1% penicillin / streptomycin (Gibco Invitrogen) before use. Before the experiment, cells (2.5x10 5 Cells / well) were seeded in 96-well plates and centrifuged at 2500 rpm for 2 min. The supernatant was discarded and the pelleted cells were washed twice with 200 μL of DPBS-5% FBS buffer pre-warmed at 37 °C. Each washing step was followed by centrifugation at 2500 rpm for 2 min. The aptamers were denatured at 85 °C for 5 min and immediately placed on an ice block at 4 °C for 5 min. The arrays were then diluted in two different concentration ranges: 30, 100 and 300 nM (primary testing of five aptamer candidates) and 30, 50, 75 and 100 nM (repeat staining with ARAA-00100001 and ARAA-01700001), followed by the addition of 100 nM phycoerythrin-labeled streptavidin (StreptaVidin-PE, eBioscience) to each solution. Cells were resuspended in aptamer dilutions (100 μL / well) and incubated for 30 min at 37°C in a humidified 5% CO atmosphere.

[0073] As a control, cells were incubated with PNE peptide (Alexa Fluor 488-labeled, Provepharm, Marseille, France), streptavidin-PE (eBioscience) or the respective buffers without additional reagents. After incubation, cells were centrifuged at 2500 rpm for 2 min and the supernatant with unbound sequences was discarded. Pelleted cells were washed with DPBS-5% FBS buffer (200 μL / well) and centrifuged twice to remove all weakly and nonspecifically attached sequences. Cells were then washed with 1 mg / mL salmon sperm DNA solution (100 μL / well) at 37 °C in a 5% CO2 humidified atmosphere. After 30 min, salmon sperm was removed by centrifugation at 2500 rpm for 2 min and cells were washed twice more with DPBS-5% buffer (200 μL / well) followed by centrifugation. The DNA sequence-bound cells were then fixed (BD CellFix solution #340181) and fluorescent positive cells were counted by flow cytometry (attunenXt; Invitrogen, Inc.) on the YL-1 channel.

[0074] Figure 14A shows the results of binding studies to HEK293T, HEK293T-CAR CD19, HEK293T-CAR PNE. For aptamers ARA-00100001 and ARAA-01700001, dose-dependent binding to CAR PNE-positive HEK293T cells was observed at the highest concentration tested without reaching signal saturation. The intensity of the signal was stronger than the PNE peptide control. Residual binding of both aptamers to CAR PNE-negative cells was observed at concentrations above 50 nM. ARAA-05200001, ARAA-0060095, ARAA-01300011 did not show differentiation between the three HEK293T cell lines indicating that these three aptamers were not specific for CAR PNE. The binding studies with aptamers ARA-00100001 and ARAA-01700001 were repeated over a wider range of concentrations, confirming the results of the primary experiments (see Figures 14B and 14C).

[0075] In another experiment, the binding of the same aptamers to human PBMCs expressing CAR PNE was studied. PBMCs were isolated from blood donors as already described in Example 5 and cultured in RPMI-1640 medium (Gibco Invitrogen), supplemented with 10% FBS (Gibco Invitrogen) and 1% penicillin / streptomycin (Gibco Invitrogen) at 37°C and 5% CO2. After 2 hours of transduction with a lentiviral vector encoding for a CAR directed against the PNE peptide, the cells were kept in culture for an additional 72 hours before performing binding studies according to the method described above. As shown in Figure 16, a dose-dependent signal was measured with both aptamers, which reached a plateau at concentrations above 300 nM. The intensity of the signal was stronger than the PNE peptide control.

[0076] Both experiments show that the ARAA-00100001 and ARAA-01700001 aptamers selected against a CAR that recognizes the PNE peptide are able to specifically recognize their targets expressed on human cells engineered to express this particular chimeric antigen receptor.

[0077] [Example 8] Serum stability of aptamers specific to CAR-PNE The stability of anti-CAR PNE RNA aptamers (ARAA-00100001 and ARAA-01700001) was studied in DPBS buffer containing 5% fetal bovine serum (FBS), RPMI medium containing 10% FBS or pure FBS. The aptamers were denatured at 85 °C for 5 min and then immediately cooled to 4 °C on an ice block for 5 min. The sequences were then diluted to a final concentration of 2 µM in DPBS buffer-supplemented with 5% FBS, RPMI medium-supplemented with 10% FBS or pure FBS serum. Samples were incubated at 37 °C for 10 min, 30 min, 1 h, 2 h, 4 h or 24 h. Control samples contained freshly prepared aptamers without incubation at 37 °C. The half-life of the aptamers in each buffer was determined by migration on an agarose gel using denaturing electrophoresis as follows: aptamer samples with different incubation times were denatured with formamide-containing loading buffer (ThermoScientific, Waltham, MA, USA) at 85 °C for 5 min, and then 15 μL of each sample was placed on a freshly prepared 3% agarose gel containing SYBRsafe (Invitrogen) as an RNA staining marker. Migration of the RNA aptamers on the agarose gel was performed in 1XTBE buffer (Invitrogen) by applying 100 V for 20 min. The gel was visualized using a Bio-Rad imaging system, and the results are shown in Figure 15. Both aptamers were stable for at least 2 h under different conditions of incubation, since the intensity of the RNA signal started to decrease after 4 h.

[0078] [Example 9] Preparation of bispecific aptamers specific to PSMA and CAR-PNE The ARAA-00100001 and ARAA-01700001 aptamers were purchased from BaseClick (Neuried, Germany) as 2′F RNA oligos synthesized via standard solid-phase phosphoramidite chemistry with purification by HPLC-RP. The A10 2'F-RNA aptamer was modified by an azide group at its 3'-end for subsequent triazole-internucleotide dimerization. Biotin was added to the 5'-end of the A10 aptamer as biotin-TEG, which introduces a 16-atom mixed polarity spacer between the aptamer sequence and the biotin flag. ARAA-00100001 and ARAA-01700001 were modified at the 5'-end by an alkyne group for subsequent triazole-internucleotide dimerization. The molecular weight, purity, and integrity were verified by HPLC-MS. The procedure described in Example 1 was used to prepare the bispecific anti-PSMA A10 and anti-CAR PNE aptamers. The results of the click reaction are shown in Figure 8A.

[0079] [Example 10] Determination of affinity and specificity of anti-PSMA x anti-CAR PNE bispecific aptamers for targets expressed on cells The affinity and specificity of anti-PSMA x anti-CAR PNE aptamers targeting proteins expressed on cells was evaluated by flow cytometry. These studies were performed on PSMA-positive LNCaP (human prostate cancer-ATCC CRL-1740) and PSMA-negative PC-3 (human prostate cancer-ATCC CRL-1435) in 5% FBS-containing DPBS buffer as described in Example 3. Aptamers were tested within a single concentration range: 30, 100 and 300 nM. The results of the binding studies to PSMA-positive cells are shown in Figure 10A. Along with the A10 monomeric aptamer, two RNA / RNA aptamers were analyzed: A10 x ARAA-00100001, A10 x ARAA-01700001. For comparison, the binding of the tested reagents to PSMA-negative PC-3 cells was also measured.

[0080] Dose-dependent binding to PSMA-positive LNCaP cells was observed for A10 without reaching signal saturation at the highest concentration tested. The intensity of the signal was as strong as the antibody control. Residual binding of A10 monomer to PC-3 cells was observed only at the highest concentration tested. Both bispecific PSMA x CAR PNE aptamers showed similar binding characteristics to A10 monomer, but with reduced residual binding to PSMA-negative cells, improving specificity for target-positive cells. At each concentration tested, the signal intensity of the bispecific aptamer was superior to that measured with A10 monomer, suggesting improved affinity through heterodimerization. The results of Examples 9 and 10 suggest that heterodimerization of aptamers selected against different targets does not significantly affect the binding properties of each moiety when assessed separately.

[0081] [Example 11] Biological activity of bispecific aptamers specific for CAR-PNE and PSMA Cytotoxicity assays are performed on unstimulated peripheral blood mononuclear cells (PBMCs). Freshly prepared PBMCs are isolated from buffy coats obtained from healthy donors (French Blood Facility, Launes-Alpes department). After dilution of blood with DPBS, PBMCs are separated on a FICOLL density gradient (FICOLL-PAQUE PREMIUM 1.077 GE Healthcare), washed twice with DPBS, resuspended in RPMI-1640 medium (Gibco Invitrogen) and diluted with 5x10 6 Obtain a cell density of 10 cells / ml These PBMCs are transduced with a lentiviral vector expressing the CAR-PNE receptor. These PBMC-CAR-PNEs are used as effector cells.

[0082] LNCaP target cells are labeled with 2 μM calcein AM (Trevigen Inc, Gaithersburg, MD, USA) for 30 min at 37 °C in cell culture medium. Calcein AM fluorochrome is a dye that is trapped inside living LNCaP cells and released only upon redirected lysis. After washing twice with cell culture medium, cells are cultured at 5 × 10 5 Adjust the cell density to cells / ml and use an aliquot of 50,000 cells in 100 μl per assay reaction. A standard reaction at 37 °C / 5% CO2 lasts for 4 hours and contains 5x10 4 Calcein AM labeled target cells, 5x10 5 1000 PBMCS-CAR-PNE (E / T ratio 1:10), 20 μl of bispecific aptamer solution are used at 1 μM in a total volume of 200 μl. After the cytotoxicity reaction, the dye released in the culture medium is quantified with a fluorescence reader (VarioSkan Lux, ThermoFisher, Waltham, MA, USA) and compared with the fluorescence signal from a control reaction in which no cytotoxic compound is present and a reaction in which the fluorescence signal was determined for completely lysed cells (the aptamer was replaced by A100 reagent purchased from Chemometec, Allerod, Denmark). Based on these measurements, the specific cytotoxicity is calculated according to the formula: [Fluorescence (sample)-Fluorescence (control)] / [Fluorescence (total lysis)-Fluorescence (control)]x100.

[0083] The results of the cytotoxicity assay obtained after 4 hours of incubation in the presence of 100 nM of aptamer with a single E:T ratio of 10:1 are determined. Specific cytotoxicity is measured with the RNA / RNA aptamer A10xPNE, which induces killing of more than 30% of LNCaP cells. The control monomeric A10, lacking the PNE binding moiety, does not induce cytotoxicity. The results indicate that the designed aptamer switch can attract effector T lymphocytes to target cells, redirect the cytolytic machinery, and eliminate specific cell populations.

[0084] Example 12: Treatment of cancer in preclinical models with anti-CD3x anti-PSMA aptamers The in vivo efficacy and toxicity of different multimeric aptamer constructs are evaluated in comparison to monomeric aptamers in mice. Adult mice bearing PSMA-positive tumors are administered aptamers that specifically bind to CD3 and PSMA, and in different groups of mice, the aptamers are either monomeric or multimeric. Efficacy is assessed by measuring tumor size, tumor growth and rate, and survival rates in treated and control groups. Toxicity is assessed by the incidence of side effects in treated and control groups.

[0085] Example 13: Treating cancer in preclinical models with CAR-T aptamer switches The in vivo efficacy and toxicity of the switch aptamer constructs are evaluated in comparison to monomeric aptamers in mice. Multimeric aptamers are prepared as switches to turn on the activity of CAR T cell-based therapeutics. Tumor-bearing adult mice are first injected with T cells transduced with CAR PNEs, and then with multimeric aptamers made from anti-CAR PNE aptamers fused with PSMA, or HER2, or CD19, or CD20, or CD22 tumor-associated targets. Efficacy is evaluated by measuring tumor size, tumor growth and its rate, as well as survival rates in treated and control groups. Toxicity is evaluated by the incidence of side effects in treated and control groups.

[0086] As used herein, "consisting essentially of" permits the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. As used herein, the term "comprising" may be replaced with "consisting essentially of" or "consisting of," particularly in describing components of a composition or in describing a device component. While the present technology has been described in conjunction with certain preferred embodiments, those of skill in the art, after reading the foregoing specification, may make various modifications, substitutions of equivalents, and other alterations to the compositions and methods described herein.

[0087] References Dassie JP, et al., "Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors." Nat. Biotechnol 27, 839 (2009). Di Stasi A, et al., "Inducible apoptosis as a safety switch for adoptive cell therapy," N Engl J Med 365, 1673 (2011). McNamara JO, et al., "Cell-type-specific delivery of siRNA by aptamer-siRNA chimeras," Nat. Biotechnol 24, 1005 (2006). Rodgers DT, et al., "Switch-mediated activation and retargeting of CA-T cells to B-cell malignancies," PNAS 113, E459 (2016). Straathof KC, et al., "An inducible caspase-9 safety switch for T cell therapy," Blood 105, 4247 (2005). Tamada K, et al., "Redirecting genetically modified T cells to various cancer types using tag antibodies." Clin Cancer Res 18, 6436 (2012).Urbanska K, et al., "A universal strategy for adoptive immunotherapy of cancer by using novel T cell antigen receptors." Cancer Res 72,1844 (2012a).Urbanska K, et al., "Development of novel universal immune receptors for antigen targeting: towards infinity and beyond," OncoImmunology 1, 777 (2012b). Wu CY, et al., "Remote control of therapeutic T cells via small molecule-gated chimeric receptors," Science 350, aab4077 (2015).

Claims

1. 1. An aptamer bridge for binding a chimeric antigen receptor (CAR) to a target, the bridge comprising one or more CAR-binding aptamers and one or more target-binding aptamers, wherein the one or more CAR-binding aptamers are connected to the target-binding aptamers by one or more linkers, at least one of the one or more CAR-binding aptamers is bound to an extracellular domain of the CAR, wherein binding of the CAR-binding aptamer of the bridge to a CAR expressed on an immune cell activates the immune cell, and wherein the CAR extracellular domain is capable of specifically binding a peptide neoepitope (PNE).

2. 2. The aptamer bridge of claim 1, wherein the linker comprises or consists of a linker moiety selected from the group consisting of a covalent bond, a single-stranded nucleic acid, a double-stranded nucleic acid, a peptide, a polypeptide, an oligosaccharide, a polysaccharide, a synthetic polymer, a hydrazone, a thioether, an ester, a triazole, a nanoparticle, a micelle, a liposome, a cell, and combinations thereof.

3. An aptamer bridge as described in claim 1 or 2, which does not contain a peptide or polypeptide.

4. (i) the linker moiety comprises a single- or double-stranded nucleic acid selected from DNA, RNA, and XNA; or (ii) the linker moiety is a polysaccharide selected from the group consisting of glucan, dextran, glycogen, starch, and derivatives thereof; or (iii) the linker moiety is a synthetic polymer selected from poly(amidoamine) (PAMAM) and poly(beta amino ester) (PBAE); The aptamer bridge of claim 2.

5. The aptamer bridge of any one of claims 1 to 4, wherein the CAR extracellular domain comprises an immunoglobulin polypeptide or a fragment thereof, and the one or more CAR-binding aptamers comprise an aptamer that binds to the immunoglobulin polypeptide or a fragment thereof.

6. (i) the PNE has an amino acid sequence not found in the human proteome; or (ii) the PNE is selected from the group consisting of SEQ ID NOs: 1-7; The aptamer bridge of claim 1 .

7. 7. The aptamer bridge of any one of claims 1 to 6, wherein at least one target-binding aptamer binds to a target selected from the group consisting of CD19, CD20, CD22, CD123, BCMA, NY-ESO-1, mesothelin, PSA, PSMA, MART-1, MART-2, Gp100, tyrosinase, p53, ras, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, EGFRvIII, BRCA1 / 2, CD70, CD73, and mutated SOD.

8. The aptamer bridge of any one of claims 1 to 7, comprising two or more identical or non-identical CAR-binding aptamers.

9. The aptamer bridge of claim 8 , comprising two or more identical or non-identical target-binding aptamers.

10. (i) comprises a molecular morphology selected from the group consisting of cyclic, star-shaped, dendrimeric, linear, branched, and combinations thereof; and / or (ii) having a distance between one or more of the CAR-binding aptamers and one or more of the target-binding aptamers within the range of 70-200 angstroms; and / or (iii) binds to said CAR when expressed in a T cell, a NK cell, a B cell, or a macrophage; and / or (iv) the one or more CAR-binding aptamers and the one or more target-binding aptamers each have a binding affinity of less than 10 nM, less than 1 nM, less than 100 pM, or less than 10 pM; An aptamer bridge according to any one of claims 1 to 9.

11. The aptamer bridge of claim 10, wherein in (iii), upon binding to the CAR, it activates the T cell, NK cell, B cell, or macrophage.

12. A system for immunotherapy comprising the aptamer bridge according to any one of claims 1 to 11 and an immune cell expressing the CAR.

13. 13. A kit for providing immunotherapy to a subject comprising the aptamer bridge of any one of claims 1 to 12 and a viral vector encoding the CAR.

14. The kit of claim 13 , wherein the viral vector is a lentiviral vector.

15. The kit according to claim 13 or 14, further comprising an aptamer having a complementary sequence to the CAR-binding aptamer or a portion thereof.

16. 1. A kit for providing immunotherapy to a subject, comprising: (i) a viral vector encoding a CAR for expression in an immune cell in vivo or ex vivo; and (ii) an aptamer capable of high affinity binding to the CAR.

17. The kit of claim 16 , wherein the viral vector is a lentiviral vector.

18. 13. A kit comprising an aptamer bridge according to any one of claims 1 to 12 and a labelled and / or unlabelled CAR-binding aptamer that binds to the same CAR as the CAR-binding aptamer of said bridge but does not bind to the target bound by said bridge.

19. 13. A kit comprising an aptamer bridge according to any one of claims 1 to 12 and a labelled and / or unlabelled target-binding aptamer that binds to the same target as the target-binding aptamer of said bridge but does not bind to the CAR bound by said bridge.

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