CN112955554A

CN112955554A - Aptamer-based CAR T cell switch

Info

Publication number: CN112955554A
Application number: CN201980049961.2A
Authority: CN
Inventors: A·米奥德克; F·毛尔兰; C·包切; R·瓦扬; P·毕晓普
Original assignee: Oratinga Tnp Biology
Current assignee: Yi Shakafaguo
Priority date: 2018-07-26
Filing date: 2019-07-26
Publication date: 2021-06-11
Also published as: EP3827083A2

Abstract

The present invention provides an aptamer-based switching technology that can enhance control of use of Chimeric Antigen Receptor (CAR) -related immunotherapy. The aptamer-based switch utilizes a synthetic bridging molecule that includes a target-binding aptamer that is bound to a CAR-binding aptamer by a linker. A system comprising a CAR and a corresponding aptamer bridge, which provides an immunotherapy platform: (i) any desired antigen can be targeted by selecting target-binding aptamers of the aptamer bridge; (ii) can be redirected from one target to another by altering the target-binding aptamer; (iii) by changing the administration scheme of the aptamer bridge, the administration can be carried out according to the requirements of individual patients changing along with time; (iv) can be started or shut down rapidly or gradually; (v) can be used as a companion diagnostic for specific CAR treatments; (vi) can integrate with CAR expression in vivo or ex vivo; (vii) is non-immunogenic; (viii) the production cost is low.

Description

Aptamer-based CAR T cell switch

Background

Chimeric Antigen Receptors (CARs) utilize single-chain variable domain (scFv) antibodies to confer upon a patient's T cells the ability to recognize and kill cancer cells. However, despite early successful application, the use of CAR T cells is complicated by the difficulty of controlling the immune response in vivo, which may lead to unwanted over-release of cytokines and/or undesirable toxicity. Furthermore, off-target reactions may occur and antigen escape from the tumor may create a need for subsequent CAR T cell therapy with modified antigen specificity. Therefore, strategies need to be developed to enhance the interaction to increase specificity, redirect CARs to new antigens, or develop "off or" kill "switches to inhibit or terminate CAR T cell therapy.

Switchable CAR T cell approaches have been developed that allow for the administration of adaptive molecular switches to patients that mediate the interaction between CAR T cells and target cells. Without this switch, a CAR-mediated immune response would not occur. For example, kill switches can be used to eliminate CAR-T cells and prevent toxicity (strathof et al, Di Stasi et al). Another approach utilizes split chimeric receptors that associate in the presence of small molecule switches (Wu et al). Antibody-based switches have also been developed that mediate the interaction between the CAR and the target (Tamada et al, Urbanska et al (2012a), Urbanska et al (2012 b)). In variants of the antibody switching technique, the target-specific antibody may be integrated with a Peptide Neoepitope (PNE) that binds the scFv portion of the CAR (Rodgers et al). The PNEs are distinct from endogenous epitopes and enable modular switching approaches, where new switches can be designed by combining a single PNE and anti-PNE CAR with different target-specific antibodies. However, this approach still requires complex engineering of the antibody molecule and creates the possibility of an immune response to the switch, especially if the scFv is not fully humanized.

SUMMARY

The present invention provides an aptamer-based switching technology that results in a high degree of control when performing Chimeric Antigen Receptor (CAR) -related immunotherapy. At the most basic level, the aptamer-based switches or aptamer bridges of the invention utilize a synthetic bridge molecule that comprises a target-binding aptamer linked to a CAR-binding aptamer by a linker. A system comprising a CAR and a corresponding aptamer bridge provides an immunotherapy platform that: (i) any desired antigen can be targeted by selecting target-binding aptamers of the aptamer bridge, (ii) can be redirected from one target to another by altering the target-binding aptamers; (iii) the dosage can be determined according to the varying needs of the individual patient over time by varying the dosing regimen of the aptamer bridge; (iv) can be opened or closed rapidly or gradually; (v) can be used as a companion diagnostic for specific CAR treatments; (vi) can integrate with CAR expression in vivo or ex vivo; (vii) is non-immunogenic; (viii) has low production cost.

The present invention provides a cell-redirecting aptamer (e.g., a bispecific or multivalent aptamer) that can be used as an aptamer bridge in an aptamer-based CAR immunotherapy system, as well as for in vivo or in vitro genetic modification of cells. The aptamer bridges, cells, kits and methods of the invention are useful for a variety of uses, including as immunotherapies for the treatment of cancer (e.g., hematologic or non-hematologic, single cell or solid tumors), autoimmune diseases (e.g., arthritis, myasthenia gravis, pemphigus), neuroinflammatory diseases, ophthalmic 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 may also be used to provide diagnostic agents, kits and methods for use in such immunotherapy, including imaging, cell trafficking assays, and research and development of new immunotherapies, as well as to provide prophylaxis when combined with stem cell therapy (e.g., HSCT).

As used herein, a "chimeric antigen receptor cell" or "CAR cell" is a genetically modified cell (e.g., T cell, NK cell, monocyte, or other) that is manipulated in vitro or in vivo to express a single chain variable domain (scFv) antibody that is fused via a stem or transmembrane domain to the intracellular domain of a receptor (e.g., CD3-TCR) to confer the ability of the cell to recognize and bind one or more specific antigens and activate a cellular immune response (e.g., kill cancer cells or destroy cells infected with a virus).

As used herein, "antigen loss" or "antigen escape" may refer to any of several mechanisms that are resistant or adaptive to immunotherapy, such as down-regulation of tumor antigens or up-regulation of inhibitory ligands (e.g., PD-L1, TIM3, LAG3) that result in CAR-T cell failure, failure of CAR cells to reach their target (e.g., tumor site), immunity to the antibody portion of the CAR (e.g., T cell response to scFv, particularly if it is not fully humanized), CAR-T cell adaptation (i.e., decreased likelihood of memory self-renewal and increased propensity to failure), or antigen splicing or mutation.

Candidate aptamers are a group of nucleic acids having different sequences from which the desired aptamer is selected. The source of the candidate aptamer may be a naturally occurring nucleic acid or fragment thereof, a chemically synthesized nucleic acid, an enzymatically synthesized nucleic acid, or a nucleic acid made by a combination of the foregoing techniques.

The nucleic acid for use in the present invention may be, for example, a DNA, RNA or XNA (heterologous nucleic acid) molecule, including any chemical modification thereof, and may be single-stranded, double-stranded or a mixture of single-stranded and double-stranded.

The target is a building block that binds to the aptamer, usually a component of a biological entity. The target may be a molecule, such as a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, drug, nutrient, or growth factor, and the like. The target may be a portion of a cell or a cell, such as a T cell, NK cell, monocyte, B cell, tumor cell, or a cell that is pathologically altered (e.g., infected by a virus or other microorganism, or altered due to a genetic rearrangement). The target may also be an organ or organelle.

Binding affinity refers to the strength of an interaction between two or more structures that are capable of reversibly binding to each other, such as ligands and receptors. Binding affinity is the binding rate (k)_on) And dissociation rate (k)_off) A measure of the dynamic equilibrium of the ratio of (a) to (b), and can be measured or estimated by any known method, including determining the dissociation constant (Kd) or association constant (Ka), for example by concentration-dependent determination from binding, or determining the gibbs dissociation free energy (Δ Gd). The increase in binding affinity is due to the strengthening of intermolecular forces between interacting structures (e.g., ligand and receptor) and results in increased residence time at the binding site. Ligands exhibiting higher binding affinities may have higher "on" rates and/or lower "off rates. Enhanced binding affinity is advantageous in therapeutic products because sufficient receptor activity can be obtained with lower concentrations of ligand. Binding affinity can be determined by the collective strength of multiple affinities of a single non-covalent binding interaction in a multivalent structure. The overall binding affinity of a multivalent ligand depends on its individual binding affinity, the valency of the ligand and receptor, and the spatial arrangement of the interacting structures. However, the overall binding affinity of the multimeric construct is not just the average of the individual binding affinities of the individual aptamers. Factors that influence the affinity of the multimeric structure include the stereochemical fit between the ligand and the receptor, the size of the contact area between them and the charged groupsDistribution of groups and hydrophobic groups.

Safety refers to the collection of adverse effects associated with a drug, therapy, or intervention, and the potential for such adverse effects. Lower frequency and/or less severe adverse reactions and/or adverse events associated with the same level of therapeutic benefit may lead to better safety. Better safety 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.

An immune response refers to the induction of a humoral and/or cell-mediated response to an antigen. The humoral component may include the production of antibodies specific for the antigen, while the cell-mediated component may include the production of delayed-type hypersensitivity and toxic effector cells to the antigen. Immune cells participate in immune responses by a variety of means, including direct interaction with antigens, interaction with other cells of the immune system, and by release and/or reaction with cytokines.

Activation or stimulation of the immune system may be 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 the activation and maturation of antigen presenting cells such as dendritic cells. It may also be mediated by the blocking of inhibitory pathways, for example by the inhibition of immune checkpoint molecules.

Brief description of the drawings

FIG. 1 shows a schematic representation of several representative embodiments of aptamer bridges of the invention.

Figure 2 shows a schematic of a system and method for CAR T cell therapy using in vivo expression of the CAR platform and targeting mediated by aptamer bridges.

Fig. 3 is a schematic diagram of a method of target redirection according to the present invention.

FIG. 4 is a sequence listing of peptide neoantigens and monomeric aptamers.

Figure 5 shows some predicted secondary structures of monomeric aptamers.

FIG. 6 shows a scheme for click chemistry ligation of aptamers.

FIGS. 7A and 7B show the binding of anti-PSMA (7A) and anti-CD 3(7B) aptamers to cells expressing and not expressing the respective antigens.

FIGS. 8A and 8B show agarose gels of monomeric and dimeric (bispecific) aptamers.

FIG. 9 shows the time course of RNA aptamers in serum.

Fig. 10A and 10B show the binding affinity of bispecific aptamers to PSMA positive and negative cells.

Fig. 11A and 11B show the binding affinity of bispecific aptamers to CD3 positive and negative cells.

Figure 12 shows the cytotoxicity of bispecific aptamers against PSMA-positive cells.

FIG. 13A shows an alignment of several anti-PNE RNA aptamers.

FIG. 13B shows the predicted secondary structures of two RNA aptamers from 13A.

Figures 14A-14C show the binding of anti-CAR PNE aptamers to cells that do not express a receptor, express a CD19 CAR, or PNE CAR.

Figure 15 shows the stability of anti-CAR PNE RNA aptamer in serum.

Figure 16 shows the binding of anti-CAR PNE aptamers to Peripheral Blood Mononuclear Cells (PBMCs) transduced with CAR-PNE.

Detailed Description

The present invention provides aptamer-based switches for immunotherapy, such as CAR T cell therapy. The aptamer switch serves as a physical bridge between the CAR-expressing immune cell and the target cell (e.g., cancer cell or pathogen cell). However, aptamer switches or aptamer bridges also serve as sensitive and rapidly adapting tools to modulate the specificity and intensity of immune responses. Since both CAR binding and target binding functions are performed by aptamers, aptamer bridges can be selected quickly and adapted to the evolving needs of in vitro patients at a faster rate and at a lower cost, which is not comparable to antibody-based approaches. Furthermore, the aptamer bridges of the invention can be combined with direct injection of the CAR-encoding vector, e.g., from polymer-coated viral vector particles, and in vivo transduction of selected immune cells (i.e., transduction in an immunotherapeutic subject) and expression of the CAR, which can further save time and cost and enable advanced immunotherapy compared to traditional ex vivo transduction and CAR T cell expansion.

The cell-redirecting aptamer technologies (e.g., bispecific or multivalent binding) and aptamer bridges of the invention, as well as systems and kits containing the same, can be used in a variety of applications, including autologous or heterologous cancer treatments and immunotherapy. The cell-redirecting aptamer technology, aptamer bridge, aptamer-based CAR immunotherapy systems, kits and methods of the invention can be used for a variety of immunotherapies, including immunotherapy for the treatment of cancer (e.g., hematologic or non-hematologic, single cell or solid tumors), autoimmune diseases (e.g., arthritis, myasthenia gravis, pemphigus), neuroinflammatory diseases, ophthalmic diseases, neurodegenerative diseases (e.g., ALS, huntington's disease, alzheimer's disease), neuromuscular diseases (including duchenne's muscular dystrophy, SMA), infectious diseases (e.g., HIV, HSV, HPV, HBV, ebola, tuberculosis, cryptococcus), and metabolic diseases (e.g., type 1 diabetes). They may also be used to provide diagnostic agents, kits and methods in such immunotherapy, including imaging, cell trafficking assays, and research and development of new immunotherapy, as well as to provide prophylaxis when combined with stem cell therapy (e.g., HSCT).

Aptamer bridges can enhance immunotherapy, such as cell-mediated immune responses against cancer or infection, by improving the quality and intensity of the immune response. In addition, aptamer bridges can be used to direct generic CARs to many different targets, as well as redirect immune responses to different targets. Aptamer bridges can also be used to modulate the intensity of an immune response, or to turn a response on or off, simply by administering a different targeted aptamer bridge or adjusting the dose or time of administration of the aptamer bridge over time. A further use of aptamer bridges is to convert inhibitory ligands such as PDL-1 into an immunostimulatory effect, i.e. by replacing the target-binding aptamer of the aptamer bridge with an aptamer that binds the inhibitory ligand, thereby converting the inhibitory ligand into a CAR-binding ligand.

CAR therapy according to the invention generally begins with introducing CAR-expressing cells into a subject in need of immunotherapy. CAR cells can be obtained ex vivo, for example, by transducing cells isolated from a subject with a viral vector, or transfecting them with a plasmid or transposon, allowing the CAR to be expressed on the cells, then expanding the cells in culture and introducing the expanded cells into the subject, for example, by intravenous injection. Alternatively, a viral vector encoding the CAR, e.g., a lentiviral vector, can be administered to the subject, e.g., by intravenous injection, whereby the CAR is expressed in a desired immune cell (e.g., an autologous or allogeneic T cell, NK cell, B cell, monocyte, macrophage, or dendritic cell). In either method, once a baseline of CAR-expressing cells is established in the subject, CAR cells will multiply more when activated by binding of the target to the scFv portion of the CAR.

Administering a suitable aptamer bridge to a subject containing a sufficient number of CAR cells will elicit an immune response against the target-binding aptamer-specific target of the aptamer bridge upon binding of the CAR-binding aptamer of the aptamer bridge to the CAR. The intensity of the resulting immune response, which may vary over time, is a function of the number of CAR cells in the subject, the contact of the CAR cells with the target, and the concentration of aptamer bridges on the target. The intensity of the immune response can be modulated up or down by modulating the number of CAR cells, and/or modulating the concentration of the bridge by increasing or decreasing the amount of bridge administered or the time interval between administrations.

In the treatment methods of the invention, the total amount of CAR cells in the subject is estimated prior to administration of the aptamer bridge to begin immunotherapy, and a dosing regimen (e.g., dose, number of doses, and/or dosing interval) is designed (e.g., using an algorithm) to produce the desired intensity and/or duration of the immune response. The intensity of the immune response can be monitored using known methods, e.g., using Fluorescence Activated Cell Sorting (FACS) analysis to quantify the number and type of certain activated immune cells in a blood sample from a patient, or by determining the level of certain cytokines in such a sample. If the immune response is too strong, potentially dangerous to the patient, the intensity of the immune response may be reduced by reducing the amount or frequency of dosing of the aptamer bridge, or stopping dosing altogether, or by administering a "kill" switch comprising a single CAR-binding aptamer (that disrupts the binding of the aptamer bridge to the CAR), a peptide or antibody that binds to the CAR and disrupts the binding of the aptamer bridge, a single target-binding aptamer (that disrupts the binding of the aptamer bridge to the target), or a soluble domain or epitope from the CAR or target antigen.

The aptamer bridges of the invention can be used to redirect CAR cells to different targets. This may be useful where the first target has antigen escape or a weak response to the first target, requiring different or additional targets to be pursued. In one embodiment of the method, a different (e.g., second or later) aptamer bridge is administered to the subject, having the same or similar CAR-binding aptamer as the first aptamer bridge, but a different target-binding aptamer. In another embodiment, a conventional CAR that directly targets a first target (i.e., without the use of a CAR switch) is expressed on a cell of a subject and an aptamer bridge is administered to the subject to redirect the CAR response to a different (second or higher) target; the aptamer bridge will include a CAR-binding aptamer specific for a target-binding CAR and a target-binding aptamer, wherein the two aptamers are bound by a linker. The method may be applied several times (e.g., 2, 3, 4, 5 or more times) sequentially or simultaneously to different targets in the same subject to modulate immunotherapy or to find the optimal target or combination of targets. One embodiment of the aptamer bridge of the invention is conjugated to a detectable label for imaging or quantification of CAR cells or targets in a subject. For example, the bridge can include a linker attached to the CAR-binding aptamer or target-binding aptamer¹⁸Part F for PET scanning. The same method can be carried out using a single aptamer (CAR binding or target binding) for treatment in an aptamer bridge. For example, the method can be used to quantify CAR cells in a subject before or during the start of treatment.

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

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 Ligangs, Lewis-Y, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, Her3, EGFRvIII, BRCA1/2, CD70, CD73, CD16A, CD40, VEGF-beta, VEGF, TGF-beta, CD32B, CD79B, cMet, PCSK9, IL-4RA, IL-17, IL-23, 4-1BB, LAG-3, CTLA-4, PD-L1, PD-1, 40, or an OX mutation. Components of the aptamer bridge aptamers can also specifically bind to combinations of these targets. In some embodiments, the target is an antigen of an infectious agent, such as gag, reverse transcriptase, tat, HIV-1 envelope protein, circumsporozoite protein, HCV non-structural protein, hemagglutinin; aptamer bridges can also specifically bind to combinations of these targets.

In a preferred embodiment, the CAR-binding aptamer is selected to specifically bind to the extracellular domain of a CAR having affinity for a Peptide Neoepitope (PNE), i.e., an anti-PNE CAR. Since PNE is an epitope that is not present in the subject, immune cells expressing an anti-PNE CAR are not activated by endogenous biomolecules, but wait for the administration of an aptamer bridge to the subject, which acts as a "turn on" switch for the immune cells and targets the CAR-expressing cells to the desired antigen or cell type bearing the antigen. Immune activation and in vivo expansion of CAR-expressing immune cells can be shut down by administering to the subject a CAR-binding aptamer or target-binding aptamer (in any form) containing a bridge of a peptide or monomer form of PNE, either of which will terminate target activation of CAR-expressing immune cells.

The PNE may be any peptide epitope not found in the host proteome (e.g., not found in the human proteome), and thus an anti-PNE CAR may be obtained. An example of a preferred PNE is a peptide fragment of the GCN4 transcription factor from Saccharomyces cerevisiae (Saccharomyces cerevisiae) (NYHLENEVARLKKL, SEQ ID NO: 1). Rodgers et al describe CAR binding to GCN4 with high affinity (Kd ═ 5.2pM) and comprising a 52SR4 single chain antibody. Other PNEs suitable for use with CARs and corresponding aptamer bridges include: (i) n-terminal 15-polypeptide ESQPDPKPDELHKSS (SEQ ID NO: 2) of Staphylococcal (Staphylococcus) enterotoxin B, paired with antibodies bound thereto, and described in Clin. vaccine immunol.17(11): 1708-1717; (ii) deoxynivalenol (deoxynivalenol), an escherichia coli mycotoxin, is paired with scFv conjugated thereto and is described in Protein expr. purif.35(1): 84-92; (iii) HPV-16 protein E5, paired with an antibody that binds thereto, described in biomed. res.int.2018; 2018: 5809028; (iv) a rabies virus Protein and scFv to which it binds, as described in Protein Expression and Purification 86(2012) 75-81; (v) an influenza a matrix protein (influenza a matrix protein), paired with an scFv that binds thereto, and described in Bioconjugate chem.2010,21, 1134-; (vi) amino acid 134-145 of the HBV pre S2 protein (PRVRGLYFPAGG, SEQ ID NO: 3), paired with scFv bound thereto, is described in Viral Immunol.2018May 30; (vii) VP 3 peptide of duck hepatitis virus type 1, paired with scFv described in J.of viral Methods 257(2018) 73-78; (viii) peptide of glycoprotein D from bovine herpes virus 1 (MEESKGYEPP, SEQ ID NO: 4), with Appl Microbiol Biotechnol. 2017 Dec; 101(23-24) 8331-8344; (ix) peptides comprising amino acid 159 of the South African type 2 (South African terrorises 2, SAT2) foot-and-mouth disease Virus VP1 protein, paired with scFv conjugated thereto, are described in Virus Research 167(2012) 370-379; (x) The peptide fragment of Salmonella typhimurium OmpD (DRTNNQVKA, SEQ ID NO: 5), paired with the scFv bound thereto, is described in Veterinary Microbiology 147(2011) 162-169; (xi) Peptides of transferrin from E.coli, paired with scFv bound thereto, described in Journal of Biotechnology 102(2003) 177/189; (xii) The peptide at the N-terminus of the grapevine leaf roll associated virus No. 3 coat protein (AQEPPRQ, SEQ ID NO: 6), paired with the scFv bound thereto, is described in Arch.Virol, (2008)153: 1075-1084; (xiii) The N protein peptide of SARS-CoV (PTDSTDNNQNGGRNGARPKQRRPQ, SEQ ID NO: 7), paired with scFv bound thereto, is 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, paired with a scFv conjugated thereto, and described in j.virol.2004 apr; 3792-3796; (xv) Peptides from the HCV NS3 helicase domain amino acids 1363-1454, paired with scFv bound thereto, are described in J.hepatology 37(2002)660-668, J Virol 1994; 68: 4829-4836 and Arch Virol 1997; 142: 601-610.

Other examples of generic CARs that can be paired with the aptamer bridge of the invention are described in j.autoimmun.2013may.42: 105-16; blood Cancer J.2016Aug,6(8): e 458; Oncotarget.2017Dec 12,8(65): 108584-108603; oncotarget 2017May 9,8(19): 31368-31385; oncotarget 2018Jan 26,9(7): 7487-7500; and WO 2016030414.

The linking moiety of the aptamer bridge may simply be one or more covalent bonds between individual aptamers, or may be a synthetic or naturally occurring polymer, such as a hydrocarbon, polyether, polyamine, polyamide, nucleic acid, peptide, carbohydrate, or lipid. In certain embodiments, the linking moiety is not a peptide. In certain embodiments, the aptamer bridge is free of peptides, and free of polypeptides and proteins. The linking moiety may also take the form of a nanoscale structure (e.g., a polymer, protein, nanoparticle, nanotube, nanocrystal, nanowire, nanobelt, nanocrystal, micelle, or liposome), or a microscale structure (e.g., a microbead or cell), or a larger structure (e.g., a solid support). Preferably, the linking moiety is a biodegradable polymer. The linking moiety may be a polymer that is linear, branched, cyclic, or a combination of these structures. The linking moiety may also serve as a backbone for a dendritic structure, or a hub or star structure (e.g., a core structure to which two or more aptamers are bound). For non-covalent binding, two or more separate aptamers may bind directly between aptamers through non-covalent interactions or through interaction with a linking moiety. The non-covalent interaction may be, for example, one or more hydrogen bonds, ionic bonds, hydrophobic bonds, van der waals interactions, or a combination thereof. High affinity binding pairs, such as streptavidin-biotin, can be used to non-covalently link aptamers in aptamer bridges.

The linker or linking moiety may be any chemical moiety that covalently or non-covalently links together the monomeric aptamer units. The linker may comprise or consist of, for example, an oligonucleotide, a polynucleotide, a peptide, a polypeptide or a carbohydrate. The linker may comprise or consist of a cellular receptor, a ligand or a lipid. The linker may comprise or consist of a hydrocarbon chain, or a 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 may be a single covalent bond, or may include one or more ionic, hydrogen, hydrophobic, or van der waals interactions. The linker may comprise a disulfide bond, a heparin or heparan sulfate derived oligosaccharide (glycoaminoglycan), a chemical cross-linker, a hydrazone, a thioether, an ester or a triazole. The linker may be cleaved by an enzyme, allowing release of individual aptamers through the aptamer bridge and/or termination of CAR-target interactions. The linker may have a net positive, negative or neutral charge. The linker can be flexible or rigid as desired to ensure that the functional properties of each monomeric aptamer unit are retained in the multimeric construct and to facilitate binding to the CAR and target, or interaction thereof. The linker may comprise a flexible moiety, for example a polymer of 5-20 glycine and/or serine residues. The linker may also comprise a rigid defined structure, such as a polymer of glutamic acid, alanine, lysine and/or leucine. The connector may include a hinge portionOr a spacer portion. The linker may comprise a substituted or unsubstituted C₂-C₅₀A chain or ring structure, a polyethylene glycol polymer (e.g., hexaethylene glycol), or a modified or unmodified oligonucleotide or polynucleotide. The linker may comprise a heparin or heparan sulfate derived oligosaccharide (glycoaminoglycan), a chemical cross-linker, a peptide, a polypeptide, a hydrazone, a thioether, or an ester.

C₂-C₅₀The linker may comprise a backbone of 2 to 50 carbon atoms (saturated or unsaturated, linear, branched, or cyclic), 0 to 10 aryl groups, 0 to 10 heteroaryl groups, and 0 to 10 heterocyclic groups, optionally comprising ether linkages (e.g., one or more alkylene glycol units including, but not limited to, one or more ethylene glycol units-O- (CH2 CH)₂O) -); one or more 1, 3-propane diol units; an amine, an amide or a thioether. Each backbone carbon atom may be independently unsubstituted (i.e., contain only-H substituents) or may be selected from C₁To C₃Alkyl, -OH, -NH₂、-SH、-O-(C₁To C₆Alkyl), -S- (C)₁To C₆Alkyl), halogen, -OC (O) (C)₁To C₆Alkyl) and-NH- (C)₁To C₆Alkyl) is substituted with one or more groups. In some embodiments, the linker is C₂-C₂₀Linker and C₂-C₁₀Linker and C₂-C₈Linker and C₂- C₆Linker and C₂-C₅Linker and C₂-C₄Linker or C₃A linker, wherein each carbon may be independently substituted as described above.

In certain embodiments, non-covalent bonds are present between aptamers, e.g., mediated by ionic bonds, hydrogen bonds, hydrophobic bonds, van der waals interactions, or combinations thereof, without any intervening linking moieties linking the individual aptamers. A single multimeric aptamer construct may also use a mixture of covalent bonds (linking a particular aptamer through an intermediate linker moiety) and non-covalent bonds, with no intermediate linking moieties at other binding sites between aptamers.

The linker optionally may have one or more functions. For example, in some embodiments, the linker is sensitive to temperature and/or pH, meaning that the linker changes conformation or is cleaved within a predetermined temperature and/or pH range.

Each CAR-binding and target-binding portion of the aptamer bridge may comprise a single aptamer, or may comprise two or more identical or different aptamer units. Using two or more identical ligands, or different ligands that bind to the same target, binding affinity can be greatly enhanced by the binding of the same target or the cooperativity of binding differently presented, particularly for targets, e.g., cells, that have multiple copies of a given target molecule. Identical monomeric aptamers may have identical nucleotide sequences (100% identical), or they may 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.

Each CAR-binding and target-binding portion of the aptamer bridge may comprise two or more separate aptamers that are not identical and bind to the same or different targets. Different aptamers may differ in structure, nucleotide sequence, and/or binding specificity or binding affinity. Such an aptamer bridge can specifically bind to two different sites on the target, e.g., different epitopes of the target protein or different surface proteins of the target cell, or different CARs, thereby increasing affinity and/or specificity for the target, or increasing 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 by a combination of actions involving different target cells and/or different CAR expressing cells.

The function of a CAR cell can be improved by directing it to two or more antigens on a target cell, for example two or more antigens on a given tumor cell (e.g., CD19 and CD22, CD19 and CD123, or CD19 and BCMA). This approach can help overcome the loss of tumor antigens and improve the persistence of the response. By linking two or more aptamers together in a target binding moiety, the aptamer bridge can be engineered to recognize a CAR and two or more antigens on the same target. This approach is similar to engineered T cells expressing both tumor-associated antigen receptors simultaneously, but exceeds the dual target limitations of antibodies, and offers lower cost and greater flexibility than antibody-based CAR switches.

The aptamer bridge constructs of the invention can be used in immunotherapeutic compositions. One embodiment of such a composition includes an aptamer bridge and an excipient. The aptamer bridge may be conjugated to other molecules, or to structures such as liposomes, micro or nanoparticles, receptors, or cell-targeting molecules, or oral, topical, or other agents to facilitate uptake, distribution, and/or absorption of the agent. In some embodiments, the aptamer bridge is encapsulated, conjugated or otherwise associated with a therapeutic agent delivery vehicle. In some embodiments, the therapeutic agent delivery vector is or includes a gene delivery vector, such as a viral vector. The viral vector may 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 preferred embodiments, the vector encoding the CAR is first administered to the subject, and in some CAR cell expansion embodiments, an initial baseline of CAR expression is established in the subject prior to administration of the aptamer bridge to the subject to activate an immune response against the target. CAR expression and optionally the expansion of cells expressing the CAR can be detected and/or quantified in a subject, for example, by administering a labeled form of a CAR-binding aptamer (e.g., the CAR-binding portion of an aptamer bridge), and then determining the binding label in peripheral blood cells or determining the distribution of the label in vivo.

Component aptamers of the aptamer bridge may comprise any desired modification to the structure or sequence of the aptamer. The modification may comprise a substitution introduced in one or more ribose or deoxyribose moieties of the candidate aptamer. The modification may comprise substitution of one or more phosphate moieties of the candidate sequence. The modification may comprise a substitution in one or more purine or pyrimidine moieties of the candidate aptamer, or a substitution with a non-natural or rare natural nucleotideNatural nucleotides of aptamers are selected. Chemical modifications can be introduced into the internucleotide phosphate linkages, for example, replacing the internucleotide phosphates with phosphorothioates or boronates, adding biotin, azide or alkyne groups. Chemical modifications can be introduced at the C2 'site of the ribose ring, such as the introduction of fluorine, LNA (locked nucleic acid) units or 2' -O-alkyl modifications. One or more nucleosides of an aptamer can include modifications selected from the group consisting of sugar modifications at the 2' -position (e.g., 2' -amino (2' -NH)₂) 2 '-fluoro (2' -F) or 2 '-0-methyl (2' -OMe)), a modification of a cytosine exocyclic amine, a modification of an internucleoside linkage, or a modification of 5-methyl-cytosine. Aptamers may include a 3' end cap, a 5' end cap, and/or inverted deoxythymidine at the 3' end. Rare nucleotides such as 2-thiouridine (s2U), pseudouridine (Ψ), and dihydrouridine (D), or unnatural nucleotides such as Peptide Nucleic Acid (PNA), morpholino, Locked Nucleic Acid (LNA), ethylene Glycol Nucleic Acid (GNA), or Threose Nucleic Acid (TNA) may be substituted for the common nucleotides. Non-natural nucleic acids (xenogenic nucleic acids (XNA)) may also be included.

The component aptamers of the aptamer bridge may be obtained using suitable methods for preparing or selecting aptamers against a target. Aptamers can be identified, for example, by systematic evolution of ligands by exponential enrichment (SELEX). SELEX is described, for example, in U.S. patent 5,270,163, which is incorporated herein by reference. Briefly, SELEX begins with a variety of nucleic acids (i.e., candidate aptamer sequences) containing a variable nucleotide sequence that is contacted with a target. Unbound nucleic acids are separated from those that form aptamer-target complexes. The aptamer-target complexes are then dissociated, the nucleic acids are amplified, and the steps of binding, separating, dissociating, and amplifying are repeated through as many cycles as desired to produce a population of aptamers with progressively increasing affinity for the target. The cycle of selection and amplification can be repeated until there is no significant improvement in binding affinity after further cycles of repetition.

The cycle of selection and amplification may be interrupted before individual aptamers are identified. In this case, a population of aptamers is identified that can provide important information about the sequences, structures or motifs that allow the aptamers to bind to the target. Such a population of candidate aptamers may also inform which portions of the aptamers are not critical for target binding. This information may direct the generation of other aptamers to the same target. The aptamers thus produced can be used as input for a new round of SELEX, possibly resulting in aptamers with better binding affinity or other features of interest.

In some embodiments, candidate aptamer sequences are generated that contain multimeric aptamer constructs, such as candidate aptamer bridges, which are then further selected as multimeric constructs. Multimeric candidate aptamer constructs can be prepared by linking a single candidate aptamer moiety to a linking moiety, and optionally using such constructs as inputs for one or more rounds of SELEX. In some embodiments, individual aptamers are independently selected by one or more rounds of SELEX and eventually ligated together with a linking moiety. Thus, multimerization of monomeric aptamer as well as multimeric aptamer constructs can be performed before, during, or after the SELEX procedure.

To select the component aptamers of the aptamer bridge, the selection of aptamers that bind to the CAR may be performed differently than aptamers that bind to the target on the target. The aptamer binding to the target cell can be selected by binding the candidate aptamer to an intact target cell, a recombinant cell, a pathogen cell, a virus-like particle, a purified target protein, a peptide epitope, a membrane prepared from the target cell, or a recombinant cell containing an antigen surface protein. However, selection of aptamers that bind to anti-PNE CARs can be performed by contacting the candidate aptamers with anti-PNE CARs expressed on cells in the presence and absence of PNE. Thus, according to this approach, aptamers with the desired binding affinity to anti-PNE CARs will be those whose binding to the CAR in the absence of PNE can be replaced by the addition of a PNE-containing peptide. However, aptamers may also be selected using peptides from the CAR region that are different (e.g., adjacent) to the PNE binding site, and aptamers selected in this manner may not be substituted by PNE, but may provide high affinity for binding to anti-PNE CARs. Furthermore, to facilitate successful immunotherapy, CAR-expressing cells, e.g., CAR-expressing T cells, should be monitored during aptamer selection. To this end, candidate CAR-binding aptamers may be selected using candidate CAR-binding aptamers and candidate target-binding aptamers installed in aptamer switches.

The component aptamers of the aptamer bridge can be of any desired length. In some embodiments, a component aptamer comprises at least about 15 nucleotides. In some embodiments, the component aptamer comprises up to about 100 nucleotides. The length of the aptamer bridge can determine the distance between cells that interact through the bridge. Thus, the length of the bridge is preferably 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.

Techniques can be used to identify or generate aptamers with any desired binding affinity. In preferred embodiments, one or more selected aptamers have a K binding to the target_dFrom about 1pM to about 10nM [ for high affinity (specific) binding]Or from about 100nM to about 10. mu.M (for low affinity binding). In some embodiments, the aptamer has a K_dFrom about 1pM to about 10. mu.M, from about 1pM to about 1. mu.M, from about 1pM to about 100nM, from about 100pM to about 10. mu.M, from about 100pM to about 1. mu.M, from about 100pM to about 100nM, from about 1nM to about 10. mu.M, from about 1nM to about 1. mu.M, from about 1nM to about 200nM, from about 1nM to about 100nM, from about 500nM to about 10. mu.M, or from about 500nM to about 1. mu.M. In some embodiments, the binding affinity of the component aptamers within the aptamer bridge is 4 to 50 times higher than the affinity of the monomeric candidate aptamer. In some embodiments, the affinity of a component aptamer of an aptamer bridge to one or more targets can be fine-tuned by varying the degree of multimerization of the aptamer bridge, the component aptamer or linker included in the aptamer bridge. In certain embodiments, the CAR-binding portion and/or the target-binding portion of the aptamer bridge is multivalent, meaning that it comprises two or more separate aptamers that bind to the same or different sites on the CAR or target. Multivalent aptamer constructs have the advantage of higher binding specificity and/or higher binding affinity to CARs or targets. Multimeric aptamers can have a much higher binding affinity than single aptamers because binding of multimeric complexes can exhibit positive synergy or positive allosteric effects between the individual binding sites. Careful selection and combination of multimeric aptamer bridge moieties by limiting deleterious or toxic effects and enhancing effective therapeutic efficacyThe ability to bind to sites improves safety.

In some embodiments, the aptamer of the aptamer bridge is stabilized and/or multimerized by complexation with another compound, such as biotin-avidin or polyethylene glycol. In some embodiments, aptamers are synthesized using a solid support.

The aptamer bridge disclosed by the invention can be used for preventing or treating proliferative or infectious diseases. One aspect of the invention is a method of preventing or treating cancer or infection by administering an aptamer bridge, or, alternatively, an immunological formulation or therapeutic delivery vehicle comprising an aptamer bridge.

The invention also provides a method of inducing or enhancing an immune response against a cancer or infectious disease in a subject by administering the aptamer bridge, or, alternatively, an immunological formulation or therapeutic agent delivery vehicle comprising the aptamer bridge.

The invention further provides methods of specifically binding an immune cell expressing a CAR to a target cell in which an immune response or an enhanced immune response is desired. Administration of an aptamer bridge having binding specificity for both the CAR and the molecular target on the target cell results in activation of the CAR-expressing immune cell and initiation or enhancement of an immune response against the target cell.

Examples

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 that has been selected for 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, and CELTIC _ core are DNA aptamers (SEQ ID NOS: 9, 10, and 11), ARACD3-3700006 and ARACD3-0010209 are RNA aptamers (SEQ ID NOS: 12 and 13), all of which were previously selected against human CD 3. These DNA or 2' -deoxy-2 ' -fluoro-thymidine modified RNA (2' F-RNA) aptamers were purchased from baseclick (neuroied, Germany) as single stranded oligonucleotides purified by HPLC-RP synthesized by standard solid phase phosphoramidite chemistry. The anti-CD 3 aptamer was unable to activate cytokine secretion or surface marker expression even when bound to the costimulatory anti-CD 28 antibody, and was different from the anti-CD 3 monoclonal antibody (data not shown).

The a10 aptamer was modified with an azide group at its 3' end for subsequent triazole internucleotide dimerization. Biotin was added as biotin-TEG to the 5' end of the a10 aptamer, which introduced a 16 atom mixed polar spacer between the aptamer sequence and the biotin tag. Cy 5-labeled a10 was also synthesized. CELTIC _1s, CELTIC _19s, CELTIC _ core, ARACD3-3700006 and ARACD3-0010209 are modified at their 5' ends with alkyne groups for subsequent triazole nucleotide dimerization. Molecular weight, purity and integrity were verified by HPLC-MS. The affinity and specificity of a10 against PSMA RNA aptamers were evaluated on PSMA-positive and PSMA-negative cells (fig. 7A).

anti-PSMA a10 and anti-CD 3 aptamers were heterodimerized using Oligo2 Click kit L (baseclick, neuroid, Germany) at 45 ℃ for 60 minutes by a copper-catalyzed Click reaction according to the manufacturer's instructions. The reaction products were separated by gel electrophoresis, migrated on a 3% agarose gel at 100V for 30 min in 1 XTBE buffer (Invitrogen). The gels were visualized using a Bio-Rad imaging system and the results are shown in FIGS. 8A and 8B. Gel sections corresponding to the dimeric aptamers were excised from the gel and extracted for nucleic acids by passive elution in 25mM NaCl TE buffer for 72 hours at 8 ℃. Bispecific aptamer dimers were recovered by standard sodium acetate precipitation, resuspended in sterile water and stored at-20 ℃ until use.

Example 2 functional stability of PSMA-specific aptamer A10

The stability of the a10 RNA aptamer was measured in Dulbecco Phosphate Buffered Saline (DPBS) containing 5% FBS or FBS alone. Biotinylated aptamers were denatured at 85 ℃ for 5 minutes and then immediately cooled on ice for 5 minutes to 4 ℃. Aptamers were then diluted to a final concentration of 2 μ M in DPBS supplemented with 5% FBS or pure FBS. Incubating the sample at 37 ℃ for 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 24 hours; control samples contained freshly prepared aptamers that were not incubated at 37 ℃. 100nM streptavidin-PE was then added to each solution and aptamers were incubated with PSMA-positive LNCaP cells (human prostate cancer-ATCC CRL-1740). Aptamer a10 half-life in DPBS buffer containing 5% FBS or pure FBS was then determined using flow cytometry on the YL-1 channel based on the number of fluorescence positive cells as a function of incubation time at 37 ℃. The measurement results are shown in fig. 9. Aptamer a10 incubated in DPBS buffer containing 5% serum remained stable for 24 hours. When tested in pure serum, half of the binding activity was lost within the first 2 hours of incubation.

Example 3 determination of affinity and specificity of bispecific aptamers against PSMA and CD3 for cell expression targets

The affinity and specificity of the anti-PSMA and anti-CD 3 bispecific aptamers to the target protein expressed on the cells was assessed by flow cytometry. These studies were performed on CD 3-positive Jurkat (acute T cell leukemia human cell line-ATCC TIB-152), CD 3-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 by co-incubation with biotinylated RNA/DNA aptamers in SELEX buffer, or with RNA/RNA aptamers in DPBS buffer supplemented with 5% FBS. Prior to use, cells were cultured in RPMI-1640 medium (Gibco Invitrogen) supplemented with 10% fbs (Gibco Invitrogen) and 1% Penicillin (Penicillin)/streptomycin (Steptomycin) (Gibco Invitrogen). Jurkat, Ramos, LNCaP and PC-3 cells (2.5X 10) were used prior to the experiment⁵One cell/well) were seeded in 96-well plates and centrifuged at 2500rpm for 2 minutes. The supernatant was discarded, and the precipitated cells were washed twice with 200. mu.L of Selex or DPBS-5% FBS buffer preheated at 37 ℃. Each washing step was followed by centrifugation at 2500rpm for 2 minutes. Aptamers were denatured at 85 ℃ for 5 minutes and immediately placed on ice cubes at 4 ℃ for 5 minutes. The test samples were then diluted in two different concentration ranges: 3. 10, 30, 100 and 300nM (CD3 binding assay); and, 30, 100 and 300nM (PSMA binding assay), then 100nM phycoerythrin-labeled streptavidin (streptavidin-PE, eBioscience) was added to each solution. Resuspending Jurkat, Ramos, LNCaP and PC-3 cells in aptamer diluent(100. mu.L/well) in 5% CO₂Incubate at 37 ℃ for 30 minutes in a humid atmosphere. As controls, cells were incubated with CD3 monoclonal antibody (PE-labeled, OKT3 human anti-CD 3, Invitrogen), PSMA monoclonal antibody (Alexa Fluor 488-labeled, GCP-05 human anti-PSMA, Invitrogen), PE-streptavidin, or their respective buffers without added reagents. After incubation, cells were centrifuged at 2500rpm for 2 minutes and the supernatant with unbound sequences was discarded. The precipitated cells were washed with SELEX or DPBS-5% FBS buffer (200. mu.L/well) and centrifuged twice to remove all weakly and non-specifically attached sequences. Then, 1mg/mL salmon sperm DNA solution (100. mu.L/well) was used at 37 ℃ with 5% CO₂Cells were washed in a humid atmosphere. After 30 minutes, the salmon sperm solution was removed by centrifugation at 2500rpm for 2 minutes, and the cells were additionally washed twice with SELEX or DPBS-5% buffer (200. mu.L/well) and then centrifuged. Jurka, Ramos, LNCaP and PC-3 cells with attached DNA or RNA sequences were then fixed (BD CellFIX solution #340181) and fluorescence-positive cells were counted on the YL-1 channel by flow cytometry (AttunnXT; Invitrogen, Inc.).

The results of the binding studies with PSMA-positive cells are shown in fig. 10A and 10B. Three RNA/DNA aptamers (A10 XCTIC _1s, A10 XCTIC _19s, A10 XCTIC _ core) and two RNA/RNA aptamers (A10 XCARCD 3-3700006 and A10 XCARCD 3-0010209) were analyzed together with A10 monomer. For comparison, binding of test agents to PSMA-negative PC-3 cells was also measured. Dose-dependent binding to PSMA-positive LNCaP cells was observed with a10, whereas signal saturation was not reached at the highest concentration tested. The signal intensity was as strong as the antibody control. Residual binding of the a10 monomer to PC-3 cells was only observed at the highest concentration tested. All bispecific PSMA × CD3 aptamers showed similar binding properties to the a10 monomer, but with reduced residual binding to PSMA-negative cells, specificity for target-positive cells increased. For each concentration tested, the signal intensity of the bispecific aptamer was superior to that measured for the a10 monomer, indicating that heterodimerization results in an increase in affinity.

In another experiment, the binding of the same aptamer to CD3 positive Jurkat and CD3 negative Ramos cells was studied. See fig. 11A and 11B. As expected, the a10 aptamer did not bind to both cell lines. Residual binding of anti-CD 3 monomer to Ramos cells was only observed at the highest concentration tested. All PSMA × CD3 bispecific aptamers showed similar dose-dependent binding, but with strongly reduced residual binding to CD3 negative cells, had excellent specificity for target positive cells. For each concentration tested, the signal intensity of the bispecific aptamer was lower than that measured for the anti-CD 3 monomer, indicating that heterodimerization results in a decrease in affinity.

Taken together, these results indicate that after heterodimerization, the binding properties of aptamers selected against different targets are not disrupted when evaluated alone due to steric hindrance. Depending on the chosen partner (partner), it is even possible to improve the specificity and affinity for the respective target after dimerization.

Example 4 measurement of binding of bispecific aptamers targeting PSMA and CD3 by surface plasmon resonance

Binding affinity measurements were performed using a BIAcore T200 instrument (GE Healthcare). To analyze the interaction between aptamers and CD3 and PSMA proteins, 300 resonance units of biotinylated aptamers were immobilized on the S series Sensor chip sa (series S sensors chips sa) (GE Healthcare) according to the manufacturer' S instructions (GE Healthcare). DPBS buffer was used as running buffer. The interaction was measured by the "Single Kinetics Cycle" mode at a flow rate of 30. mu.l/min and by injecting different concentrations of human CD3 ε/γ, CD3 ε/δ, IgG1-Fc and PSMA (Acro biosystems). The highest aptamer concentration used was 300 nM. Other concentrations were obtained by 3-fold dilution. All kinetic data were evaluated using BIAcore T200 evaluation software.

K for monomeric and bispecific aptamers_DComparison of the values shows that dimerization does not interfere with the binding properties of each subunit to its specific target. Simultaneous recording of PSMA and CD3 ε/γ may also be performed by a manual injection mode (flow rate of 10 μ l/min) and by injecting a solution of the first target at saturation concentration first, followed by a solution of the second target at saturation concentrationAnd (6) mixing. A second injection with the reverse sequence was performed. In both sequences, each injection resulting in the same intensity response indicates that both arms of the bispecific aptamer are able to bind the second target while the binding site of the first antigen is occupied. Monomers that failed to respond to injection of both target solutions indicated that bispecific aptamers could bind both targets simultaneously.

Example 5 biological Activity of bispecific aptamers specific for PSMA and CD3

Cytotoxicity assays were performed on unstimulated Peripheral Blood Mononuclear Cells (PBMCs). From a healthy donor (

Sang，Division

) Freshly prepared PBMCs were isolated from the buffy coat. After diluting the blood with DPBS, PBMC were separated with FICOLL density gradient (FICOLL-PAQUE PREMIUM 1.077GE Healthcare), washed twice with DPBS, resuspended in RPMI-1640 medium (Gibco Invitrogen) to obtain 5X 10⁶Cell density of individual cells/ml. These PBMCs were used as effector cells.

LNCaP target cells were labeled with 2. mu.M calcein-AM (Trevigen Inc, Gaithersburg, Md., USA) for 30 minutes at 37 ℃ in cell culture medium. calcein-AM fluorescent dye is a dye that can be trapped inside living LNCaP cells and only be released upon redirected lysis. After washing 2 times in cell culture medium, the medium was adjusted to 5X 10 in RPMI-1640 medium⁵Cell density of one cell/ml and 50,000 cells per assay reaction using 100 μ l aliquots. Standard reaction at 37 deg.C/5% CO₂The duration of the treatment was 4 hours, and a total volume of 200. mu.l of 5X 10 was used⁴ 5X 10 target cells labeled with calcein AM⁵PBMC (E/T ratio 1:10) and 1. mu.M of 20. mu.l of bispecific aptamer solution. After the cytotoxic reaction, the dye released in the medium is quantified by a fluorescence reader (Varioskan Lux, ThermoFisher, Waltham, MA, USA) and reacted with a control for the fluorescent signalIn comparison, no cytotoxic compound was present in the control reaction and its fluorescence signal was dependent on the presence of fully lysed cells (where the aptamers were replaced by a100 reagents purchased from chemimetec, Allerod, denmark). Based on these readings, specific cytotoxicity was calculated according to the following formula: [ fluorescence intensity (sample) -fluorescence intensity (control)]/[ fluorescence intensity (Total lysis) -fluorescence intensity (control)]×100。

The cytotoxicity assay results obtained after 4 hours incubation in the presence of 100nM aptamer are shown in figure 12, where the single E: T ratio is 10: 1. No to weak specific cell killing activity (< 10%) was observed with PSMA × CD3 bispecific RNA/DNA aptamers. Excellent specific cytotoxicity was measured with RNA/RNA aptamers a10 xarcard 3-3700006 and a10 xarcard 3-0010209, which induced killing of 40-50% of LNCaP cells. Control monomer a10, lacking a CD3 binding moiety, did not induce any cytotoxicity.

These results indicate that engineered aptamer switches are able to recruit effector T lymphocytes to target cells to redirect their cytolytic mechanisms and eliminate specific cell populations.

Example 6 anti-CAR PNE aptamers

Pool and primers

Initial RNA library templates and primers were synthesized as single-stranded DNA from IDT (Coralville, IA, USA): 5 '-CCTCTCTATGGGCAGTCGGTGAT- (N20) -TTTCTGCAGCGATTCTTGTTT- (N10) -GGAGAATGAGGAACCCAGTGCAG-3', 5'-TAATACGACTCACTATAGGGCCTCTCTATGGGCAGTCGGTGAT-3' (forward primer), 5'-CTGCACTGGGTTCCTCATTCTCC-3' (reverse primer). Two short "blocking" sequences (purchased from IDT) complementary to the 5 '-and 3' -constant primer regions were synthesized to minimize the effect of the primers on secondary structure: 5'-ATCACCGACTGCCCATAGAGGG-3' (forward block sequence), 5'-CTGCACTGGGTTCCTCTCC-3' (reverse block sequence). Another biotinylated "capture" sequence complementary to the constant central region of the library was also synthesized by IDT: 5' -Biotin-GTC-PEG-6 spacer-CAAGAATCGCTGCAG-3. All materials were arranged on a 250nmol scale and desalted and purified.

RNA articles for RNA aptamer selection using 2 '-fluoro- (2' F-) pyrimidine pairsThe library is modified to improve its stability in the end use application. The T7 primer was combined with the library template sequence and primer extension was performed using Titanium Taq DNA polymerase (Clontech; Mountain View, Calif., USA). Then use

The primer extension material was transcribed using the T7 transcription kit (Epicentre; Madison, Wis., USA), purified using denaturing polyacrylamide and 8M urea (Sequel NE reagent, part A and part B) from American bioanalytical company (Natick, MA, USA). During the selection procedure, the library was reverse transcribed using SuperScript IV reverse transcriptase (Invitrogen; Carlsbad, Calif., USA) and amplified using titanium Taq DNA polymerase from Clontech, according to the manufacturer's instructions. During selection, the library was amplified using the following PCR protocol (95 ℃ for 10 seconds, 60 ℃ for 30 seconds, 95 ℃ for 60 seconds of initial hot start activation). Then use

The T7 transcription kit transcribes the RNA library and purifies it on polyacrylamide gel (PAGE). The gel elution buffer used for library recovery after overnight purification at 4 ℃ was prepared from the following solutions: 0.5M NH4OAc, 1mM EDTA (both from Teknova), 0.2% SDS (from Amresco), pH 7.4.

RNA aptamer selection

Screening of the RNA library was performed by six rounds of selection using the Melting-Off format. Rounds 1-6 selection were performed on a stable HEK293T cell line (human embryonic kidney-ATCC CRL-1573) expressing GCN4(S25R14) CAR (chimeric antigen receptor) against a cell surface peptide neo-epitope (PNE), and a negative selection step (cell SELEX) (cell-SELEX) was performed with stably transduced HEK293T cells carrying an anti-CD 19 CAR. The HEK293T Cell line was taken from American Type Cell Collection (American Type Cell Collection) and cultured in DMEM medium (Gibco-Invitrogen) supplemented with 10% FBS (Gibco-Invitrogen) and 1% penicillin/stamycin (Gibco-Invitrogen). All selections were performed in DMEM medium supplemented with 10% serum medium, and each round of SELEX included the following steps: RN (relay node)The library a is immobilized on streptavidin-coated magnetic beads, counter-selected, incubated with the target, reverse transcribed to recognize the sequence of the target, PCR amplified, and transcribed into RNA. Before each round, each whole Streptavidin-coated magnetic bead (MyOne Streptavidin T1 Dynabeads)^TMTypically, Dynabead is present at 20 μ g per day^TMUsing 1pmole biotinylated material, the amount depends on the stringency required) was pre-washed three times with 200 μ L PBS-T (final concentration 0.01% tween 20, pH 7.4) wash buffer. In addition, trypsin treatment collected target cells and anti-target cells, centrifugation at 5000 × g, and washing twice with DPBS buffer, followed by suspension in 200 μ L of DPBS buffer; the number of cells used in each round is shown in table 1. The RNA library was renatured in serum-free DMEM medium (denaturation at 90 ℃ for 1 min, annealing at 60 ℃ for 5 min, and annealing at 23 ℃ for 5 min) and the library molar amount of the primer blocking sequence and the capture sequence was twice the library molar amount. This was done to minimize the effect of the constant primer region on aptamer secondary structure, capture the library with magnetic beads via streptavidin-biotin binding interactions, and protect the aptamer ends from exonuclease.

After renaturation and primer blocking, the library was captured on magnetic beads by incubation for 15 min at room temperature. The magnetic beads were then separated from the solution and washed three times with 200 μ L of selection buffer at 37 ℃ to eliminate any remaining PBS-T and non-specifically bound library species. The beads with the immobilized RNA pool were then incubated at 200. mu.L of 10⁶A30 minute counter-selection incubation at 37 ℃ in the individual anti-target cell preparations resulted in the release of non-specific sequences from the magnetic beads. The non-specific library components were then discarded and the beads were washed 6 times with 200 μ L of selection buffer over the course of one hour, 7 minutes each (7 minutes in DPBS at 37 ℃, magnetic separation 3 minutes, supernatant discarded and this process repeated) to reduce the probability of non-specific library components surviving to respond during the forward selection step. The forward selection consisted of incubation of the magnetic bead RNA library with 200 μ Ι _ of positive cell preparation for 60 min at 37 ℃ in 1-4 rounds, 30 min in 5-6 rounds, and parallel evaluation (cell count in table 1). Once the forward selection is completeAnd separating and recovering the supernatant containing the recognition target sequence from the magnetic beads. The supernatant was then subjected to a second magnetic separation to ensure that the magnetic beads had been completely removed. Targeted HEK293T CAR PNE cells were centrifuged at 5000 × g and washed once with 200 μ Ι _ DPBS buffer to remove weakly bound aptamer species. The library was recovered from the cells by heat denaturation at 70 ℃. The recovered library from each round was subjected to protein precipitation, ethanol precipitation and sample concentration using MPC reagent (Lucigen Corp, middlleton, WI, USA) and purified by 10% denaturing PAGE using 8M urea. The library was then reverse transcribed using SuperScript IV reverse transcriptase according to the manufacturer's instructions

Amplification with Taq DNA polymerase and use according to manufacturer's instructions

Transcription was performed with the T7 transcription kit. The transcripts were purified by 10% denaturing polyacrylamide gel electrophoresis (PAGE) using 8M urea. The gel sections were cut, eluted in gel elution buffer overnight at 4 ℃ and the concentration of the RNA library was calculated by measuring the absorbance of NanoDrop-1000 at 260 nm. During successive rounds of SELEX, the concentration of the RNA library and the number of target cells gradually decreased. Additional parallel assessments and "cross-fitness tests" were also performed during the post-selection bioinformatic analysis to help identify good aptamer candidates.

Table 1: SELEX conditions for each round of RNA aptamer selection against CAR PNE

Aptamer candidates were selected by next generation sequencing using the MiniSeq 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 (Li ex Belgium) as single-stranded oligonucleotides purified by HPLC-RP by standard solid-phase phosphoramidite chemistry. Biotin was added to the 5' end of the aptamer as biotin-TEG, which introduced a 16 atom mixed polar spacer between the aptamer sequence and the biotin tag. Molecular weight, purity and integrity were verified by HPLC-MS. The nucleic acid sequences of these aptamers are shown in FIG. 13A.

Example 7 determination of anti-CAR Affinity and specificity of PNE aptamers to 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 on cells were assessed by flow cytometry. These studies were performed on HEK293T, HEK293T-CAR CD19 (HEK 293T cell line transduced with lentiviral vectors encoding CARs directed against the CD19 antigen) and HEK293T-CAR PNE (HEK 293T cell line transduced with lentiviral vectors encoding CARs directed against the PNE peptide) cells by incubating them with biotinylated aptamers in DPBS buffer supplemented with 5% FBS. Prior to use, cells were cultured in DMEM medium (Gibco Invitrogen) supplemented with 10% fbs (Gibco Invitrogen) and 1% penicillin/streptomycin (Gibco Invitrogen). Prior to the experiment, cells (2.5X 10)⁵One cell/well) were seeded in 96-well plates and centrifuged at 2500rpm for 2 minutes. The supernatant was discarded, and the precipitated cells were washed twice with 200. mu.L of DPBS-5% FBS preheated at 37 ℃. Each washing step was followed by centrifugation at 2500rpm for 2 minutes. Aptamers were denatured at 85 ℃ for 5 minutes and immediately placed on ice cubes at 4 ℃ for 5 minutes. The sequence was then diluted in two different concentration ranges: 30100 and 300nM (preliminary testing of 5 aptamer candidates); and, 30, 50, 75 and 100nM (repeated staining with ARAA-00100001 and ARAA-01700001), then 100nM phycoerythrin-labeled streptavidin (streptavidin-PE, eBioscience) was added to each solution. Cells were resuspended in aptamer dilution (100. mu.L/well) and incubated at 5% CO₂Incubate at 37 ℃ for 30 minutes in a humid atmosphere. As a control, cells were incubated with PNE peptide (Alexa Fluor 488-labeled, Provepharm, Marseille, France), streptavidin-PE (eBioscience), or their respective buffers without added reagents.After incubation, cells were centrifuged at 2500rpm for 2 minutes and the supernatant with unbound sequences was discarded. The precipitated cells were washed with DPBS-5% FBS buffer (200. mu.L/well) and centrifuged twice to remove all weakly and non-specifically attached sequences. Then, 1mg/mL salmon sperm DNA solution (100. mu.L/well) was used at 37 ℃ with 5% CO₂Cells were washed in a humid atmosphere. After 30 minutes, the salmon sperm solution was removed by centrifugation at 2500rpm for 2 minutes, and the cells were washed twice more with DPBS-5% buffer (200. mu.L/well) and then centrifuged. Cell fixation with attached DNA sequences (BD CellFIX solution #340181) was then fixed and fluorescence positive cells were counted on YL-1 channel by flow cytometry (attunnxt; Invitrogen, Inc.).

The results of the binding studies with HEK293T, HEK293T-CAR CD19, and HEK293T-CAR PNE are shown in figure 14A. Dose-dependent binding to CAR-PNE positive HEK293T cells was observed with aptamers ARAA-00100001 and ARAA-01700001, whereas signal saturation was not reached at the highest tested concentration. The signal intensity was stronger than that of 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, and ARAA-01300011 did not show any differential staining between the three HEK293T cell lines, indicating that these three aptamers are not specific for the CAR PNE. The binding studies with aptamers ARAA-00100001 and ARAA-01700001 were repeated over a broader concentration range, confirming the results of the preliminary experiments (see fig. 14B and 14C).

In another experiment, the binding of the same aptamers to human PBMCs expressing CAR PNE was studied. PBMCs were isolated from blood donors as 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 ℃ with 5% CO₂Culturing under the condition. After 2 hours of transduction with lentiviral vector encoding CAR against PNE peptide, cells were cultured for a further 72 hours before binding studies were performed as described above. As shown in figure 16, dose-dependent signals were measured for both aptamers at a plateau above 300nM concentration. The signal intensity was stronger than that of the PNE peptide control.

Both sets of experiments showed that the ARAA-00100001 and ARAA-01700001 aptamers selected against the PNE peptide-recognizing CARs were able to specifically recognize their targets expressed on human cells that had been engineered to express this particular chimeric antigen receptor.

Example 8 serum stability of CAR-PNE specific aptamers

Stability against 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. Aptamers were denatured at 85 ℃ for 5 minutes and then immediately cooled on ice to 4 ℃ for 5 minutes. The sequences were then diluted to a final concentration of 2 μ M in DPBS buffer supplemented with 5% FBS, RPMI medium supplemented with 10% FBS, or pure FBS serum. Incubating the sample at 37 ℃ for 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 24 hours; control samples contained freshly prepared aptamers that were not incubated at 37 ℃. The half-life of the aptamers in their respective buffers was determined by migration on an agarose gel using the method of denaturing electrophoresis as follows: after mixing aptamer samples for different incubation times with formamide-containing loading buffer (ThermoScientific, Waltham, MA, USA) and denaturing at 85 ℃ for 5 minutes, 15 μ L of each sample was placed on a freshly prepared 3% agarose gel containing sybrsafe (invitrogen) as a marker for RNA staining. Migration of RNA aptamers on agarose gels was performed in 1 XTBE buffer (Invitrogen) by applying 100V voltage over 20 min. The gels were visualized using the Bio-Rad imaging system and the results are shown in FIG. 15. Since the intensity of the RNA signal starts to decrease after 4h, both aptamers are stable for at least 2h under different incubation conditions.

Example 9 preparation of bispecific aptamers against PSMA and CAR-PNE

ARAA-00100001 and ARAA-01700001 aptamers were purchased from baseclick (Neuried, Germany) as HPLC-RP purified 2' -F RNA oligonucleotides synthesized by standard solid phase phosphoramidite chemistry.

The A102 'F-RNA aptamer was modified at its 3' end with an azide group for subsequent triazole internucleotide dimerization. Biotin was added as biotin-TEG to the 5' end of the a10 aptamer, which introduced a 16 atom mixed polar spacer between the aptamer sequence and the biotin-marker. ARAA-00100001 and ARAA-01700001 were modified at their 5' ends with an alkyne group for subsequent triazole internucleotide dimerization. Molecular weight, purity and integrity were verified by HPLC-MS.

The procedure described in example 1 was used to prepare bispecific anti-PSMA a10 and anti-CAR PNE aptamers. The result of the click reaction is shown in FIG. 8A.

Example 10 determination of anti-PSMA x anti-CAR Affinity of PNE bispecific aptamers for targets expressed on cells Force and specificity

The affinity and specificity of the anti-PSMA x anti-CAR-PNE aptamers to the target protein expressed by the cells was examined 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 DPBS buffer containing 5% FBS as described in example 3. Aptamers were tested at single concentration ranges of 30, 100 and 300 nM.

The results of the study for binding to PSMA-positive cells are shown in fig. 10A. Two RNA/RNA aptamers, A10 × ARAA-00100001 and A10 × ARAA-01700001, and A10 monomeric aptamers were analyzed. For comparison, the binding of the test agent to PSMA-negative PC-3 cells was also measured.

Dose-dependent binding to PSMA-positive LNCaP cells was observed with a10, whereas signal saturation was not reached at the highest concentration tested. The signal intensity was as strong as the antibody control. Residual binding of the a10 monomer to PC-3 cells was only observed at the highest concentration tested. The two bispecific PSMA × CAR-PNE aptamers showed similar binding properties to the a10 monomer, but increased specificity for target positive cells as residual binding to PSMA negative cells decreased. For each concentration tested, the signal intensity of the bispecific aptamer was superior to that of the a10 monomer, indicating that heterodimerization results in an increase in affinity.

In summary, the results from examples 9 and 10 show that heterodimerization of aptamers selected against different targets does not significantly affect the binding properties of each moiety when evaluated separately.

Example 11 biological Activity of bispecific aptamers specific for CAR-PNE and PSMA

The cytotoxicity assays were performed on unstimulated Peripheral Blood Mononuclear Cells (PBMCs). From a healthy donor (

Sang，Division

) Freshly prepared PBMCs were isolated from the buffy coat. After diluting the blood with DPBS, PBMC were separated by FICOLL density gradient (FICOLL-PAQUE PREMIUM 1.077GE Healthcare), washed twice with DPBS, resuspended in RPMI-1640 medium (Gibco Invitrogen) to obtain 5X 10⁶Cell density of individual cells/ml. These PBMCs were transduced with a lentiviral vector expressing the CAR-PNE receptor. These PBMC-CAR-PNE were used as effector cells.

LNCaP target cells were labeled with 2. mu.M calcein-AM (Trevigen Inc, Gaithersburg, Md., USA) in cell culture medium for 30 min at 37 ℃. calcein-AM fluorescent dye is a dye that can be trapped inside living LNCaP cells and only be released upon redirected lysis. After washing 2 times in cell culture medium, the medium was adjusted to 5X 10 in RPMI-1640 medium⁵Cell density of one cell/ml and 50,000 cells per assay reaction using 100 μ l aliquots. Standard reaction at 37 deg.C/5% CO₂The duration of the treatment was 4 hours, and a total volume of 200. mu.l of 5X 10 was used⁴ 5X 10 target cells labeled with calcein AM⁵PBMCs-CAR-PNE (E/T ratio 1:10) and 20. mu.l of bispecific aptamer solution. After the cytotoxic reaction, the dye released in the medium is quantified by a fluorescence reader (Varioskan Lux, ThermoFisher, Waltham, MA, USA) and compared with the fluorescence signal of a control reaction, which is free of cytotoxic compounds and whose fluorescence signal depends on the fully lysed cells (which are)Medium aptamer was replaced by a100 reagent purchased from chemimetec, Allerod, denmark). Based on these readings, specific cytotoxicity was calculated according to the following formula: [ fluorescence intensity (sample) -fluorescence intensity (control)]/[ fluorescence intensity (Total lysis) -fluorescence intensity (control)]×100。

In the presence of 100nM aptamer, the binding was performed at 10:1, single E: t is determined from the cytotoxicity results obtained after 4 hours of incubation. Specific cytotoxicity was determined using RNA/RNA aptamer a10 × PNE, which induced killing of greater than 30% of LNCaP cells. Control monomer a10 lacking the PNE binding moiety did not induce any cytotoxicity.

Example 12 treatment of cancer with anti-CD 3 × anti-PSMA aptamer in preclinical models

The therapeutic efficacy and toxicity of the different multimeric aptamer structures compared to the monomeric aptamers in mice were evaluated. Aptamers that specifically bind to CD3 and PSMA, which may be monomeric or multimeric in different groups of mice, were administered to adult mice bearing PSMA-positive tumors. The efficacy was evaluated by measuring tumor size, tumor growth, growth rate and survival rate of the treated and control groups. Toxicity was assessed by the incidence of adverse reactions in the treated versus control groups.

Example 13 treatment of cancer in preclinical models using CAR-T aptamer switch

The switch aptamer constructs were evaluated for efficacy and toxicity in mice compared to the monomeric aptamers. Multimeric aptamers were made as switches that would initiate the activity of CAR T cell-based therapeutics. Tumor-bearing adult mice are first injected with T cells transduced with CAR-PNE, and then infused with multimeric aptamers made from anti-CAR PNE aptamers fused to PSMA, HER2, CD19, CD20, or CD22 tumor-associated targets. The efficacy was evaluated by measuring tumor size, tumor growth, growth rate and survival rate of the treated and control groups. Toxicity was assessed by the incidence of adverse reactions in the treated versus control groups.

As used herein, "consisting essentially of allows for the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claims. Any recitation herein of the term "comprising," particularly in the context of components of a composition or elements of a device, is interchangeable with "consisting essentially of or" consisting of.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in the compositions and methods set forth herein may be made, and equivalents may be substituted, as well as other changes, after reading the foregoing description.

Reference to the literature

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 siRNAs with aptamer-siRNA chimeras. Nat.Biotechnol 24,1005(2006).

Rodgers DT,et al.,Switch-mediated activation and retargeting of CAR-T cells for 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 gene-modified T cells toward various cancer types using tagged antibodies.Clin Cancer Res 18,6436(2012).

Urbanska K,et al.,A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor.Cancer Res 72,1844(2012a).

Urbanska K,et al.,Development of a novel universal immune receptor for antigen targeting: To infinity and beyond,OncoImmunology 1,777(2012b).

Wu CY,et al.,Remote control of therapeutic T cells through a small molecule-gated chimeric receptor,Science 350,aab4077(2015).

Claims

1. An aptamer bridge for binding a Chimeric Antigen Receptor (CAR) to a target, the aptamer bridge comprising one or more CAR-binding aptamers and one or more target-binding aptamers, wherein the one or more CAR-binding aptamers are bound to the target-binding aptamers by one or more linkers, and wherein at least one of the one or more CAR-binding aptamers binds to an extracellular domain of a CAR.

2. The aptamer bridge of claim 1, wherein the CAR extracellular domain is capable of specifically binding a peptide neo-epitope (PNE).

3. The aptamer bridge of claim 1 or 2, wherein binding of the CAR-binding aptamer of the aptamer bridge to a CAR expressed on an immune cell activates the immune cell.

4. The aptamer bridge of claim 1 or 2, wherein binding of the CAR-binding aptamer of the aptamer bridge to the CAR expressed on the immune cell does not activate the immune cell.

5. The aptamer bridge of claim 3 or 4, wherein the immune cell is a T cell, NK cell or macrophage and the binding results in destruction of target cells bound by target binding aptamers of the aptamer bridge.

6. The aptamer bridge of any preceding claim, 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.

7. An aptamer bridge according to any preceding claim, wherein the aptamer bridge is free of immunoglobulin peptides or fragments thereof.

8. The aptamer bridge of any preceding claim, wherein the aptamer bridge is free of peptides or polypeptides.

9. The aptamer bridge of claim 6, wherein the linker moiety comprises a single-or double-stranded nucleic acid selected from DNA, RNA, and XNA.

10. The aptamer bridge of claim 6, wherein the linker moiety is an immunoglobulin polypeptide or a fragment thereof.

11. The aptamer bridge of claim 6, wherein the linker moiety is a polysaccharide selected from the group consisting of dextran, glycogen, starch, and derivatives thereof.

12. The aptamer bridge of claim 6, wherein the linker moiety is a synthetic polymer selected from poly (amidoamine) (PAMAM) and poly (beta amino ester) (PBAE).

13. The aptamer bridge of any preceding claim, wherein the CAR extracellular domain comprises an immunoglobulin polypeptide, a fragment thereof, or a derivative thereof.

14. The aptamer bridge of claim 10, wherein the immunoglobulin polypeptide is a single chain Fv.

15. The aptamer bridge of claim 2, wherein the PNE has an amino acid sequence that is not found in the human proteome.

16. The aptamer bridge of claim 2, wherein the PNE is selected from the group consisting of SEQ ID NOs 1-7.

17. The aptamer bridge of any preceding claim, wherein at least one of the target-binding aptamers binds 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.

18. The aptamer bridge of any preceding claim, wherein the aptamer bridge comprises two or more CAR-binding aptamers that are the same or different.

19. The aptamer bridge of claim 18, comprising two or more target-binding aptamers that are the same or different.

20. The aptamer bridge of any preceding claim, wherein the aptamer bridge comprises a molecular form selected from the group consisting of cyclic, star, dendritic, linear, branched, and combinations thereof.

21. The aptamer bridge of any preceding claim, wherein there is a distance of about 70 to about 200 angstroms between one or more of the CAR-binding aptamers and one or more of the target-binding aptamers.

22. The aptamer bridge of any preceding claim, wherein when expressed in a T cell, NK cell, B cell or macrophage it binds to the CAR.

23. The aptamer bridge of claim 22, wherein the T cell, NK cell, B cell, or macrophage is activated upon binding to the CAR.

24. The aptamer bridge of any preceding claim, wherein the one or more CAR-binding aptamers and the one or more target-binding aptamers each have a binding affinity of less than 10nM, preferably less than 1nM, more preferably less than 100pM or less than 10 pM.

25. A system for immunotherapy comprising the aptamer bridge of any preceding claim and an immune cell expressing the CAR.

26. The system of claim 25, wherein the immune cell is activated by binding to a CAR-binding aptamer of the aptamer bridge.

27. Use of the system of claim 25 or 26 to enhance an immune response to a cancer, pathogen, or inflammatory cell or protein.

28. A kit for providing immunotherapy to a subject, the kit comprising the aptamer bridge of any of claims 1-24 and a viral vector encoding the CAR.

29. The kit of claim 28, wherein the viral vector is a lentiviral vector.

30. The kit of claim 28 or 29, wherein the kit further comprises an aptamer having a complementary sequence to the CAR-binding aptamer, or a portion thereof.

31. A kit for providing immunotherapy to a subject, the kit 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.

32. The kit of claim 31, wherein the viral vector is a lentiviral vector.

33. A kit comprising an aptamer bridge of any one of claims 1 to 24; and, a labeled and/or unlabeled CAR-binding aptamer that binds to the CAR-binding aptamer of the aptamer bridge to the same CAR, but does not bind to the target bound by the aptamer bridge.

34. A kit comprising an aptamer bridge of any one of claims 1 to 24; and, a labeled and/or unlabeled target-binding aptamer that binds to the target-binding aptamer of the aptamer bridge to the same target, but does not bind to the CAR bound by the aptamer bridge.

35. A method of immunotherapy in a subject in need thereof, the method comprising the steps of:

(a) administering to a subject a population of cells expressing a CAR, wherein the CAR comprises an extracellular domain capable of specifically binding a Peptide Neoepitope (PNE); and

(2) administering to a subject an aptamer bridge of any one of claims 1 to 24;

wherein the aptamer bridge binds to a CAR-expressing cell in the subject and induces an interaction between the CAR-expressing cell and the target.

36. The method of claim 35, further comprising:

(a1) administering to the subject a labeled monospecific aptamer that binds to the CAR, and determining the total amount of CAR or CAR-expressing cells within the subject.

37. The method of claim 36, wherein step (b) is performed only when the amount of CAR or CAR-expressing cells reaches or exceeds a predetermined amount required for the expected success of the immunotherapy.

38. The method of claim 37, wherein the amount of CAR or CAR-expressing cells falls below the predetermined amount and step (a1) is repeated after a period of time.

39. The method of any one of claims 35 to 38, wherein the target is on a target cell and the interaction results in death of the target cell.

40. The method of any one of claims 35 to 38, wherein the interaction results in an enhanced immune response in the subject.

41. The method of any one of claims 35 to 38, wherein the interaction results in immune tolerance in the subject.

42. The method of any one of claims 35 to 38, wherein the interaction results in a reduced or terminated immune response in the subject.

43. The method of claim 40, wherein the enhanced immune response is against a cancer, a pathogen, or an inflammatory cell or protein.

44. The method of any one of claims 35 to 38, wherein the intensity or duration of the immune response is modulated by the dose or duration of administration of the aptamer bridge in step (b).

45. A method according to any one of claims 35 to 44 wherein the magnitude or duration of the immune response is limited by the further steps of:

(c) administering to the subject an aptamer having a sequence complementary to the sequence of the CAR-binding aptamer or a portion thereof.

46. The method of any one of claims 35 to 45, further comprising the steps of:

(a0) transducing a cell from a subject with a viral vector that expresses a CAR in the cell.

47. The method of claim 46, wherein the transduction is performed in vitro (ex vivo).

48. The method of claim 46, wherein the transduction is performed in a subject (in vivo).