Dual-specific individualization app

Abstract

Provided herein are bispecific personalized aptamers that induce cell death in cancer cells and methods of use thereof.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 027,629, filed May 20, 2020, and 63 / 121,079, filed December 3, 2020, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Aptamers are short, single-stranded nucleic acid oligomers that can bind to specific target molecules. Aptamers are typically selected from large random pools of oligonucleotides in an iterative process.

[0003] Aptamer-based therapeutics offer numerous advantages over conventional antibody-based therapeutics, including their rapid chemical generation, their amenability to chemical modification, their high stability, and their lack of immunogenicity. Thus, aptamers capable of selectively targeting and killing cancer cells would have great potential as anti-cancer therapeutics. Summary of the Invention

[0004] Provided herein are bispecific personalized aptamers useful as cancer therapeutic agents, as well as pharmaceutical compositions comprising such bispecific personalized aptamers, and methods for producing and using such aptamers. In certain embodiments, the bispecific personalized aptamers provided herein are cancer therapeutic agent molecular species that are composed of three functionally distinct parts: (1) a cancer cell target-specific part that can bind to and induce cytotoxicity against target cancer cells; (2) an immune cell-engaging part; and (3) a CpG motif.

[0005] In certain aspects, the compositions and methods disclosed herein provide and facilitate customized cancer therapy for patients, treating them with a personalized solution optimized for the unique set of drug targets and conditions presented by each patient, as reflected by fresh tumor tissue samples. In certain embodiments, the bispecific personalized aptamers disclosed herein consist of two arms. One aptamer arm targets the tumor of an individual subject. This tumor-targeting arm is a functional aptamer selected for its ability to both bind to target cancer cells and specifically induce cell death in those tumor cells. This portion is variable and custom-made for each individual patient. The second aptamer arm targets immune effector cells, thereby functioning as an "engager" and causing tumor cell lysis by immune cells. This latter immunomodulatory arm is designed to be shared between different patients. In embodiments, the two aptamer arms of the bispecific structure are bridged together by nucleobase hybridization of single-stranded overhangs of complementary sequences. This hybridization domain is CpG-rich and designed to induce toll-like receptor 9 (TLR9)-mediated antigen-presenting cell (APC) stimulation and increase tumor antigen uptake. Thus, in certain embodiments, the bispecificity of the disclosed aptamers combined with their TLR9 agonist activity makes them valuable components in a multifaceted approach to cancer treatment.

[0006] In certain aspects, provided herein are bispecific personalized aptamers comprising a cancer cell-binding chain that selectively binds to and / or kills cancer cells (e.g., breast cancer cells, colorectal cancer cells), including by inducing apoptosis. The bispecific personalized aptamer also comprises an immune effector cell-binding chain that promotes cancer cell lysis, e.g., via T cell- or natural killer (NK) cell-mediated cytotoxicity. In some embodiments, the cancer cell-binding chain is linked to the immune effector cell-binding chain by a CpG-rich TLR9 agonist sequence that induces TLR9-mediated APC stimulation and / or increased tumor antigen uptake. In some aspects, provided herein are pharmaceutical compositions comprising such bispecific personalized aptamers, methods of using such bispecific personalized aptamers to treat cancer and / or kill cancer cells, and methods of making such bispecific personalized aptamers.

[0007] In certain embodiments, provided herein are bispecific personalized aptamers comprising: (a) a cancer cell-binding chain that specifically binds to a target expressed on a cancer cell; (b) a TLR9 agonist CpG motif; and (c) an immune effector cell-binding chain that specifically binds to an immune effector cell, wherein the cancer cell-binding chain is linked to the immune effector cell-binding chain by a CpG motif.

[0008] In some embodiments, the cancer cell-binding chain induces cell death (e.g., apoptosis) when contacted with cancer cells. In some embodiments, the cancer cells are patient-derived cancer cells. The cancer cells can be solid tumor cells (e.g., breast cancer cells or colorectal cancer cells), sarcoma cells (e.g., soft tissue sarcoma cells), or blood cancer cells (e.g., lymphoma cells). The cancer cell-binding chain induces cell death when contacted with cancer cells in vitro or in vivo. In some embodiments, the immune effector cell-binding chain mediates cancer cell lysis via T cell- or NK cell-mediated cytotoxicity. In some embodiments, the cancer cell-binding chain and the immune effector cell-binding chain are linked together by hybridization of the 5' sequence of the cancer cell-binding chain to the 5' sequence of the immune effector cell-binding chain. In some embodiments, the 5' sequence of the cancer cell-binding chain hybridizes to the 5' sequence of the immune effector cell-binding chain to form a TLR9 agonist sequence. In some embodiments, the TLR9 agonist sequence comprises a double-stranded region of a CpG motif. In some embodiments, the CpG motif induces TLR9-mediated APC stimulation and / or increased tumor antigen uptake. In some embodiments, the TLR9 agonist sequence induces an anti-tumor immune response. In some embodiments, the TLR9 agonist sequence induces IFNα secretion, IL6 secretion, and / or B cell activation.

[0009] In some embodiments, the CpG motif is a double-stranded nucleic acid sequence comprising a sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 63-66. In some embodiments, the CpG motif is a double-stranded nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 63-66.

[0010] In certain embodiments, the CpG motif is a double-stranded nucleic acid sequence comprising at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22) consecutive nucleotides of any one of SEQ ID NOs: 63-66. In some embodiments, the CpG motifs provided herein have a sequence consisting essentially of SEQ ID NOs: 63-66. In certain embodiments, the CpG motifs provided herein have a sequence consisting essentially of SEQ ID NOs: 63-66.

[0011] In certain embodiments, the CpG motif is 35 nucleotides or less in length (e.g., 34 nucleotides or less, 33 nucleotides or less, 32 nucleotides or less, 31 nucleotides or less, 30 nucleotides or less, 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, 25 nucleotides or less, 24 nucleotides or less, 23 nucleotides or less, or 22 nucleotides or less in length).

[0012] In certain embodiments, the cancer cell-binding chain is a personalized aptamer chain selected (e.g., selected using the aptamer selection methods provided herein) to bind to and / or kill tumor cells obtained from an individual patient. In some embodiments, the cancer cell-binding chain binds to a cancer antigen. In certain embodiments, the cancer antigen is selected from major histocompatibility complex (MHC) tumor-associated antigen (TAA) peptide complex, prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), tyrosinase, glycoprotein 100 (gp100), melanoma antigen 1 recognized by T cells (MART-1) / melan-A, heat shock protein 70 (HSP70)-2-m, human leukocyte antigen (HLA)-A2-R17OJ, human papillomavirus 16 (HPV16)-E7, mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2) / neu, or mammaglobin-A. In some embodiments, the cancer cell-binding chain comprises a nucleic acid sequence that is at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the cancer cell-binding chain comprises a nucleic acid sequence that is at least one of SEQ ID NOs: 43-62 or 107-115.

[0013] In certain embodiments, the cancer cell-binding chain comprises at least 30 (e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60) contiguous nucleotides of any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the cancer cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the cancer cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115.

[0014] In certain embodiments, the cancer cell-binding strand is 120 nucleotides or less in length (e.g., 115 nucleotides or less, 110 nucleotides or less, 105 nucleotides or less, 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 69 nucleotides or less, 68 nucleotides or less, 67 nucleotides or less, 66 nucleotides or less, 65 nucleotides or less, 64 nucleotides or less, or 63 nucleotides or less). In certain embodiments, the cancer cell-binding strand is about 63 nucleotides in length.

[0015] In some embodiments, the cancer cell-binding strand is 53-73 nucleotides in length. In certain embodiments, the cancer cell-binding strand is 58-68 nucleotides in length. In certain embodiments, the cancer cell-binding strand is approximately 63 nucleotides in length. In some embodiments, the cancer cell-binding strand comprises a cancer targeting moiety approximately 40 nucleotides in length. In certain embodiments, the cancer cell-binding strand comprises a CpG-complementary motif of approximately 23 nucleotides.

[0016] In some embodiments, the immune effector cell binding chain binds to a T cell (e.g., CD8 + In some embodiments, the immune effector cell-binding chain binds to an antigen expressed by a target cell, such as a human T cell, a NK cell, a B cell, a macrophage, a dendritic cell, a neutrophil, a basophil, or an eosinophil. In some embodiments, the immune effector cell-binding chain binds to an immune effector cell antigen selected from CD16, Notch-2, other Notch family members, KCNK17, CD3, CD28, 4-1BB, CTLA-4, ICOS, CD40L, PD-1, OX40, LFA-1, CD27, PARP16, IGSF9, SLC15A3, WRB, and GALR2.

[0017] In some embodiments, the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the immune effector cell-binding chain comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-42, 88-106, or 116.

[0018] In certain embodiments, the immune effector cell-binding chain comprises at least 20 (e.g., at least 25, at least 30, at least 35, at least 40, at least 45, at least 50) contiguous nucleotides of any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the immune effector cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. In certain embodiments, the immune effector cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116.

[0019] In certain embodiments, the immune effector cell-binding chain is 120 nucleotides or less in length (e.g., 115 nucleotides or less, 110 nucleotides or less, 105 nucleotides or less, 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 74 nucleotides or less, or 73 nucleotides or less). In certain embodiments, the immune effector cell-binding chain is about 73 nucleotides in length.

[0020] In some embodiments, the immune effector cell-binding chain is 63-83 nucleotides in length. In specific embodiments, the immune effector cell-binding chain is 68-78 nucleotides in length. In specific embodiments, the immune effector cell-binding chain is approximately 73 nucleotides in length. In some embodiments, the immune effector cell-binding chain comprises a cancer targeting moiety approximately 50 nucleotides in length. In specific embodiments, the immune effector cell-binding chain comprises a CpG complementarity motif of approximately 23 nucleotides.

[0021] In some embodiments, the bispecific individualized aptamer comprises a combination of two strands, including one strand selected from any one of SEQ ID NOs: 1-42, 88-106, or 116, and the other strand selected from any one of SEQ ID NOs: 43-62 or 107-115. For example, in certain embodiments, the paired strands are selected from SEQ ID NOs: 29 and 54, 29 and 50, 32 and 50, 33 and 48, 41 and 49, 34 and 59.

[0022] In some embodiments, the bispecific individualized aptamers provided herein comprise one or more chemical modifications. In some embodiments, the bispecific individualized aptamers are chemically modified with polyethylene glycol (PEG) (e.g., linked to the 5' or 3' end of the aptamer). In some embodiments, the bispecific individualized aptamers comprise a 5' end cap. In certain embodiments, the aptamers comprise a 3' end cap (e.g., inverted thymidine, biotin). In some embodiments, the bispecific individualized aptamers comprise one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) end caps. 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions) at 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 positions. In certain embodiments, bispecific individualized aptamers comprise locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'deoxy-2'fluoro-D-arabinonucleic acid (2'-F ANA) sugars in their backbone.

[0023] In certain embodiments, the aptamer comprises one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 The double-stranded CpG motif contains 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54) methylphosphonate and / or phosphorothioate (PS) internucleotide linkages. In certain embodiments, the double-stranded CpG motif contains partial PS modifications. In certain embodiments, five nucleotides from the 5' end of the double-stranded CpG motif are modified. In other embodiments, five nucleotides from both the 5' and 3' ends of the double-stranded CpG motif are modified. In certain embodiments, the double-stranded CpG motif contains full PS modifications. In certain embodiments, the bispecific individualized aptamer comprises one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54) triazole internucleotide linkages. In certain embodiments, the bispecific individualized aptamers are modified (e.g., at their 5' ends) with cholesterol or dialkyl lipids. In some embodiments, the bispecific individualized aptamers comprise one or more modified bases.

[0024] In certain embodiments, the bispecific individualized aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or enantiomeric L-DNA aptamers). In some embodiments, the bispecific individualized aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or enantiomeric L-RNA aptamers). In some embodiments, the bispecific individualized aptamers comprise a mixture of DNA and RNA.

[0025] In certain aspects, provided herein are pharmaceutical compositions comprising the bispecific personalized aptamers provided herein (e.g., therapeutically effective amounts of the bispecific personalized aptamers). In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0026] In certain embodiments, the pharmaceutical composition is for use in treating cancer. In some embodiments, the cancer is a solid tumor (e.g., breast cancer). In certain embodiments, the cancer is a carcinoma (e.g., colorectal cancer).

[0027] In certain aspects, provided herein are methods of treating cancer in a subject, comprising administering to the subject a bispecific personalized aptamer (e.g., a therapeutically effective amount of a bispecific personalized aptamer) and / or pharmaceutical composition provided herein. In some embodiments, administration is parenteral (e.g., subcutaneous). Administration can be intratumoral or peritumoral injection. In some embodiments, two or more doses are administered. In certain embodiments, at least 10-12 doses are administered. In some embodiments, administration of two or more doses to a subject is separated by at least one day.

[0028] In some embodiments, the cancer is a solid tumor (e.g., breast cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, bladder cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, or colorectal cancer). In some embodiments, the solid tumor is accessible to intratumoral administration. In certain embodiments, the cancer is a sarcoma (e.g., soft tissue sarcoma). In certain embodiments, the cancer is a hematological cancer (e.g., lymphoma). In certain embodiments, the subject has undergone chemotherapy.

[0029] In some embodiments, the therapeutic methods provided herein further comprise administering to the subject an additional cancer therapy. In some embodiments, the additional cancer therapy comprises chemotherapy. In certain embodiments, the additional cancer therapy comprises radiation therapy. In some embodiments, the additional cancer therapy comprises surgical removal of the tumor. In certain embodiments, the additional cancer therapy comprises administering to the subject an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an anti-CTLA4 antibody).

[0030] In certain aspects, provided herein are methods for killing cancer cells, comprising contacting the cancer cells with a bispecific personalized aptamer provided herein. In some embodiments, the cancer cells are killed by apoptosis, necrosis, immunological cell death (ICD), autophagy, or necroptosis. In some embodiments, the cancer cells are solid tumor cells (e.g., breast cancer cells or colorectal cancer cells), sarcoma cells (e.g., soft tissue sarcoma cells), or blood cancer cells (e.g., lymphoma cells). In some embodiments, the cancer cells are killed when contacted with the bispecific personalized aptamer in vitro. In certain embodiments, the cancer cells are killed when contacted with the bispecific personalized aptamer in vivo (e.g., in humans and / or animal models).

[0031] In certain aspects, provided herein are methods for making bispecific personalized aptamers. In some embodiments, the methods include: (1) synthesizing a cancer cell-binding chain; (2) synthesizing an immune effector cell-binding chain; and (3) hybridizing both chains to form the bispecific personalized aptamer.

[0032] In some embodiments, cancer cell-binding chains are identified using a systematic evolution of ligands by exponential enrichment (SELEX) process. In certain embodiments, multiple rounds (e.g., three rounds) of binding SELEX are performed with targeted cancer cells to identify aptamers that bind to cancer cell targets. Certain embodiments also include the steps of: (a) contacting cancer cells with a plurality of particles having a library of aptamer clusters immobilized on their surfaces ("aptamer cluster particles"), wherein at least a subset of the immobilized aptamer clusters bind to at least a subset of the cancer cells, thereby forming cell-aptamer cluster particle complexes; (b) incubating the cell-aptamer cluster particle complexes for a period of time sufficient to allow at least a portion of the cancer cells in the cell-aptamer cluster particle complexes to perform a cellular function; (c) detecting cell-aptamer cluster particle complexes that are performing the cellular function (e.g., using a functional reporter added to the reaction either before or after the aptamer cluster particle complexes are formed); (d) separating cell-aptamer cluster particle complexes that comprise cancer cells performing the cellular function detected in step (c) from other cell-aptamer cluster particle complexes; and (e) detecting the cell-aptamer cluster particle complexes that comprise cancer cells performing the cellular function detected in step (c) from other cell-aptamer cluster particle complexes. A functional SELEX assay is performed through a process including: (a) amplifying the aptamers in the separated cell-aptamer cluster particle complexes to generate a functionally enriched population of aptamers; and (b) identifying the enriched population of aptamers through sequencing, thereby identifying the cancer cell-binding chains.

[0033] In some embodiments, steps (c) and (d) are performed using a flow cytometer. In some embodiments, the methods described herein further include a step of separating aptamer cluster particles from target cells in the cell-aptamer cluster particle complexes separated in step (d). In some embodiments, the methods described herein further include a step of dissociating aptamers from particles in the separated aptamer cluster particles. In some embodiments, the methods described herein further include a step (e') after step (e) and before step (f): (i) forming aptamer cluster particles from the functionally enriched population of aptamers of step (e); and (ii) repeating steps (a) to (e) using the newly formed aptamer cluster particles to generate a further functionally enriched population of aptamers. In some embodiments, step (e') is repeated at least twice (e.g., at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times). In some embodiments, step (e') further comprises applying restrictive conditions during successive rounds of enrichment. In some embodiments, the restrictive conditions are selected from (i) reducing the total number of particles, (ii) reducing the copy number of aptamers per particle, (iii) reducing the total number of target cells, (iv) reducing the incubation time, and (v) introducing errors into the aptamer sequence by amplifying the aptamer population using an error-prone polymerase. In some embodiments, the further enriched population of aptamers in step (e') has reduced sequence diversity, for example, by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times, compared to the library of aptamer clusters in step (a).In some embodiments, each round of step (e') enriches the population of aptamers for aptamers that alter cellular function, e.g., by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 fold. In some embodiments, the time is from about 10 minutes to about 5 days (e.g., from about 1.5 hours to about 72 hours, or from about 1.5 hours to about 24 hours).

[0034] In some embodiments, the cancer cells are incubated with a reporter of cellular function before, during, or after contacting the cancer cells with the aptamer cluster particles. In some embodiments, the cancer cells are contacted with the reporter of cellular function before, during, or after step (b). In some embodiments, the reporter of cellular function is a fluorescent dye. In some embodiments, the cellular function is cell viability, cell death (e.g., apoptosis, unprogrammed cell death), or cell proliferation. In some embodiments, the methods described herein further comprise isolating cancer cells from a patient prior to step (a). In some embodiments, the cancer cells are isolated from a tumor biopsy or surgical resection.

[0035] In some embodiments, the method comprises synthesizing (e.g., chemically synthesizing) a cancer cell-binding strand comprising a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the method comprises synthesizing a cancer cell-binding strand comprising a nucleic acid sequence of any one of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the method comprises synthesizing a cancer cell-binding strand comprising a nucleic acid sequence comprising at least 30 (e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60) consecutive nucleotides of any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the method comprises synthesizing a cancer cell-binding chain having a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the method comprises synthesizing a cancer cell-binding chain having a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115.

[0036] In some embodiments, the method comprises synthesizing (e.g., chemically synthesizing) an immune effector cell-binding chain comprising a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the method comprises synthesizing an immune effector cell-binding chain comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-42, 88-106, or 116. In certain embodiments, the method comprises synthesizing an immune effector cell-binding chain comprising a nucleic acid sequence comprising at least 20 (e.g., at least 25, at least 30, at least 35, at least 40, at least 45, at least 50) consecutive nucleotides of any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the method comprises synthesizing an immune effector cell-binding chain having a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. In certain embodiments, the method comprises synthesizing a nucleic acid having a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116.

[0037] In some embodiments, the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain further comprise a complementary 5' sequence. In some embodiments, step (3) comprises hybridizing the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain. In some embodiments, the complementary 5' sequence comprises a CpG motif.

[0038] In some embodiments, the complementary 5' sequence comprises a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 63-66. In some embodiments, the complementary 5' sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 63-66. In certain embodiments, the complementary 5' sequence comprises a nucleic acid sequence comprising at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22) contiguous nucleotides of any one of SEQ ID NOs: 63-66. In some embodiments, the complementary 5' sequence has a sequence consisting essentially of SEQ ID NOs: 63-66. In certain embodiments, the complementary 5' sequence has the sequence of SEQ ID NOs: 63 to 66. In certain embodiments, the double-stranded CpG motif comprises a partial PS modification.

[0039] In certain aspects, provided herein are methods of treating cancer in a subject comprising administering to the subject a bispecific personalized aptamer generated using the methods described herein.

[0040] [Table 1] JPEG0007753256000002.jpg227162JPEG0007753256000003.jpg225163JPEG0007753256000004.jpg227162JPEG0007753256000005.jpg213162 [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 shows a schematic representation of a bispecific personalized aptamer showing three distinct domains. [Figure 2]FIG. 1 depicts the personalized aptamer selection process funnel. [Figure 3] 3A-3C show three modes of action (MoA) in solid tumors for intratumorally administered bispecific personalized aptamers and their downstream systemic effects (FIG. 3D). [Figure 4] FIG. 1 illustrates key steps in the personalization process for each patient. [Figure 5] Figure 1 shows a schematic diagram of the CTL-binding Cell-SELEX process. Rounds 1 and 2 were performed using cells from donor #1 (labeled blue). Rounds 3, 4, and 6 were performed using cells from donor #2 (labeled cyan). Negative selection was performed after rounds 3 and 4 using CD8-negative cells from donor #1 and donor #2, respectively. The final round, round 7, was repeated three times: once under "normal" conditions (i.e., 3x washes and a short incubation time), once with a long incubation time before the final wash ("long wash"), and finally with twice the number of washes ("6x wash"). Round 7 was performed using cells from donor #3. [Figure 6] Figure 6 shows a comparative binding SELEX assay. Isolated CD8 T cells were incubated with random library 2.6 or one of the binding SELEX results from rounds 4, 6, or 7 tagged with Cy5 at 37°C for 1 hour. Cy5 fluorescence intensity was assayed using flow cytometry. Figure 6A shows a histogram of Cy5 fluorescence intensity for each round. Figure 6B shows the fold change for each round relative to the initial library, the random 2.6 library. [Figure 7-1]Figure 7 shows the results of next-generation sequencing (NGS) analysis. Figure 7A shows the relative abundance of individual sequences in the different rounds sequenced (R2, R5, R6, and R7). The top 100 most abundant sequences in the final enriched library, R7, are displayed in gray. The top 10 most abundant sequences are displayed in color. Figure 7B shows the R7-bound-unbound ratio of individual sequences identified after "long wash" stringency plotted against their relative abundance in R7. Selected sequences are displayed in color. [Figure 7-2] Figure 7 shows the results of next-generation sequencing (NGS) analysis. Figure 7C shows the R7-bound-to-unbound ratio of individual sequences at 6x wash stringency plotted against their relative abundance at R7. Selected sequences are indicated by color. Figure 7D shows the R7-bound-to-unbound ratio of individual sequences at "long wash" stringency plotted against their R7-bound-to-unbound ratio at 6x wash stringency. [Figure 8] Figure 1 shows the initial screening of putative aptamers for binding to CD8 cells via flow cytometry. Fluorescence of isolated T cells was measured after each wash cycle for a total of three washes. Results were normalized to a "random" aptamer in each wash. N=1 or 2. [Figure 9] FIG. 1 depicts the structure predicted by NUPACK (Zadeh et al. (2011) J. Comput. Chem. 32:170-173) of CTL3, a promising CD8 cell-binding candidate. [Figure 10] Figure 1 shows that CTL3 binds to PBMCs. The CTL3 aptamer showed significantly higher binding affinity to total PBMCs compared to the control aptamer. Cy5-labeled CTL3, random aptamer sequence (RND), and polyT aptamer, each at 250 nM, were tested for their binding after 1 hour (hr) of incubation at 4°C. Unstained cells represented cells without aptamer. N=3. [Figure 11-1]Figure 11 shows CTL3 binding to different PBMC subpopulations. CTL3 bound to lymphocytes, but no significant binding to monocytes was observed (Figures 11A and 11B). CTL3 bound equally to CD8-positive and -negative cells (Figures 11C and 11D). Cy5-labeled CTL3, RND, and polyT aptamers, each at 250 nM, were tested for their binding after 1 hour of incubation at 4°C. Unstained cells represented cells without aptamer. N=3. [Figure 11-2] Continued from Figure 11-1. [Figure 12] Figure 12 shows CTL3 binding compared to a scrambled sequence. The CTL3 aptamer showed significant binding affinity to PBMCs (Figure 12A) and CD8 T cells (Figure 12B) compared to the control scrambled (SCR) aptamer. Cy5-labeled CTL3 and CTL3 SCR aptamers, each at 250 nM, were tested for their binding after 1 hour of incubation at 4°C. Unstained cells represent cells without aptamer. N=3. [Figure 13] Figure 1 shows that CTL3 bound to isolated CD8 T cells. Cy5-labeled CTL3, RND, and polyT aptamers, each at 250 nM, were tested for their binding to isolated CD8 cells after 1 hour of incubation at 4°C. Unstained cells represented cells without aptamer. [Figure 14] Figure 14 shows that CTL3 bound to activated and expanded pan T cells. CTL3, RND, and polyT aptamers were tested for their binding to activated and expanded pan T cells 11 days after initial activation. CTL3 bound to both CD8-positive (Figure 14A) and -negative cells (Figure 14B) when compared to control aptamers. Cy5-labeled CTL3, RND, and polyT aptamers, each at 250 nM, were tested after 1 hour of incubation at 4°C. Unstained cells represented cells without aptamer. N=1. [Figure 15]Figure 1. Description of Integral Molecular's Membrane Proteome Array (MPA). The MPA is a high-throughput cell-based platform for identifying membrane protein targets of ligands. Membrane proteins were expressed in human cells on 384-well microplates, and ligand binding was detected by flow cytometry, allowing for sensitive detection of both specific and off-target binding. [Figure 16] FIG. 1 shows membrane protein array screening using CTL3. [Figure 17] Figure 1 shows target hit validation for CTL3 aptamers by serial dilution. [Figure 18] Figure 18 shows a schematic diagram of thermofluorescence analysis (TFA) of aptamer-protein binding. Intercalator fluorescence is low in the melt-free state (left) and high in the folded aptamer or protein-bound state (center, right). Protein binding adds stability and increases the aptamer melting temperature (i.e., Tm, bound > Tm, unbound). Figure 18 is adapted from Hu, Kim and Easley (2016) HHS Public Access. 7:7358-7362. [Figure 19] Quantitative protein detection using TFA at 100 nM CTL3. Increasing concentrations of Notch2 and CD160 were used as controls. Total fluorescence (left) and fluorescence curve derivative (right) are shown. [Figure 20] Figure 1 shows the sequences evaluated for binding to recombinant Notch2. CTL3 and two scrambled DNA sequences were evaluated for their binding to recombinant Notch2. [Figure 21] Figure 21 shows quantitative protein binding detection using TFA. Tm profile curves were generated using 100 nM CS with increasing concentrations of human recombinant Notch2 (green, Figure 21A), mouse recombinant Notch2 (purple, Figure 21B), and rat recombinant Notch2 (orange, Figure 21C). [Figure 22] FIG. 1 shows a schematic diagram of the CD3ε binding SELEX process. [Figure 23-1] Figure 23B shows a comparative binding SELEX assay. Binding assays were performed using the initial random library (Rnd Lib) and library enrichment pools from rounds 3 (R3), 6 (R6), 9 (R9), and 11 (R11) against the target protein CD3ε-bead complex (black) or the control protein IgG1 (gray). After incubation and washing, library DNA was eluted, and the concentration in the supernatant was assessed via real-time PCR. A standard curve was performed using the random library (top). Binding of the Cy5 fluorescently labeled library to Jurkat T cell lines and pan-B cells was demonstrated by flow cytometry (Figure 23B). Dot plots and histogram graphs are shown. Flow data quantification of Cy5 median fluorescence intensity (MFI) is shown. [Figure 23-2] Continuation of Figure 23-1. [Figure 24-1] Figure 24 shows the results of next-generation sequencing (NGS) analysis. Figure 24A shows the analysis of single aptamer sequences from the 8th, 9th, 10th, and 11th SELEX round enrichment libraries on a dot plot, with the X-axis representing the average P negative and the Y-axis representing the average P positive. The diagonal line represents the threshold between specific binder aptamers and nonspecific low-binding aptamer sequences. The top five candidates selected for further consideration are shown with their names. Figure 24B shows the sequence logo display (using GLAM2 software) of the shared motifs of the top 14 specific binder aptamers (top) and the top four selected aptamers (bottom). [Figure 24-2] Figure 24C shows the results of next-generation sequencing (NGS) analysis. Figure 24C shows the secondary structure analysis (mfold) of the five selected candidates. The motif nucleotide positions are highlighted with red asterisks. [Figure 25]Figure 1 shows aptamer sequences that bind to target proteins by HPLC. Folded Cy5-labeled aptamer candidates were assayed for recombinant human CD3ε (hCD3ε) binding. Aptamers were incubated with hCD3e or negative control IgG1 for 1 hour at 37°C. PolyT was used as a negative control sequence. [Figure 26-1] Figure 26 shows CS6 binding to T cells as demonstrated by flow cytometry. Jurkat cells and Kasumi-1 cells were incubated with CpG'-Cy5-labeled CS6, CS7, and CS8c and analyzed by flow cytometry (Figure 26A). Jurkat cells and Daudi cells were incubated with CpG'-Cy5-labeled CS6, CS7, and CS8c and analyzed by flow cytometry. MFI quantification is shown below (Figure 26B). [Figure 26-2] Figure 26 shows CS6 binding to T cells as demonstrated by flow cytometry. Isolated pan-T cells and pan-B cells were incubated with CpG'-Cy5-labeled CS6 and analyzed by flow cytometry. Dot plots of Cy5 (X axis) / SSC (Y axis) and MFI quantification of T cells and B cells are shown (Figure 26C). [Figure 27] Figure 10: CS6 Effective Concentration: Jurkat cells were incubated with serially diluted concentrations of CpG'-Cy5 labeled CS6 and analyzed by flow cytometry to determine the EC50 of the compounds. [Figure 28] FIG. 1 shows the binding of CS6 to either the target protein hCD3ε (top) or a non-specific IgG control protein (bottom) by SPR sensograms. [Figure 29] FIG. 1 shows that bispecific aptamers act as T cell engagers and stimulate CD69 upregulation. [Figure 30]Figure 30 shows a schematic representation of a bispecific personalized aptamer showing three distinct domains, highlighting the double-stranded hybridization domain that functions as a TLR9 agonist (Figure 30A). The chemical structure of a phosphodester bond compared to a phosphorothioate modification (adapted from Pohar et al. (2017) Sci. Rep. 7:14598) (Figure 30B). (i) List of 22 base pair (bp) CpG bridge sequences: CpG1 (SEQ ID NO: 63), CpG1' (SEQ ID NO: 64), CpG2 (SEQ ID NO: 65), and CpG2' (SEQ ID NO: 66). (ii) Shows PS variations of bispecific personalized aptamers showing different monomer sequences: CpG1|CTL3 (SEQ ID NO: 28), 5PS-CpG1|CTL3 (SEQ ID NO: 29), 10PS-CpG1|CTL3 (SEQ ID NO: 30), FullPS-CpG1|CTL3 (SEQ ID NO: 31), CpG1'|VS12 (SEQ ID NO: 49), 5PS-CpG1'|VS12 (SEQ ID NO: 50), 10PS-CpG1'|VS12 (SEQ ID NO: 51), FullPS-CpG1'|VS12 (SEQ ID NO: 52). The phosphodiester backbone is shown in light gray. The PS backbone is indicated by an asterisk (Figure 30C). [Figure 31] Figure 31 shows the effect of introducing a CpG1 motif into a bispecific aptamer on its ability to induce tumor cell death. Killing assays against NK cells and CD8 T cell engagers using a CpG-containing bispecific personalized aptamer (Figure 31A) and different PS modifications, e.g., CTL3 and VS12 monomers both have five PS modifications at their 5' ends for CTL3|5PS-CpG1-5PS|VS12 (Figure 31B). HCT116 cells were co-cultured with PBMCs for 72 hours with three doses of 100 μM bispecific personalized aptamer. The lethality was analyzed by flow cytometry on HCT116 cells. [Figure 32]Figure 32 shows that the CpG / TLR9 agonist motif of bispecific aptamers alters immune responses in both humans and mice. Pan-B cells were isolated and seeded in 96-well plates (200,000 cells / well) for 24 hours. Cells were treated with vehicle, polyT-polyT (50 μM) as a negative control, 5 μM oligodeoxynucleotide ODN-2395 (Roda et al. (2005) J. Immunol. 175:1619-1627), a cell culture-tested ODN used as a positive control, and bispecific aptamer CTL3-VS12 (50 μM). After 24 hours of treatment, cells were harvested and analyzed for CD86 expression by flow cytometry. A representative donor from one of three is shown (Figure 32A). Splenocytes from BALB / c mice were isolated (n=3) and seeded in 96-well plates (500,000 cells / well). The cells were treated for 48 hours with vehicle, ODN negative control (5 μM), ODN 2395 (5 μM) as a positive control, and bispecific aptamer CTL3-VS12 (50 μM). After 48 hours of treatment, the cells were centrifuged, the supernatant was collected, and IL-6 secretion was analyzed using an IL-6 ELISA kit (Figure 32B). PBMCs were co-cultured with HCT-116 cells for 48 hours and treated with 50 μM ODN 2395 with (positive control) or without (negative control) PS modification, and with dsCpG2 as a standalone sequence or in the context of a bispecific aptamer. The cell culture medium was collected and analyzed for IFN-α using an ELISA kit (Figure 32C). [Figure 33-1] FIG. 33 shows that CpG motifs (SEQ ID NOs: 63 and 64) in either single-chain form or in the bispecific aptamer structure alter IL-6 secretion (FIG. 33A) and costimulatory molecule expression (FIG. 33B). [Figure 33-2] Continuation of Figure 33-1. [Figure 34] FIG. 1 shows that CpG motifs (SEQ ID NOs: 63 and 64) in bispecific entities act in a dose-dependent manner. [Figure 35]FIG. 1 shows functional enrichment of DNA libraries for activation of apoptosis in HCT116 (colorectal cancer) cells. [Figure 36] FIG. 1 shows bioinformatics analysis of the final enriched functional library after NGS. [Figure 37] FIG. 1 shows multiple doses of the top aptamer candidates for cytotoxicity. [Figure 38] Figure 38A shows functional enrichment results for the MCF7 cell line. Comparative functional assay showing enriched libraries from the initial (F3.1), fifth (F3.5), sixth (F3.6), and final (F3.7) rounds of enrichment incubated with the MCF7 cell line for 2 hours. Annexin V positive staining was measured via flow cytometry and normalized to the initial round of enrichment (F3.1). Total Annexin V levels are shown above the bars for the first and final rounds of enrichment (Figure 38A). Sequencing results are presented in a scatter plot, with each dot representing a single sequence. The X-axis indicates the tendency of the sequence to induce Annexin V binding on MCF7 cells (P positive), and the Y-axis indicates the tendency of the sequence to induce Annexin V binding to negatively selected cells, healthy donor-derived PBMCs (P negative). Green-colored dots represent sequences selected for individual screening via high-content fluorescence microscopy (Figure 38B). [Figure 39]Figure 39 shows high-content screening of individual aptamers by time-lapse fluorescence microscopy. Representative images at t=14 hours from the initial screening of aptamer leads VS13 (SEQ ID NO: 45), VS16 (SEQ ID NO: 46), and VS19 (SEQ ID NO: 47), all at 50 μM concentration, compared to vehicle, random oligonucleotide, and staurosporine. Cell nuclei were stained with Hoechst 33258 (blue) and Annexin V (pink) (Figure 39A). Scatter plots depict the analysis of lead aptamers. The X-axis shows the total percent of cells positive for Annexin V at t=14 hours. The Y-axis shows the fold increase in Annexin V at t=14 hours compared to t=0 hours for each aptamer. Top leads are highlighted in pink, negative controls in green, and positive controls (staurosporine) in red (Figure 39B). [Figure 40] Figure 40A shows potency and specificity confirmation for the final MCF7 and aptamer leads. Dose-dependent (50, 100, and 200 μM) viability of MCF7 cells incubated with lead aptamers (red line), VS13 (right panel), and VS16 (left panel) was assessed over 48 hours and compared to poly-T aptamer control (dotted line) and PBMC (blue line) at doses administered once daily. Viability was measured using an XTT assay and plotted as fold over vehicle control (Y-axis) (Figure 40A). Scatter plot summaries showing MCF7 viability (Y-axis) and PBMC viability (X-axis) for the lead aptamers were examined. The positive control (staurosporine) is indicated by a red circle. Vehicle and untreated controls are indicated by light green circles. The six lead aptamers are indicated by dark blue hexagons for the 200 μM dose, blue diamonds for the 100 μM dose, and light blue triangles for the 50 μM dose level. PolyT controls are indicated by dark green symbols: hexagons, diamonds, and triangles for 200 μM, 100 μM, and 50 μM, respectively. VS13 and VS16 are indicated by "13" and "16" (Figure 40B). [Figure 41] FIG. 1 shows functional enrichment results for the A549 cell line. [Figure 42]FIG. 1 shows validation for the final A549 variable chain aptamer lead. [Figure 43] FIG. 1 shows CRC organoid formation. [Figure 44] Figure 44A shows functional enrichment results for CRC13 organoids (Figure 44A) and efficacy validation for final CRC13 variable chain aptamer leads (Figure 44B). [Figure 45] Figure 1 shows a schematic illustration of bispecific personalized aptamer formulation using the CTL3|CpG1|VS12 example. Each arm is reconstituted to a concentration of 2 mM and subjected to a rapid temperature ramp, i.e., immediate cooling of the solution from 95°C to 4°C, followed by aptamer folding and subsequent mixing and hybridization, resulting in a bispecific entity with a final concentration of 1 mM. [Figure 46] Figure 46 shows a cytotoxicity assay mediated by bispecific personalized aptamers engaging either natural killer (NK) cells or cytotoxic T lymphocytes (CTLs). HCT116 cells and peripheral blood mononuclear cells (PBMCs) from two healthy donors were co-cultured for 72 hours. Natural killer and CTL bispecific personalized aptamers were administered once daily at 100 μM for a total of three doses, followed by a Live / Dead dye assay. Figure 46A shows the lethality of HCT116 cells, and Figure 46B shows the lethality of PBMCs. Vehicle and polyT||polyT dimers are used as negative controls. Mitomycin (10 μM) and anti-CD3 / anti-CD28 antibodies (1 μg / mL), administered as a single dose, are positive controls. n=2. [Figure 47] Figure 1 shows bispecific personalized aptamers targeting cancer cells in a dose-dependent manner. Four concentrations of each bispecific personalized aptamer were tested: 10, 25, 50, and 100 μM. HCT116 cells were co-cultured with PBMCs in the presence of the bispecific personalized aptamers at the indicated concentrations for 72 hours. Lethality was analyzed by flow cytometry. n=2. [Figure 48]Figure 1 shows killing assay data for bispecific personalized aptamers using PBMCs and HCT116 or MCF10a cells. Either HCT116 or MCF10a cells were co-cultured with PBMCs for 72 hours. CTL bispecific personalized aptamers were administered once daily at 100 μM for a total of three doses, followed by a Live / Dead dye assay. Lethality was analyzed by flow cytometry. Benchmark criteria for bispecific personalized aptamer selection are highlighted by boxes. [Figure 49] Figure 49 shows that bispecific individualized aptamers induce higher lethality than each monomer. HCT116 cells were co-cultured with PBMCs for 72 hours with 100 μM bispecific individualized aptamers or three doses of monomers. The lethality was analyzed by flow cytometry for HCT116 cells (Figure 49A) and PBMCs (Figure 49B). n=14. [Figure 50] Figure 1 shows killing assay data for CTL3||VS12 and CTL6||VS12 bispecific personalized aptamers. HCT116 cells were co-cultured with PBMCs for 72 hours with 100 μM bispecific personalized aptamer or 3 doses of monomer. Lethality was analyzed by flow cytometry. n=3. [Figure 51] FIG. 1 shows that bispecific personalized aptamers induce tumor cell death in vitro. [Figure 52]Figure 52 shows that bispecific personalized aptamers induced cytotoxicity in MCF7 cells cocultured with PBMCs. PBMCs were primed with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (400 U / mL) for 4 days prior to coculture setup. Primed immune cells were cocultured with MCF7 cells at a 5:1 effector:target ratio and incubated with 100 μM bispecific aptamers CTL3||VS13, CTL3||VS16, and CTL3||VS19 for 48 hours. PolyT dimer (polyT||polyT) and vehicle were used as negative controls. Lethality was measured via Live-Dead Zombie staining (flow cytometry) (Figure 52A). Viability was measured via XTT and normalized to vehicle control (Figure 52B). n=4 PBMC donors. [Figure 53] Figure 53 shows the in vivo efficacy of the CD16||VS12 bispecific personalized aptamer. Female immunodeficient female NOD scid gamma (NSG™) mice were implanted subcutaneously (SC) with HCT116 tumor cells mixed with human PBMCs and subsequently treated with 100 mg / kg polyT or 100 mg / kg NK engager bispecific personalized aptamer (priming dose, highlighted by triangle) for a total of 12 doses administered SC. Tumor volumes were measured up to day 32 and shown as mean ± SEM (Figure 53A). Tumor weights were assessed at the end of survival (day 33). Results are expressed as mean ± SEM (Figure 53B). Figure 53C shows Kaplan-Meier survival analysis of the bispecific personalized aptamer (Figure 53C). *(p≦0.05) and **(P≦0.01) indicate significant differences. [Figure 54]Figure 1 shows the in vivo efficacy of CTL6||VS12 bispecific personalized aptamers produced by two different suppliers. Female NSG™ mice were implanted SC with HCT-116 tumor cells mixed with human PBMCs and subsequently treated with 100 mg / kg T cell engager bispecific personalized aptamer (represented as squares) for a total of 12 doses administered SC. HCT116 tumor volumes were measured up to day 27 (mean ± SEM shown). * indicates significant difference (p≦0.05). [Figure 55] Individual HCT116 tumor volumes in vehicle- and CTL6||VS12-treated mice are shown. Open shapes represent deaths. [Figure 56] Figure 1 shows HCT116 tumor volumes on day 27. Comparison between different treatment groups. * indicates significant difference (p≦0.05). [Figure 57] Figure 57A shows HCT116 tumor volumes for CTL3||VS12-treated, polyT||polyT, vehicle, and untreated mouse groups monitored over the 22 days of the study. Tumors were weighed at the end of the in-life period (Figure 57B). Statistical T-tests were performed. **(p≦0.005) and ***(P≦0.001) indicate significant differences. [Figure 58] FIG. 1 depicts Kaplan-Meier survival analysis of CTL3||VS12-treated mice. [Figure 59] Figure 59A shows the in vivo efficacy of an exemplary bispecific T cell engager aptamer composed of the CS6 aptamer (SEQ ID NO: 116) hybridized with an HCT116 colon cancer cell line-targeting aptamer sequence (designated VS12; SEQ ID NO: 50). Female NSG mice were implanted SC with HCT-116 tumor cells mixed with human PBMCs and subsequently treated with the T cell engager bispecific personalized aptamer for a total of 10 doses administered SC. HCT116 tumor volume was monitored for CS6-VS12-treated, polyT-polyT (nonspecific DNA aptamer), and vehicle mouse groups (Figure 59A). Individual mouse growth curves are depicted in Figure 59B. *** indicates significant differences (P≦0.001). [Figure 60] Figure 1 depicts Kaplan-Meier survival analysis of treated mice. ** indicates significant difference (P≦0.01). [Figure 61] Figure 61 shows the in vivo efficacy of CTL3|5PS-CpG1|VS16 (CTL3-VS16) in a xenograft MCF7 tumor model. MCF7 tumor volume was measured for 18 days after CTL3-VS16 or vehicle treatment. Mean tumor volume ± SEM is presented (n=6) (Figure 61A). Individual tumor volume increases compared to the day of randomization are plotted (Figure 61B). Statistical T-tests were performed. ** indicates significant differences (P≦0.005). [Figure 62]

[0023] Figure 1 shows the in vivo efficacy of an exemplary bispecific T cell engager aptamer composed of the CS6 aptamer (SEQ ID NO: 116) hybridized to a 4T1 breast cancer cell line-targeting aptamer sequence (designated VS32; SEQ ID NO: 111). Female Balb / c mice were implanted with 4T1 tumor cells SC into both flanks. When primary tumors reached a size of 50 mm3, treatment with the T cell engager bispecific personalized aptamer was initiated using an intratumoral route of administration. Primary and secondary tumor volumes were monitored for CS6-VS12 treatment with or without anti-PD1. DETAILED DESCRIPTION OF THE INVENTION

[0042] overview The methods and compositions provided herein are based, in part, on the development of bispecific, personalized aptamer entities composed of two arms. One aptamer arm is designed to be variable between different patients and bind to a unique target on the surface of the patient's tumor cells. The second aptamer arm is designed to engage effector immune cells and cause tumor cell lysis. This latter, immunomodulatory arm is designed to be shared between different patients. In some embodiments, the two arms are bridged by double-stranded DNA. This DNA "bridge" can have toll-like receptor 9 (TLR9) agonist activity, leading to increased uptake and engulfment of tumor antigens by antigen-presenting cells and the secretion of inflammatory cytokines. The specificity of the aptamer combined with effector cell engagement and TLR9 agonist activity makes bispecific, personalized aptamers promising candidates for a multifaceted approach to cancer treatment. The platform described herein also enables the creation of customized cancer therapeutics for treating patients with personalized solutions.

[0043] Thus, in certain aspects, provided herein are bispecific personalized aptamers comprising a cancer cell-binding chain that selectively binds to and / or kills cancer cells (e.g., breast or colorectal cancer cells), including by inducing apoptosis, ICD, necrosis, necroptosis, and / or autophagy. The bispecific personalized aptamer also comprises an immune effector cell-binding chain that mediates cancer cell lysis via T cell- or NK cell-mediated cytotoxicity. In some embodiments, the cancer cell-binding chain is linked to the immune effector cell-binding chain by a CpG motif that induces TLR9-mediated antigen-presenting cell (APC) stimulation and / or increased tumor antigen uptake. In some aspects, provided herein are pharmaceutical compositions comprising such bispecific personalized aptamers, methods of using such bispecific personalized aptamers to treat cancer and / or kill cancer cells, and methods of making such bispecific personalized aptamers.

[0044] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here. The articles "a" and "an" are used herein to refer to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0045] As used herein, the term "aptamer" refers to a short (e.g., less than 200 bases) single-stranded nucleic acid molecule (ssDNA and / or ssRNA) that can specifically bind to a target molecule (e.g., a protein or peptide, or a topographical feature on a target cell).

[0046] The terms "bind" or "interact" refer to an association, which may be a stable bond between two molecules, e.g., an aptamer and a target, due to electrostatic, hydrophobic, ionic, pi-stacking, coordination, van der Waals, covalent and / or hydrogen bonding interactions, e.g., under physiological conditions.

[0047] As used herein, two nucleic acid sequences are "complementary" or "complementary" to one another if they form base pairs with each other at every position.

[0048] The terms "alter" or "altering," when used with respect to a functional property or biological activity or process (e.g., enzymatic activity or receptor binding), refer to the ability to up-regulate (e.g., activate or stimulate), down-regulate (e.g., inhibit or suppress), or otherwise change the nature of such property, activity, or process. In certain instances, such alterations may be contingent on the occurrence of a specific event, such as activation of a signaling pathway, and / or may be manifest only in particular cell types.

[0049] As used herein, "specific binding" refers to the ability of an aptamer to bind to a single target. Typically, an aptamer binds to a single target at a specific binding site within a range of about 10 -7 Less than M, about 10 -8 Less than M or about 10 -9 K less than M D and is significantly less (e.g., at least 2-fold less, at least 5-fold less, at least 10-fold less, at least 50-fold less, at least 100-fold less, at least 500-fold less, or at least 1000-fold less) than its affinity for binding to its nonspecific, unrelated target (e.g., BSA, casein, or unrelated cells such as HEK293 cells or colon cancer cells). D and binds to the target.

[0050] The terms "oligonucleotide" and "nucleic acid molecule" refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified, such as by conjugation with a labeling component.

[0051] Bispecific personalized aptamers In certain aspects, provided herein are bispecific personalized aptamers comprising: (a) a cancer cell-binding chain that specifically binds to an antigen expressed on a cancer cell; (b) a CpG motif; and (c) an immune effector cell-binding chain that binds to an immune effector cell, wherein the cancer cell-binding chain is linked to the immune effector cell-binding chain by a CpG motif.

[0052] In some embodiments, the cancer cell-binding chain is capable of inducing cell death (e.g., apoptosis) in a cancer cell (e.g., a human cancer cell) when contacted with the cancer cell. In some embodiments, the cancer cell is a patient-derived cancer cell. In some embodiments, the cancer cell is a solid tumor cell (e.g., a breast cancer cell). In certain embodiments, the cancer cell is a carcinoma cell (e.g., a colorectal cancer cell). In some embodiments, the aptamer induces cell death when contacted with a cancer cell in vitro. In certain embodiments, the aptamer induces cell death when contacted with a cancer cell in vivo (e.g., in a human and / or animal model). In some embodiments, the cancer cell binding chain binds to a cancer antigen selected from prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), tyrosinase, gp100, MART-1 / melan-A, HSP70-2-m, HLA-A2-R17OJ, HPV16-E7, MUC-1, HER-2 / neu, mammaglobin-A, or an MHC-TAA peptide complex.

[0053] In certain embodiments, the cancer cell-binding chain comprises a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the cancer cell-binding chain comprises the nucleic acid sequence of any one of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the cancer cell-binding strand comprises at least 30 (e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69) contiguous nucleotides of any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the cancer cell-binding strand has a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the cancer cell-binding strand has a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115.

[0054] The term "identical" or "percent identity," in the context of two or more nucleic acids, means two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0055] In certain embodiments, the cancer cell-binding strand is 120 nucleotides or less in length (e.g., 115 nucleotides or less, 110 nucleotides or less, 105 nucleotides or less, 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 69 nucleotides or less, 68 nucleotides or less, 67 nucleotides or less, 66 nucleotides or less, 65 nucleotides or less, 64 nucleotides or less, or 63 nucleotides or less). In certain embodiments, the cancer cell-binding strand is about 63 nucleotides in length.

[0056] In some embodiments, the immune effector cell binding chain binds to a T cell (e.g., CD8 +The immune effector cell binding chain binds to a target expressed by a T cell, a B cell, a NK cell, a macrophage, or a dendritic cell. In certain embodiments, the immune effector cell binding chain binds to an immune effector cell antigen selected from CD16, Notch-2, other Notch family members, KCNK17, CD3, CD28, 4-1BB, CTLA-4, ICOS, CD40L, PD-1, OX40, LFA-1, CD27, PARP16, IGSF9, SLC15A3, WRB, and GALR2. In some embodiments, the immune effector cell binding chain mediates lysis of cancer cells via T cell- or NK cell-mediated cytotoxicity.

[0057] In some embodiments, the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the immune effector cell-binding chain comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-42, 88-106, or 116.

[0058] In certain embodiments, the immune effector cell-binding chain comprises at least 20 (e.g., at least 25, at least 30, at least 35, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53) contiguous nucleotides of any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the immune effector cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. In certain embodiments, the immune effector cell-binding chain provided herein has a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. In certain embodiments, the immune effector cell-binding strand is 120 nucleotides or less in length (e.g., 115 nucleotides or less, 110 nucleotides or less, 105 nucleotides or less, 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 74 nucleotides or less, or 73 nucleotides or less). In certain embodiments, the immune effector cell-binding strand is about 73 nucleotides in length.

[0059] The cancer cell-binding chain and the immune effector cell-binding chain can be linked together by hybridization of the 5' sequence of the cancer cell-binding chain to the 5' sequence of the immune effector cell-binding chain. In certain embodiments, the 5' sequence of the cancer cell-binding chain hybridizes to the 5' sequence of the immune effector cell-binding chain to form a TLR9 agonist sequence that is a CpG-rich motif. The cancer cell-binding chain and the immune effector cell-binding chain can be linked together by direct ligation to each of the two ends (e.g., 5' ends) of the double-stranded sequence. In certain embodiments, the double-stranded sequence is a CpG motif, TLR9 agonist sequence.

[0060] In some embodiments, the TLR9 agonist sequence comprises a double-stranded region comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) CpG motif nucleotide. In some embodiments, the CpG motif induces TLR9-mediated antigen-presenting cell (APC) stimulation and / or increased uptake of tumor antigens. In some embodiments, the TLR9 agonist sequence induces an anti-tumor response. In some embodiments, the TLR9 agonist sequence induces cytokine production.

[0061] In some embodiments, the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 63-66. In some embodiments, the CpG motif sequence is a double-stranded nucleic acid sequence comprising any one of SEQ ID NOs: 63-66.

[0062] In certain embodiments, the CpG motif sequence is a double-stranded nucleic acid sequence comprising at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22) consecutive nucleotides of any one of SEQ ID NOs: 63-66. In some embodiments, the CpG motif sequence provided herein has a sequence consisting essentially of SEQ ID NOs: 63-66. In certain embodiments, the CpG motif sequence provided herein has a sequence consisting of SEQ ID NOs: 63-66.

[0063] In certain embodiments, the CpG motif sequence is 35 nucleotides or less in length (e.g., 34 nucleotides or less, 33 nucleotides or less, 32 nucleotides or less, 31 nucleotides or less, 30 nucleotides or less, 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, 25 nucleotides or less, 24 nucleotides or less, 23 nucleotides or less, or 22 nucleotides or less in length).

[0064] The bispecific personalized aptamers provided herein can comprise any combination of the cancer cell-binding chains and immune cell-binding chains described herein. For example, in some embodiments, the bispecific personalized aptamers comprise a combination of a cancer cell-binding chain and an immune cell-binding chain selected from the group consisting of SEQ ID NOs: 29 and 54, 29 and 50, 32 and 50, 33 and 48, 41 and 49, or 34 and 59. In some embodiments, the bispecific personalized aptamers provided herein comprise one or more chemical modifications. Exemplary modifications are provided in Table 2.

[0065] [Table 2]

[0066] In certain embodiments, the bispecific individualized aptamer comprises a terminal modification. In some embodiments, the bispecific individualized aptamer is chemically modified with polyethylene glycol (PEG) (e.g., 0.5 to 40 kDa) (e.g., linked to the 5' end of the aptamer). In some embodiments, the bispecific individualized aptamer comprises a 5' end cap (e.g., inverted thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS). In certain embodiments, the bispecific individualized aptamer comprises a 3' end cap (e.g., inverted thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS).

[0067] In certain embodiments, the bispecific personalized aptamers provided herein comprise one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54) modified sugars. In some embodiments, bispecific personalized aptamers comprise one or more 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions). In certain embodiments, bispecific personalized aptamers comprise locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'deoxy-2'fluoro-D-arabinonucleic acid (2'-F ANA) sugars in their backbones.

[0068] In certain embodiments, the bispecific individualized aptamer comprises one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54) methylphosphonate internucleotide linkages and / or phosphorothioate (PS) internucleotide linkages.

[0069] In certain embodiments, the bispecific individualized aptamer can comprise a PS modification in the double-stranded region (e.g., CpG motif sequence). For example, the double-stranded region (e.g., CpG motif sequence) of the bispecific individualized aptamer can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 phosphorothioate (PS) internucleotide linkages on one or both strands. In some embodiments, the double-stranded region (e.g., CpG motif sequence) of the bispecific individualized aptamer can comprise a partial PS modification. In certain embodiments, five nucleotides from the 5' end of the double-stranded CpG motif sequence are modified. In other embodiments, five nucleotides from both the 5' end and the 3' end of the double-stranded CpG motif sequence are modified. In certain embodiments, the double-stranded CpG motif sequence comprises a full PS modification.

[0070] In certain embodiments, an aptamer comprises one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54) triazole internucleotide linkages. In certain embodiments, aptamers are modified (eg, at their 5' ends) with cholesterol or dialkyl lipids.

[0071] In some embodiments, the aptamer comprises one or more modified bases (eg, BzdU, naphthyl, tryptamino, isobutyl, 5-methylcytosine, alkyne (dibenzocyclooctyne), azide, maleimide).

[0072] In certain embodiments, the aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or enantiomeric L-DNA aptamers). In some embodiments, the aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or enantiomeric L-RNA aptamers). In some embodiments, the aptamers comprise a mixture of DNA and RNA.

[0073] Pharmaceutical Composition In certain aspects, provided herein are pharmaceutical compositions comprising the bispecific personalized aptamers provided herein (e.g., therapeutically effective amounts of the bispecific personalized aptamers). In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral administration (e.g., subcutaneous administration).

[0074] In certain embodiments, the pharmaceutical composition is for use in treating cancer. In some embodiments, the cancer is a solid tumor (e.g., breast cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, bladder cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, or colorectal cancer). In some embodiments, the solid tumor is accessible to intratumoral administration. In certain embodiments, the cancer is a carcinoma. In certain embodiments, the cancer is a sarcoma (e.g., soft tissue sarcoma). In certain embodiments, the cancer is a hematological cancer (e.g., lymphoma).

[0075] A "pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an active agent to a subject and the absorption of the active agent by the subject, and can be included in the compositions described herein without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, phosphate-buffered saline, MgCl, KCl, CaCl, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, starch, fatty acid esters, lipids, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such formulations can be sterilized and, if necessary, can be mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or flavoring agents that do not adversely react with the compositions described herein. Those skilled in the art will recognize that other excipients are useful.

[0076] Treatment method In some embodiments, the present invention provides a method for treating cancer, comprising administering a pharmaceutical composition comprising one or more bispecific personalized aptamers described herein.In certain embodiments, the cancer is breast cancer.In some embodiments, the cancer is colorectal cancer.Therefore, in certain aspects, the present invention provides a method for delivering the bispecific personalized aptamer and / or pharmaceutical composition described herein to a subject.

[0077] In certain embodiments, the pharmaceutical compositions and aptamers described herein can be administered as monotherapy or in conjunction with any other conventional anti-cancer treatment, such as, for example, radiation therapy and surgical resection of the tumor. These treatments may be administered as needed and / or as directed, and can occur before, simultaneously with, or after administration of the pharmaceutical compositions, dosage forms, and kits described herein.

[0078] In certain embodiments, the method comprises multiple administrations of aptamers. Each individual administration can comprise any number of administrations (e.g., multiple administrations) of two or more, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21, 22, 23, 24, or 25 administrations. In some embodiments, at least 8, 9, 10, 11, 12, 13, 14, or 15 administrations are included. For the treatment methods and other monitoring methods provided herein, those skilled in the art can easily determine the number of administrations to be performed, or whether it is desirable to perform one or more additional administrations, according to methods known in the art. Thus, the methods provided herein include methods of providing one or more administrations of a bispecific personalized aptamer to a subject, where the number of administrations is determined by monitoring the subject, and based on the results of the monitoring, it can be determined whether or not to provide one or more additional administrations. The determination of whether or not to administer one or more additional administrations can be made based on various monitoring results, including, but not limited to, signs of tumor growth or inhibition of tumor growth, the appearance of new metastases or inhibition of metastases, the subject's anti-aptamer antibody titer, the subject's anti-tumor antibody titer, the subject's general health, and / or the subject's body weight.

[0079] The time period between doses can be any of a variety of time periods. In some embodiments, doses can be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or 1, 2, 3, or 4 weeks. The time period between doses can be a function of any of a variety of factors, including an acceptable regimen for intratumoral administration, monitoring steps, as described with respect to the number of doses, the time period for a subject to mount an immune response, and / or the time period for a subject to clear the bispecific personalized aptamer. For example, the time period can be a function of the time it takes for a subject to mount an immune response; for example, the time period can be longer than the time it takes for a subject to mount an immune response, e.g., more than about 1 week, more than about 10 days, more than about 2 weeks, or more than about 1 month; for example, the time period can be shorter than the time it takes for a subject to mount an immune response, e.g., less than about 1 week, less than about 10 days, less than about 2 weeks, or less than about 1 month. In another example, the time period can be a function of the time it takes for a subject to remove the bispecific individualized aptamer; for example, the time period can be longer than the time it takes for a subject to remove the bispecific individualized aptamer, e.g., more than about 1 day, more than about 2 days, more than about 3 days, more than about 5 days, or more than about 1 week.

[0080] The dosage of the bispecific personalized aptamer described herein is the amount of bispecific personalized aptamer effective for a particular patient, composition, and administration method to achieve a desired therapeutic response while minimizing toxicity to the patient or at the maximum feasible dose. Effective dosage levels can be determined using the methods described herein and depend on a variety of factors, including the activity of the administered individual composition (i.e., the efficacy of the arm selected for personalization, the distribution and expression level of the individualized aptamer target), the route of administration, the time of administration, the excretion rate of the individual compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the individual composition used, the age, sex, weight, disease, general health and medical history of the patient receiving treatment, and the size of the target lesion injected for intratumoral administration, all of which are well known in the medical field. Generally, an effective dose of a cancer therapeutic agent is the amount of the therapeutic agent that is the lowest effective dose to produce a therapeutic effect. Such an effective dose generally depends on the above factors.In some embodiments, the amount of ethanol per dose is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 ,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0 mg / kg; or a total of 50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg of aptamer or pharmaceutical composition is administered (e.g., administered intratumorally).

[0081] Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal), or injection. Injection administration includes intravenous (IV), intralesional, intratumoral, peritumoral, intramuscular (IM), and subcutaneous (SC) administration. The compositions described herein can be administered in any form and by any effective route, including oral, parenteral, enteral, intravenous, intratumoral, intravesical, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ocular, nasal (intranasal), topical, parenteral, e.g., aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., transvaginal and perivaginal)), implantable, intravesical, pulmonary, duodenal, gastric, and intrabronchial. In some embodiments, the bispecific personalized aptamers described herein are administered orally, rectally, topically, intravesically, by injection into or near a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In some embodiments, administration is parenteral (e.g., subcutaneous). Administration can be by intratumoral or peritumoral injection.

[0082] Dosage regimens can take any of a variety of forms and amounts, and can be determined by those skilled in the art based on known clinical factors. As is known in the medical arts, the dosage for any one patient can depend on numerous factors, including the subject's species, size, body surface area, age, sex, immune competence, tumor size, general health and specific biomarkers, the particular bispecific personalized aptamer administered, the duration and route of administration, the type and stage of disease, e.g., tumor size, and other compounds, such as drugs, administered concomitantly.

[0083] The therapeutic methods described herein are believed to be suitable for treating primary tumors, secondary tumors, or metastases, as well as tumor or cancer recurrence. The dosage of the pharmaceutical compositions described herein can be appropriately set or adjusted depending on the dosage form, administration route, severity or stage of the target disease, etc.

[0084] In some embodiments, the dose administered to a subject is sufficient to prevent cancer, delay the onset of cancer, delay or stop the progression of cancer, or prevent the recurrence of cancer, reduce tumor burden, or contribute to the subject's disease-free survival, time to progression, or overall survival. Those skilled in the art will recognize that the dosage will depend on various factors, such as the potency of the particular compound used, as well as the age, species, disease, and weight of the subject. The size of the dosage will also be determined by the route, timing, and frequency of administration, as well as the presence, nature, and extent of any adverse side effects associated with the administration of the particular compound, and the desired physiological effect.

[0085] Appropriate dosages and administration schedules can be determined by conventional dose-response range-finding techniques known to those skilled in the art. Generally, treatment is initiated with a small dose that is less than the optimal dose of the compound. The dose is then gradually increased until the optimal effect under the circumstances is achieved. Effective dosages and treatment protocols can be determined by routine methods, such as starting with a low dose in experimental animals, increasing the dose while confirming efficacy, and systematically changing the administration schedule. Animal experiments are often used to determine the maximum tolerated dose (MTD) of a biologically active substance per kg body weight. Those skilled in the art routinely estimate dosages for efficacy in other species, including humans, while avoiding toxicity.

[0086] In accordance with the above, dosages of the aptamers described herein for therapeutic use may vary depending on the particular aptamer, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the treating clinician or practitioner, among other factors that will affect the selected dosage. Generally, the dosage should be sufficient to result in slowing, and preferably regression, of tumor growth, and most preferably complete regression of the cancer.

[0087] Examples of cancers that can be treated by the methods described herein include hematological malignancies, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, fetal cell leukemia, eosinophilic leukemia, Gross' leukemia, Reeder's cell leukemia, Schilling's leukemia, stem cell leukemia, and subleukemic leukemia. Anaplastic cell leukemia, Hairy cell leukemia, Hemocytoblastic leukemia, Hemocytoblastic leukemia, Histiocytic leukemia, Stem cell leukemia, Acute monocytic leukemia, Leukopenic leukemia, Lymphatic leukemia, Lymphoblastic leukemia, Lymphocytic leukemia, Lymphogenous leukemia, Lymphoid leukemia, Lymphosarcoma cell leukemia, Mast cell leukemia, Megakaryocytic leukemia, Micromyeloblastic leukemia, Monocytic leukemia, Myeloblastic leukemia, Myelogenous leukemia, Myelomonocytic leukemia, Naegeli leukemia, Plasma cell cell) leukemia, plasmacytic leukemia, promyelocytic leukemia, acinic cell carcinoma, lobular carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar epithelial carcinoma, basal cell carcinoma, basal cell carcinoma basocellulare), basal cell carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma comedonal carcinoma, endometrial carcinoma, cribriform carcinoma, armor-like carcinoma, skin cancer, columnar cell carcinoma, columnar cell carcinoma, ductal carcinoma, hard carcinoma, embryonal carcinoma, encephaloid carcinoma carcinoma), epidermoid carcinoma, adenoid carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, globular cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, beaded carcinoma, telangiectatic carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, choriocarcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematogenous carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline cell carcinoma, adrenal-like carcinoma, infantile embryonal carcinoma, carcinoma in situ (cancer in the normal location, carcinoma in situ), intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kurticsky cell (neuroendocrine cell) carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma carcinoma, melanoma, soft carcinoma, mucinous carcinoma, carcinoma muciparum, mucous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, medullary carcinoma, renal cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromatous sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcomaReticulum cell sarcoma, rhabdomyosarcoma, serous cystic sarcoma, synovial sarcoma, telangiectatic sarcoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic islet cell tumor, malignant carcinoid, precancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, neuro These include, but are not limited to, blastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subungual melanoma, superficial spreading melanoma, plasmacytoma, colon cancer, and rectal cancer.

[0088] In some embodiments, the methods and compositions described herein relate to the treatment of sarcoma. The term "sarcoma" generally refers to a tumor composed of an embryonic connective tissue-like entity, generally consisting of closely packed cells organized in a fibrillar, heterogeneous, or homogeneous entity. Sarcomas include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma, and sarcoma. sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromatous sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0089] Additional examples of neoplasms that can be treated using the methods and compositions described herein include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant insulinoma, malignant carcinoid, precancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma, among others.

[0090] In some embodiments, the cancer to be treated is melanoma.The term "melanoma" is intended to mean tumors arising from the melanocyte system of the skin and other organs.Non-limiting examples of melanoma include Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral lentigo melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0091] Specific categories of tumors that can be treated using the methods and compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, and metastatic cancers of all of the above. Individual tumor types include hepatocellular carcinoma, liver cancer, hepatoblastoma, rhabdomyosarcoma, esophageal cancer, thyroid cancer, ganglioneuroblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, invasive ductal carcinoma of the breast, papillary adenocarcinoma, melanoma, lung squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well-differentiated, moderately differentiated, poorly differentiated, or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, adrenal gland tumor (Gravitz tumor, hypernephroma), adrenal gland-like adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, and embryonal carcinoma. Cancer, Wilms' tumor, testicular cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, and large cell lung cancer), bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon cancer, rectal cancer, hematopoietic malignancies (including all types of leukemia and lymphoma, such as acute myeloid leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.).

[0092] Cancers addressed in certain embodiments also include precancerous lesions, such as actinic keratosis, moles (dysplastic nevi), actinic cheilitis (farmer's lip), skin horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, iron deficiency dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis, and cervical dysplasia.

[0093] Cancers addressed in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal, or mesenchymal origin, including, but not limited to, cholangiomas, colon polyps, adenomas, papillomas, cystadenomas, hepatocellular adenomas, hydatidiform moles, renal tubular adenomas, squamous cell papillomas, gastric polyps, hemangiomas, osteomas, chondromomas, lipomas, fibromas, lymphangiomas, leiomyomas, rhabdomyomas, astrocytomas, nevi, meningiomas, and ganglioneuromas.

[0094] In certain embodiments, the cancer is a solid tumor (e.g., breast cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, bladder cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, or colorectal cancer). In some embodiments, the solid tumor is accessible to intratumoral administration. In certain embodiments, the cancer is a sarcoma (e.g., soft tissue sarcoma). In certain embodiments, the cancer is a hematological cancer (e.g., lymphoma).

[0095] Methods for identifying cancer cell targeting chains In some embodiments, cancer cell binding chains are identified through the SELEX process. In certain embodiments, to identify aptamers that bind to cancer cell targets, multiple rounds (for example, three rounds) of binding SELEX are carried out using targeted cancer cells. Certain embodiments also include the steps of: (a) contacting cancer cells with a plurality of particles having a library of aptamer clusters immobilized on their surfaces ("aptamer cluster particles"), wherein at least a subset of the immobilized aptamer clusters bind to at least a subset of the cancer cells, thereby forming cell-aptamer cluster particle complexes; (b) incubating the cell-aptamer cluster particle complexes for a period of time sufficient to allow at least a portion of the cancer cells in the cell-aptamer cluster particle complexes to perform a cellular function; (c) detecting cell-aptamer cluster particle complexes that are performing the cellular function; (d) separating cell-aptamer cluster particle complexes that include cancer cells performing the cellular function detected in step (c) from other cell-aptamer cluster particle complexes; (e) amplifying the aptamers in the separated cell-aptamer cluster particle complexes to generate a functionally enriched population of aptamers; and (f) A functional SELEX assay is performed through a process comprising identifying an enriched population of aptamers through sequencing, thereby identifying cancer cell-binding chains. In a specific embodiment, a cell death reporter is added after incubation of cancer cells with aptamer cluster particles, but before detecting cell-aptamer cluster particle complexes that undergo cellular function.

[0096] In some embodiments, steps (c) and (d) are performed using a flow cytometer. In some embodiments, the methods described herein further include separating aptamer cluster particles from target cells in the cell-aptamer cluster particle complexes separated in step (d) via thermal denaturation. In some embodiments, the methods described herein further include dissociating aptamers from particles in the separated aptamer cluster particles. In some embodiments, the methods described herein further include, after step (e) and before step (f), the step (e'): (i) forming aptamer cluster particles from the functionally enriched population of aptamers of step (e); and (ii) repeating steps (a) to (e) using the newly formed aptamer cluster particles to generate a further functionally enriched population of aptamers.

[0097] In certain embodiments, enriching the population of functional aptamers comprises applying restrictive conditions (e.g., reducing the total number of particles) during successive rounds. In some embodiments, the population of aptamers in each additional round of screening is functionally enriched by at least 1.1 times (e.g., about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times). The number of rounds of enrichment can be as many as desired. For example, in some embodiments, the number of rounds is at least 2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100).

[0098] The library of aptamer cluster particles can be incubated with cancer cells under any conditions that allow cell-aptamer cluster particle complexes to form and the aptamer cluster particles to exert their effect on the cancer cells. Conditions include, but are not limited to, a controlled time period, an optimal temperature (e.g., 37°C), and / or an incubation medium (e.g., cancer cell culture medium). The incubation period can be about 10 minutes to about 5 days, about 30 minutes to about 4 days, about 1 hour to about 3 days, about 1.5 hours to about 24 hours, or about 1.5 hours to about 2 hours. In some embodiments, the incubation period can be, for example, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0099] Cancer cells and aptamer cluster particles were incubated at a ratio of 10:1 to 1:2000 (e.g., 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:33, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, Can be mixed in the following ratios: 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000. The cell-aptamer cluster particle complexes that are formed can comprise between about 1 and 50 particles per cancer cell (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 particles per cancer cell). In certain embodiments, the cell-aptamer cluster particle complexes formed comprise about 2 to 10 particles per cancer cell. In some embodiments, the aptamer cluster particles in the cell-aptamer cluster particle complexes formed comprise about 1 to 10 clusters per particle (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 clusters per particle). In certain embodiments, the aptamer cluster particles in the cell-aptamer cluster particle complexes formed comprise about 1 to 6 clusters per particle.

[0100] In some embodiments, the cancer cells are labeled with and / or comprise a detectable label. The cancer cells can be detectably labeled directly (e.g., via a direct chemical linker) or indirectly (e.g., using a detectably labeled cancer cell-specific antibody). In some embodiments, the cancer cells can be labeled by incubating the cancer cells with a detectable label under conditions in which the detectable label is internalized by the cells. In some embodiments, the cancer cells are detectably labeled before performing the aptamer screening methods described herein. In some embodiments, the cancer cells are labeled during the aptamer screening methods provided herein. In some embodiments, the cancer cells are labeled after binding to the aptamer clusters (e.g., by contacting the bound target with a detectably labeled antibody). In some embodiments, any detectable label can be used. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties, and / or colorimetric moieties. In some embodiments, the cancer cells described herein are linked to, contain and / or are bound by a fluorescent moiety.Examples of fluorescent moieties include, but are not limited to, allophycocyanin (APC), fluorescein, fluorescein isothiocyanate (FITC), phycoerythrin (PE), Cy3 dye, Cy5 dye, peridinin-chlorophyll protein conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 661, Alexa Fluor 662, Alexa Fluor 663, Alexa Fluor 664, Alexa Fluor 665, Alexa Fluor 666, Alexa Fluor 668, Alexa Fluor 669, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 669, Alexa Fluor 669, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 661, Alexa Fluor 662, Alexa Fluor 663, Alexa Fluor 664, Alexa Fluor 665, Alexa Fluor 666, Alexa Fluor 668, Alexa Fluor 669 ... 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet.

[0101] In some embodiments, the cancer cells contacted with the aptamer-cluster particles are live / viable, hi other embodiments, the cancer cells contacted with the aptamer-cluster particles are fixed or in suspension.

[0102] In some embodiments, the cancer cells are human cancer cells or patient-derived cancer cells. In some embodiments, the cells are derived from any cancerous or precancerous tumor. Non-limiting examples of cancer cells include cancer cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lymph node, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testis, tongue, salivary gland, or uterus. Additionally, the cancer may be of the following histological types, including, but not limited to, neoplasia, malignancy, carcinoma, undifferentiated, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, calcifying cell carcinoma (pilomatrix). carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, gastrinoma, malignant, cholangiocarcinoma, hepatocellular carcinoma, mixed hepatocellular and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, adenocarcinoma, familial polyposis coli, solid carcinoma, carcinoid tumor, malignant, bronchiolar and alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe cell carcinoma, acidophilic carcinoma, acidophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-encapsulating sclerosing carcinoma, adrenocortical carcinoma, endometrioid carcinoma carcinoma), skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, cerumen gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, breast cancer, acinic cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, thymoma, malignant, ovarian stromal tumor, malignant, theca cell tumor, malignant, granulosa cell tumor, malignant, male germ cell tumor, malignant, Sertoli cell carcinoma, Leydig cell tumor, malignant, lipid cell tumortumor), malignant, paraganglioma, malignant, extramammary paraganglioma, malignant, pheochromocytoma, glomous sarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell melanoma, blue nevus, malignant, sarcoma, fibrosarcoma, fibrous histiocytoma, malignant, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, malignant, mixed Müllerian tumor, nephroblastoma, hepatoblastoma , carcinosarcoma, mesenchymoma, malignant, Brenner tumor, malignant, phyllodes tumor, malignant, synovial sarcoma, mesothelioma, malignant, dysgerminoma, embryonal carcinoma, teratoma, malignant, ovarial goiter, malignant, choriocarcinoma, mesonephroma, malignant, angiosarcoma, hemangioendothelioma, malignant, Kaposi's sarcoma, hemangiopericytoma, malignant, lymphangiosarcoma, osteosarcoma, parosteal osteosarcoma, chondrosarcoma, chondroblastoma, malignant, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, odontogenic tumor, malignant, ameloblastoma, ameloblastoma, malignant, ameloblastic fibrosarcoma , pinealoma, malignant, chordoma, glioma, malignant, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal, cerebellar sarcoma, soft tissue sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, meningioma, malignant, neurofibrosarcoma, schwannoma, malignant, granular cell tumor, malignant, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, paragranuloma anuloma), malignant lymphoma, small lymphocytic type, malignant lymphoma, large cell, diffuse, malignant lymphoma, follicular, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0103] In some embodiments, the detectable label is a fluorescent dye.Non-limiting examples of fluorescent dye include but are not limited to calcium-sensitive dye, cell tracer dye, lipophilic dye, cell proliferation dye, cell cycle dye, metabolite-sensitive dye, pH-sensitive dye, membrane potential-sensitive dye, mitochondrial membrane potential-sensitive dye, and redox potential dye.In certain embodiments, cancer cells are labeled with activation-related marker, oxidative stress reporter, angiogenesis marker, apoptosis marker, autophagy marker, immunological cell death marker, cell viability marker, or marker for ion concentration.

[0104] In some embodiments, the cancer cells are labeled prior to exposing the cancer cells to the aptamer. In some embodiments, the cancer cells are labeled after exposing the cancer cells to the aptamer. In one embodiment, the cancer cells are labeled with fluorescently labeled antibodies, antibody fragments, and artificial antibody-based constructs, fusion proteins, sugars, or lectins. In another embodiment, the cancer cells are labeled with fluorescently labeled antibodies, antibody fragments, and artificial antibody-based constructs, fusion proteins, sugars, or lectins after exposing the cancer cells to the aptamer.

[0105] In certain embodiments, the cellular function is cell death. Exemplary cell death reporters include, but are not limited to, those targeting cleaved / activated caspase-3, 7, 8, or 9, annexin V, mitochondrial membrane potential, calreticulin, heat shock proteins, ATP, and HMGB1.

[0106] [Table 3]

[0107] In some embodiments, the reporter of cell function is an antibody. In certain embodiments, the antibody is labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, allophycocyanin (APC), fluorescein, fluorescein isothiocyanate (FITC), phycoerythrin (PE), Cy3 dye, Cy5 dye, peridinin-chlorophyll protein conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 661, Alexa Fluor 662, Alexa Fluor 663, Alexa Fluor 664, Alexa Fluor 665, Alexa Fluor 666, Alexa Fluor 668, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 667, Alexa Fluor 668, Alexa Fluor 669 ... 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet. In some embodiments, the cellular function is cell proliferation and the antibody binds to a proliferation marker (eg, Ki67, MCM2, PCNA).

[0108] In some embodiments, the cellular function is tumor antigen expression and the antibody binds to a tumor antigen (e.g., prostate specific antigen (PSA), prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), alpha-fetoprotein (AFP), NY-ESO-1, MAGEA-A3, WT1, hTERT, tyrosinase, gp100, MART-1, melanA, B-catenin, CDC27, HSP70-2-m, HLA-A2-R17OJ, AFP, EBV-EBNA, HPV16-E7, MUC-1, HER-2 / neu, mammaglobin-A).

[0109] In some embodiments, the library can be, for example, synthesized de novo or can be the output of a previous selection process, which can include one or more positive selection cycles, one or more negative selection cycles, or both, in any combination and order.

[0110] The prepared library is loaded onto particles such as beads. Emulsion PCR (ePCR) amplification converts each single sequence from the initial library into clusters of at least, for example, 10,000 copies of the same sequence. The library of aptamer cluster particles is then incubated with cancer cells. The cancer cells can be labeled with a fluorescent dye prior to introduction into the aptamer cluster particles for the purpose of reporting biological or chemical effects on the cancer cells. The cancer cells and the library of aptamer cluster particles are incubated for a period of time to allow the effect to occur. A fluorescent dye or marker for reporting biological or chemical effects (e.g., cell apoptosis) can then be added to the cancer cells. In some embodiments, a reporter is added to the cells before incubation. In some embodiments, the reporter is added during incubation. In certain embodiments, the reporter is added after incubation. In some embodiments, a second reporter is used (e.g., before incubation) to mark cells that express the desired phenotype (e.g., apoptosis) regardless of the incubation process with the aptamer. In certain embodiments, the second reporter helps distinguish false positives. In some embodiments, a second (or third) reporter is used (e.g., a reporter that functions via a different mechanism) to confirm that the detected phenotype is not a false positive. Subsequently, the action-positive clusters are sorted from the action-negative clusters, and the corresponding functional aptamer sequences are analyzed. The sorted positive clusters can also be amplified and immobilized on the surface of particles as an initial library for additional rounds of screening. A portion of the enriched functional aptamers after each round of screening are subjected to output sampling and comparative functional analysis before identifying the aptamers by sequencing.

[0111] Additional methods for generating aptamer libraries and immobilized aptamer clusters, as well as methods for screening aptamer libraries to identify aptamers that specifically alter target cell function (e.g., aptamers that induce cancer cell apoptosis), are described in PCT Application No. PCT / IB2019 / 001082, which is incorporated herein by reference.

[0112] Immune effector cell-binding chains can be identified using methods known to those skilled in the art. For example, immune effector cell-binding chains can be identified using the Cell-SELEX binding process described in the Examples and Figures of this disclosure. Immune effector cell-binding chains can also be identified from the literature.

[0113] Methods for producing bispecific personalized aptamers In certain aspects, a method for producing a bispecific personalized aptamer is provided herein. In some embodiments, the method includes the steps of: (1) synthesizing a cancer cell-binding chain; (2) synthesizing an immune effector cell-binding chain; and (3) linking both chains to form a bispecific personalized aptamer. The two chains can be linked via complementary sequence hybridization, covalent bond, or PEG crosslinking.

[0114] After identifying cancer cell binding chain and immune effector cell binding chain, both chains can be synthesized by methods well known to those skilled in the art.For example, the synthesis of different aptamers can be carried out by well-established automated solid-phase phosphoramidite chemistry.According to the programmed sequence, one nucleotide is added in each synthesis cycle that consists of a series of steps.

[0115] Briefly, the synthesis cycle begins with the removal of the acid-labile 5'-dimethoxytrityl protecting group (DMT, "trityl") from the hydroxyl function of the terminal, support-bound nucleoside by UV-controlled treatment with an organic acid. The exposed, highly reactive hydroxyl group is now available to react with the next protected nucleoside phosphoramidite building block in the coupling step, thereby forming a phosphite triester backbone. The acid-labile phosphite triester backbone is then oxidized to a stable pentavalent phosphate trimester. If phosphorothioate modification at a specific backbone position is desired, the acid-labile phosphite triester backbone is sulfurized in this step instead of the oxidation process, generating a P=S bond rather than a P=O bond. Subsequently, all unreacted 5'-hydroxyl groups are acetylated ("capped") to block these sites during the subsequent coupling step and avoid internal mismatch sequences. Following the capping step, the cycle begins again with removal of the DMT protecting group and subsequent coupling of the next base according to the desired sequence. Finally, the oligonucleotide is cleaved from the solid support and all protecting groups are removed from the backbone and bases.

[0116] In some embodiments, the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain further comprise a complementary 5' sequence. In some embodiments, the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain further comprise a complementary 3' sequence. In some embodiments, step (3), i.e., linking both chains to form a bispecific personalized aptamer, comprises hybridizing the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain. In some embodiments, the complementary 5' or 3' sequence comprises one or more CpG motifs. In a preferred embodiment, the complementary 5' or 3' sequences of the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain hybridize to form a double-stranded CpG-rich sequence.

[0117] In some embodiments, the complementary 5' sequence comprises a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 63-66. In some embodiments, the complementary 5' sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 63-66. In certain embodiments, the complementary 5' sequence comprises a nucleic acid sequence comprising at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22) contiguous nucleotides of any one of SEQ ID NOs: 63-66. In some embodiments, the complementary 5' sequence has a sequence consisting essentially of SEQ ID NOs: 63-66. In a particular embodiment, the complementary 5' sequence has the sequence consisting of SEQ ID NOs: 63-66.

[0118] In some embodiments, the method comprises synthesizing (e.g., chemically synthesizing) a cancer cell-binding strand comprising a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the method comprises synthesizing a cancer cell-binding strand comprising a nucleic acid sequence of any one of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the method comprises synthesizing a cancer cell-binding strand comprising a nucleic acid sequence comprising at least 30 (e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60) consecutive nucleotides of any one of SEQ ID NOs: 43-62 or 107-115. In some embodiments, the method comprises synthesizing a cancer cell-binding chain having a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115. In certain embodiments, the method comprises synthesizing a cancer cell-binding chain having a sequence consisting essentially of SEQ ID NOs: 43-62 or 107-115.

[0119] In some embodiments, the method comprises synthesizing (e.g., chemically synthesizing) an immune effector cell-binding chain comprising a nucleic acid sequence at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the method comprises synthesizing an immune effector cell-binding chain comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-42, 88-106, or 116. In certain embodiments, the method comprises synthesizing an immune effector cell-binding chain comprising a nucleic acid sequence comprising at least 20 (e.g., at least 25, at least 30, at least 35, at least 40, at least 45, at least 50) consecutive nucleotides of any one of SEQ ID NOs: 1-42, 88-106, or 116. In some embodiments, the method comprises synthesizing an immune effector cell-binding chain having a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. In certain embodiments, the method comprises synthesizing a nucleic acid having a sequence consisting essentially of SEQ ID NO: 1-42, 88-106, or 116. [Example]

[0120] Example 1: Bispecific personalized aptamers A. Representative structures of bispecific personalized aptamers In some embodiments, the personalized cancer therapeutics described herein are composed of a heterodimeric structure comprising three distinct domains (Figure 1). In certain aspects, the platform described herein is designed to generate customized cancer therapeutics for treating patients with personalized solutions optimized for the unique set of conditions and potential drug targets presented by each patient, as reflected by fresh sample tissue from their tumor. In some embodiments, bispecific personalized aptamers are designed to target specific neoantigens and surface molecules presented by the patient's cancer cells and promote both direct cancer cell killing and immune-related responses. In some embodiments, efficacy is achieved through three distinct modes of action (MoA) combined into a single therapeutic entity, as described below:

[0121] 1. Personalized Chain: Direct Killing of Cancer Cells with Personalized Aptamers In some embodiments, the moiety is 10 15 The aptamers are selected through a process that begins with a random pool of potential leads and is described in detail in PCT Application No. PCT / IB19 / 01082. Briefly, the personalized process is designed to identify aptamers that best promote targeted killing of cancer cells without harming healthy cells. Patient-specific chains are identified through a binding and functional enrichment process (cellular and functional SELEX), screening candidates using high-throughput microscopy, and confirming the activity and specificity of the top candidates, including selectivity testing and attempting to rule out off-target effects (Figures 2 and 3A).

[0122] 2. Immunomodulatory chain: Cancer cell lysis via T cell or NK cell mediated cytotoxicity In some embodiments, the aptamer arm is a CD3-binding aptamer disclosed herein (e.g., comprising any one of SEQ ID NOs: 88-106 or 116) (Figure 3B). This immunomodulatory arm can potentially be designed to be shared across different patients.

[0123] 3. CpG motifs with TLR9 agonist activity In some embodiments, the two aptamer arms of the bispecific structure are cross-linked by nucleobase hybridization of the single-stranded overhangs of complementary sequences. This hybridization domain is CpG-rich and designed to induce TLR9-mediated antigen-presenting cell (APC) stimulation and increased tumor antigen uptake (Figure 3C). The stimulated APC will then migrate to tumor-draining lymph nodes and cross-present the engulfed tumor antigen to cytotoxic T lymphocytes, generating an adaptive systemic anti-tumor immune response (Figure 3D).

[0124] B. Individualized Process for Each Patient In some embodiments, for cancer therapeutic platforms, the personalization process involves several key steps (Figure 4): 1. Acceptance of two primary matched samples from subjects a. Tumor biopsy b. Healthy tissue used as a negative control, which will consist of either normal tissue or peripheral blood mononuclear cells (PBMCs) from the site of the biopsy. 2. Pursuing the selection process described herein to identify personalized aptamers that induce tumor cell death while leaving healthy cells unharmed; 3. Preparation and hybridization of both strands to obtain bispecific personalized aptamers; 4. A bispecific personalized aptamer is administered to each individual subject.

[0125] Example 2: Materials and Methods for Examples 3-4 A. Material a. Random Library Random Library 2.6 was purchased from IDT. The library contains approximately 10 unique sequences flanked at the 3' and 5' ends by two unique sequences that act as primers in PCR amplification during the SELEX procedure. 15 The lyophilized library ("Lib 2.6"), which contains a large repertoire of diverse 50 nt-long random sequences, was reconstituted in ultrapure water (UPW) to a final concentration of 1 mM.

[0126] The random library sequences are: 5'TATCCGTCTGCTCTCGCTATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACGCACCTAATGTCCTACTG-3' (SEQ ID NO: 71) where N represents a random oligonucleotide selected from a mixture of evenly represented T, A, C and G nucleotides.

[0127] b. Pre-SELEX preparation Library 2.6 (Lib 2.6) underwent QC validation using an HPLC gel filtration column.

[0128] c. Library primers and caps A set of 20-nt primers and caps was purchased from IDT. The caps were used to hybridize to the library primer sites during incubation with cells to avoid possible interactions of the primer sequences with the random 50-nt sequence sites. A mixture of 3' and 5' caps was used in each SELEX round at a cap:library ratio of 3:1.

[0129] The forward primer was purchased from IDT and labeled with Cy5 at the 5' site for sequence amplification, detected by a fluorescent assay. Lyophilized primers were reconstituted in ultrapure water (UPW) to a final concentration of 100 μM.

[0130] [Table 4]

[0131] d. Aptamer folding buffer Phosphate-buffered saline (magnesium- and calcium-free) was supplemented with 1 mM magnesium chloride (MgCl2). The folding buffer was sterilized using a 0.22 μm PVDF membrane filter unit and kept at 4°C.

[0132] e. Fresh PBMC Blood samples were obtained from the Tel Hashomer Medical Center Blood Bank and PBMCs were isolated using Ficoll (Lymphoprep, Axis-Shield) density gradient centrifugation according to the manufacturer's protocol.

[0133] f. Human CD8 T cell isolation Isolation of human CD8 cells was performed according to the manufacturer's protocol. + This was done via a T cell isolation kit (Miltenyi Biotec, 130-096-495).

[0134] g. Aptamer List Each aptamer was diluted to the desired concentration with folding buffer. The aptamer was heated to 95°C for 5 minutes, then rapidly cooled on ice for 10 minutes and incubated at room temperature (RT) for 10 minutes. The folded aptamer was then added to cells suspended in culture medium. The following aptamers were used:

[0135] [Table 5]

[0136] The lyophilized aptamer was kept in the dark at RT, then reconstituted in PBS supplemented with 1 mM MgCl 2 to a concentration of 100 μM and stored at −20° C. in the dark.

[0137] B. Experimental Method a. Combinatorial SELEX Protocol Binding SELEX involving two negative selection rounds (rounds 3 and 4) was performed on CD8 isolated from three healthy donors. + Seven successive rounds of cell-based SELEX were performed. Combinatorial SELEX was performed as follows:

[0138] (1) Isolation and preparation of CD8 T cells for individual SELEX rounds Prior to each round, CD8 cells were isolated and allowed to recover for 1 hour in warm RPMI1640 (ATCC) at 37° C. Cells were then counted and seeded into 1.5 mL Eppendorf tubes at the following concentrations:

[0139] [Table 6]

[0140] (2) Initial library and round enrichment library preparation and folding protocols The library was initially reconstituted to 1 mM. The working concentration in the first round was 14.3 µM, and in rounds 2 to 7, enriched library concentrations of 0.25 to 0.5 µM were used. In each round, the following components were used:

[0141] [Table 7]

[0142] The library, which underwent DNA folding according to the following protocol, was heated to 95°C for 5 minutes, followed by quick cooling on ice for 10 minutes and incubation at room temperature (RT) for 10 minutes. After folding, to avoid nonspecific nucleotide adsorption, the following components were added and adjusted to the final volume as shown in Table 8.

[0143] [Table 8]

[0144] (3) SELEX round duration and washing conditions Once the enriched library rounds were folded, they were added to the isolated CD8 or negative cell population for the following times:

[0145] [Table 9]

[0146] After incubation, the cells were washed three times and centrifuged at 300 g for 5 minutes. The supernatant "unbound to positive" fraction was removed and kept at -20°C until NGS preparation. The cells were resuspended in binding buffer and washed again. After the third wash, the cells were resuspended in UPW or, if a negative SELEX round was to follow, in binding buffer. The cells were lysed by heating at 95°C for 10 minutes and centrifuged at maximum speed for 5 minutes at room temperature. The supernatant "bound to positive" fraction was removed and used as a template for PCR reactions. If a negative SELEX round was to follow, the bound fraction was then added to CD8-negative cells for 1 hour under the same conditions as above, and the collected fractions were designated "unbound to negative" and "bound to negative," respectively. After the negative SELEX round, the "unbound to negative" fraction was used for PCR amplification.

[0147] (4) PCR amplification protocol The "positive bound" or "negative unbound" fractions were used as templates for asymmetric PCR amplification. The PCR reaction was varied for each round. The PCR components and amplification protocol are shown in Table 10 and Table 11, respectively.

[0148] [Table 10]

[0149] [Table 11]

[0150] (5) PCR ssDNA purification The PCR products were purified using HPLC or with a PCR ssDNA gel extraction kit (QIAEX II) according to the manufacturer's protocol. After purification, the DNA concentration was measured using a NanoDrop and the DNA was diluted for a new SELEX round.

[0151] b. SELEX Library Binding Assay Protocol The isolated CD8 cells or CD8 negative cell fraction (negative control) were counted and 1 x 10 6 Cells were aliquoted into 1.5 mL Eppendorf tubes. Cells were centrifuged and washed once with binding buffer. Cells were resuspended in 225 μL of binding buffer supplemented with 0.01% azide and 0.1% tRNA, and 25 μL of prefolded Cy5-labeled aptamer was added to each treatment, followed by incubation at 37°C for 1 hour in the dark. Cells were washed four times with binding buffer supplemented with 0.01% azide and 0.1% tRNA, and fluorescence intensity was measured after each wash using flow cytometry (CytoFlex).

[0152] c. Individual aptamer binding assays Isolated CD8 cells or pan T cells, PBMCs or cell lines were counted and 1 x 10 6 Cells were aliquoted into 1.5 mL Eppendorf tubes. Cells were centrifuged and washed once with binding buffer. Cells were resuspended in 225 μL RPMI 1640 supplemented with 10% human serum, and prefolded Cy5-labeled aptamer was added to each treatment, followed by incubation on ice in the dark for 1 hour. Cells were washed four times with chilled medium, and fluorescence intensity was measured using flow cytometry (CytoFlex).

[0153] d. Thermofluorescence Analysis (TFA) To determine the binding of CTL3 to its putative target, Notch2, TFA was used. 100 nM CTL3, 1 μM SYBR green I (Sigma), and 20, 40, 80, 160, and 320 nM Fc-Notch2 human (R&D Systems) or Fc-CD160 (Abcam) were mixed together, and SYBR green and fluorescence were measured in triplicate using RT-PCR at 1°C / min from 25°C to 95°C. Subsequent experiments were performed similarly to the previous experiments using 50 nM of either CTL3 (SEQ ID NO: 3), scrambled CTL3-A (SEQ ID NO: 86), or scrambled CTL3-B (SEQ ID NO: 87); 1 μM SYBR green I (Sigma); and 25, 50, 100, and 200 nM of Fc-Notch2 human (R&D Systems), Fc-Notch2 mouse (R&D Systems), or Fc-Notch2 rat.

[0154] Example 3: Identification of candidate T cell engagers via combinatorial SELEX T cells have been established as the primary effector for cancer immunotherapy, particularly due to their abundance, killing efficiency, and proliferation capacity. T cell engagers are bispecific molecules that target a constant component of the T cell / CD3 complex at one end and a tumor-expressed ligand or antigen at the other end. This structure allows bispecific T cell engagers to physically link T cells to tumor cells, ultimately stimulating T cell activation and subsequent tumor killing (Huehls et al. (2015) Immunol. Cell Biol. 93:290-296; Ellerman D. (2019) Methods 154:102-117).

[0155] The selection of cytotoxic T lymphocyte-engaging aptamers was described herein. Cytotoxic T lymphocyte arms were generated using samples from multiple blood donors via Cell-Selex binding. The final lead was selected from the target CD8 +T cells were characterized for their binding to their putative protein targets identified via membrane protein array assays and validated via thermofluorescence analysis.

[0156] This disclosure describes a novel application of Cell-SELEX methodology. 15 We describe the identification and characterization of a cytotoxic T lymphocyte (CTL)-engaging aptamer from a random library of possible aptamers.

[0157] In the SELEX protocol, CTLs isolated from multiple healthy donors were used sequentially in iterative selection rounds to increase the likelihood of identifying aptamers targeting a broad range of ligands, as opposed to individual unique isoforms / mutants. To increase the specificity of the aptamer pool for CTLs, negative selection was added in the form of CD8-negative PBMCs. In the final round of Cell-Selex, the washing stringency of the bound aptamer population was increased, both in terms of wash time and number, to increase the affinity of potential aptamers in the final pool. After sequencing and statistical analysis of the enriched library throughout the selection process via next-generation sequencing (NGS), putative binders were individually screened for their ability to bind primary CTLs. Top leads were tested for their ability to promote targeted cancer cell cytotoxicity with the assembled structure of a bispecific aptamer bearing a cancer-targeting aptamer arm. Concurrently, a membrane protein array (MPA) platform (Tucker et al. (2018) Proc. Natl. Acad. Sci. USA 115:E4990-E4999) was used to deconvolute the putative targets of the top leads, and the target of one leading aptamer, "CTL3," was further validated using thermofluorescence analysis (Hu, Kim, & Easley (2015) Anal. Methods 7:7358-7362). The target of CTL3 has been shown to be Notch-2, a membrane signaling receptor involved in T cell-mediated antitumor immunity and T cell-based immunotherapy (Janghorban et al. (2018) Frontiers in Immunology 9:1649; Duval et al. (2015) Oncotarget 6:21787-21788; Ferrandino et al. (2018) Frontiers in Immunology 9:2165; Kelliher and Roderick (2018) Frontiers in Immunology 9:1718; Weerkamp et al. (2006) Leukemia 20:1967-1977).

[0158] Binding Cell-SELEX was performed using three healthy PBMC donors for a total of seven rounds, as shown in Figure 5. The use of multiple PBMC donors was performed to ensure robustness of aptamer binding capacity across different potential patients and to avoid targeting unique epitopes expressed only in the PBMCs of a single donor. Rounds 3 and 4 were followed by negative selection rounds using CD8-negative PBMCs from donors 1 and 2.

[0159] a. SELEX round comparison assay The libraries eluted from rounds 4, 6, and 7 were tested for their binding affinity with isolated CD8 cells. Each round was amplified using a 5' primer labeled with Cy5, followed by incubation with isolated CD8 cells for 1 hour. As shown in Figures 6A and 6B, the affinities of the libraries from rounds 4, 6, and 7 were much higher than the random initial library used in combinatorial SELEX.

[0160] b. NGS results The final round of binding Cell-SELEX was repeated two more times with increased washing stringency, once doubling the number of washes of unbound sequences ("6x wash" compared to the baseline 3x wash) and once using an increased incubation time after the final wash, thereby increasing the high K off The aptamer with the formula (I) was released into the medium and washed away ("long wash") (see Table 12).

[0161] The enriched libraries from the second, fifth, sixth and seventh rounds of the three conditions ("bound"), as well as the supernatants from each round ("unbound"), were sequenced via high-throughput sequencing using an NGS Illumina NextSeq500.

[0162] Figure 7A shows the relative abundance of the most abundant sequences, with the 10 most abundant sequences shown in color and the rest in black (100 sequences total). The results in Figure 7A show increased abundance of the top aptamers in the final enriched library, which is consistent with the increased binding results in Figures 6A and 6B.

[0163] In addition to relative abundance, two additional measurements were calculated for each sequence in the 7 enriched libraries of the final round: the proportion of sequences found in the cell-bound population compared to the unbound population (supernatant) for increased wash times (6x wash), and the proportion of sequences found in the cell-bound population compared to the unbound population (supernatant) for increased duration of the final wash (long wash).

[0164] The results of the three measurements for each sequence in the final enriched library were plotted against each other (Figures 7B-7D), and 27 sequences were selected to be individually tested for their binding affinity to synthetic and CTL (Table 13).

[0165] [Table 12]

[0166] [Table 13] JPEG0007753256000018.jpg66159

[0167] c. Individual aptamer validation Aptamers selected from the statistical analysis were synthesized with a 5' Cy5 fluorescent label and screened for their binding to isolated CD8 cells. The positive binding threshold was determined as 1.5-fold greater than random aptamer sequences (Figure 8).

[0168] Example 4: T cell engager characterization (as in Example 3) a. CTL3 sequence and structure CTL3 sequence: 5'-GCATACCTTTCGTATGCCTTTTTGACCCGTATTTTTGCCCTACCCTTCGG-3' (SEQ ID NO: 3) The predicted structure of CTL3 by Nupack software is shown in Figure 9 .

[0169] b. CTL3 binding assay via flow cytometry 1. CTL3 binding to human PBMC To visually demonstrate the binding of selected aptamers to their target cell types and to better understand their specificity, frozen human PBMCs from several different donors were thawed and stained with the CTL3 Cy5-labeled aptamer and Cy5-labeled negative control polyT and random (RND) aptamer sequences. The CTL3 aptamer showed higher binding to total PBMCs compared to the random aptamer control and the polyT aptamer (Figure 10). To better understand the specificity of the CTL3 aptamer, CD8 staining was used in conjunction with SSC / FSC to distinguish between PBMC subpopulations. Human PBMCs from three different healthy donors were tested for binding using Cy5-labeled aptamers (250 nM) followed by CD8 antibody staining.

[0170] The binding of CTL3 to lymphocyte populations was higher than that of the RND control and polyT aptamer, and no significant difference in binding between CTL3, the RND control, and the polyT aptamer to monocytic cells was observed (Figures 11A and 11B). However, within the lymphocyte population, CTL3 was found to bind to both CD8-positive and CD8-negative lymphocytes (Figures 11C and 11D).

[0171] A scrambled sequence (SCR) containing the same nucleotide ratios as CTL3 was designed, and CTL3 demonstrated binding even compared to this stringent control (Figures 12A and 12B).

[0172] 2. Binding assay using isolated CD8 cells To eliminate signal reduction due to mixed PBMC populations, CD8 T cells were isolated prior to the assay and CTL3 binding was measured directly on this subpopulation. Figure 13 displays representative results from a single experiment. Nevertheless, the results were consistent with the PBMC binding results.

[0173] 3. CTL3 Binding to Expanded and Stimulated T Cells The CTL3 aptamer was subjected to target deorphaning as described herein to identify and validate Notch2 as the aptamer target. Notch2 surface expression is dramatically regulated during T cell development and activation (Duval et al. (2015) Oncotarget 6:21787-21788; Ferrandino et al. (2018) Frontiers in Immunology 9:2165; Kelliher and Roderick (2018) Frontiers in Immunology 9:1718; Weerkamp et al. (2006) Leukemia 20:1967-1977).

[0174] To measure the dependence of CTL3 binding on the activation state of target cells, preliminary experiments were performed in which T cells were isolated from PBMCs of one donor using a pan-T isolation kit and activated with a combination of anti-CD3 (1 μg / μL) and anti-CD28 (1 μg / μL) antibodies for 48 hours, followed by IL-2 (300 units) for 9 days. Binding was measured 11 days after initial activation. Under these conditions, no significant increase in CTL3 binding capacity was observed compared to binding to whole hPBMCs or isolated CD8 T cells (Figures 14A and 14B).

[0175] c. Target deconvolution of CTL3 by membrane proteome array The membrane proteome array (MPA) is a platform developed by Integral Molecular (Philadelphia, PA, US) to profile the specificity of antibodies and other ligands targeting human membrane proteins. MPA can be used to determine target specificity and deconvolute orphan ligand targets (Tucker et al. (2018) Proc. Natl. Acad. Sci. USA 115:E4990-E4999).

[0176] The platform uses flow cytometry to directly detect ligand binding to membrane proteins expressed in unfixed cells (see Figure 15). As a result, all target proteins have native conformations and appropriate post-translational modifications.

[0177] The CTL3 aptamer was tested for reactivity against a library of over 5,300 human membrane proteins, including 94% of all single-pass, multi-pass, and GPI-anchored proteins. Identified targets were validated in a secondary screen to confirm reactivity.

[0178] A high-throughput cell-based platform is used to identify membrane protein targets of ligands. Membrane proteins are expressed in human cells in 384-well microplates, and ligand binding is detected by flow cytometry, which allows for sensitive detection of both specific and off-target binding.

[0179] Each well on the matrix plate contains 48 different overexpressed protein components. Each protein is contained in a "row" pool and a "column" pool, and is therefore represented by a unique combination of two different wells on the matrix plate. Test CS aptamers were added to the MPA matrix plate at a predetermined concentration, washed with 1x PBS, and detected by flow cytometry.

[0180] CTL3 aptamer target hits were subsequently identified by detecting binding to duplicate pooled matrix wells arising from the same transfection plate, thereby allowing specific deconvolution. The screen yielded two potential hits: KCNK17 and Notch2 (Figure 16).

[0181] To validate the protein targets identified using MPA, HEK 293T cells were transfected in a 384-well format with plasmids encoding each target or with vector alone (pUC; negative control). After 36 hours of incubation, four 4-fold dilutions of CTL3 were added to the transfected cells, followed by detection of aptamer binding using a high-throughput immunofluorescence flow cytometry assay. Mean fluorescence intensity (MFI) values ​​were determined for each aptamer dilution (Figure 17). Notch2 and KCNK17 (potassium channel subfamily K member 17) were confirmed to produce concentration-dependent binding curves substantially higher than those of the negative control vector.

[0182] d. Binding of CTL3 to recombinant Notch2 by thermofluorescence analysis Although no T cell-related literature has been found for KCNK17, the Notch pathway regulates CD8 T cells in multiple ways. For example, CD8-specific deletion of Notch2, but not Notch1, resulted in increased tumor size and reduced survival after tumor inoculation in mice, implicating the possible contribution of this receptor to antitumor immune responses (Sugimoto et al. (2010) J Immunol.; Mathieu et al. (2012) Immunol. Cell Biol. 82-88; Tsukomo and Yasutomo (2018) Front. Immunol. 9, 1-7).

[0183] To provide direct biochemical evidence that Notch2 is a binding target of CTL3, we used a thermofluorescence analysis (TFA) assay. In TFA, a DNA-intercalating dye was used to determine the binding constant between a DNA aptamer and a target protein by measuring the temperature-dependent fluorescence of aptamers labeled with the intercalating dye SYBR with or without their predicted protein binding partners (Hu, Kim, and Easley (2016) HHS Public Access. 7:7358-7362). Upon stepwise heating of the aptamer-dye solution, the double-stranded portion in the aptamer denatured, releasing the dye back into solution, greatly reducing its fluorescence. Because the aptamer 3D conformation was highly stabilized upon binding to its respective target protein, the temperature-dependent fluorescence of the aptamer-dye complex varied greatly with and without the putative protein binding partner (Figure 18).

[0184] To monitor the aptamer-protein complexes in the presence of various concentrations of either Notch2 or a nonspecific control (CD160 protein), T was measured by measuring SYBR green fluorescence during a temperature ramp. m Melting curve profiles were generated. A dose-dependent change in fluorescence associated with CTL3 was measured only upon addition of increasing concentrations of Notch2, but not CD160 (Figure 19). When viewing the total fluorescence graph, high fluorescence intensity can be seen at 25°C, but when examining the derivative of the rate of change in frequency (dF / dT) curve, the temperature-dependent intensity reached a maximum at 37°C.

[0185] CTL3-Notch2 binding was compared with two scrambled sequences containing the same base composition (designated scrambled CTL3-A and scrambled CTL3-B). Figure 20A shows that CTL3 exhibits a dose-response curve with increasing concentrations of Notch2. This phenomenon was not observed with the scrambled sequence, suggesting a specific interaction between CTL3 and Notch2 that reached saturation at 100-200 nM protein.

[0186] In conclusion, in the presence of a DNA-intercalating dye, the CTL3 aptamer bound to the Notch2 protein exhibits a change in fluorescence intensity compared to the unbound intercalated aptamer, which does not occur when CD160 or a scrambled sequence is added instead of Notch2.

[0187] In contrast to human recombinant Notch2, where the CTL3 aptamer showed clear concentration-dependent binding (Figure 21A), no such pattern was clearly demonstrated for mouse or rat Notch2, suggesting relatively low specific binding by CTL3 (Figures 21B and 21C).

[0188] Example 5: Materials and Methods for Examples 6-7 A. Material a. Random Library Random Library 9.0 ("Lib 9.0") was purchased from IDT. The library contains approximately 10 nucleotides flanked by two 20 nt unique sequences at the 3' and 5' ends that act as primers in PCR amplification during the SELEX procedure. 15 It contains a large repertoire of diverse 40 nt long random sequences. The lyophilized library was reconstituted in ultrapure water (UPW) to a final concentration of 1 mM. The random library sequences are: 5'-TCACTATCGGTCCAGACGTA-40N-TATTGCGCCGAGGTTCTTAC-3' (SEQ ID NO: 117) where N represents a random oligonucleotide selected from a mixture of evenly represented T, A, C and G nucleotides (1:1:1:1 ratio).

[0189] Pre-SELEX preparation After reconstitution, the library underwent QC validation for size exclusion using an HPLC ProSEC 300S column (Agilent).

[0190] b. Library primers and caps A set of 20-nt primers and caps was purchased from IDT (Table 14). The caps were used to hybridize to the library primer sites during incubation with cells to avoid possible interactions of the primer sequences with the random 40-nt sequence sites. A mixture of 3' and 5' caps (Table 14) was used in each SELEX round at a cap:library ratio of 3:1.

[0191] The forward primer was purchased from IDT and labeled with Cy5 at the 5' site for sequence amplification detected by a fluorescent assay. Lyophilized primers were reconstituted in ultrapure water (UPW) to a concentration of 100 μM.

[0192] [Table 14]

[0193] c. Aptamer folding buffer Phosphate-buffered saline (magnesium- and calcium-free) was supplemented with 1 mM magnesium chloride (MgCl2). The folding buffer was sterilized using a 0.22 μm PVDF membrane filter unit and kept at 4°C.

[0194] d. PBMC PBMCs were isolated using Ficoll (Lymphoprep, Axis-Shield) density gradient centrifugation according to the manufacturer's protocol. Frozen cynomolgus monkey PBMCs (NHP-PC001) were purchased from Creative Biolabs.

[0195] e. Human pan-T and B cell isolation Human pan T cell isolation was performed using a pan T cell isolation kit (Miltenyi Biotec, 130-096-535) according to the manufacturer's protocol. Human pan B cell isolation was performed using a pan B cell isolation kit (Miltenyi Biotec, 130-101-638) according to the manufacturer's protocol.

[0196] f. Antibodies, proteins and enzymes αCD3ε-FITC (catalog no. 130-113-690) / APC (catalog no. 130-113-687) / VioBlue (catalog no. 130-114-519) / APC-Vio770 (catalog no. 130-113-688), αCD4-FITC (catalog no. 130-114-531), αCD8-FITC (catalog no. 130-113-719) / PE-Vio770 (catalog no. 130-113-159), and matched isotype controls were purchased from Miltenyi Biotech. αCD3ε OKT3 clone (catalog no. 317302) was purchased from BioLegend.

[0197] Recombinant human CD3ε protein (Fc chimeric His-tag) (ab220590), recombinant cynomolgus monkey CD3ε protein (Fc chimeric His-tag) (ab220531), and recombinant mouse CD3ε protein (His-tag) (ab240841) were purchased from Abcam. Human IgG1 isotype was used for negative counterselection (InVivoMAb, BE0297). Protein G magnetic beads were purchased from ThermoFisher (88847).

[0198] Herculase II Fusion DNA polymerase (600675) used for asymmetric PCR (A-PCR) was purchased from Agilent, and real-time PCR iTaq Universal SYBRGreen Supermix (1725124) was purchased from BIO-RAD.

[0199] g. Cell lines Jurkat, Daudi, and Kasumi-1 cell lines were purchased from ATCC. Jurkat cells (ATCC TIB-152), Daudi cells (ATCC CCL-213), and Kasumi-1 cells (ATCC CRL-2724) were grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (Pen / Strep). All cells were cultured at 37°C and 5% CO2.

[0200] h. Aptamers Each aptamer was diluted to the desired concentration with folding buffer. The aptamer was heated to 95°C for 5 minutes, then rapidly cooled on ice for 10 minutes and incubated at room temperature (RT) for 10 minutes. The folded aptamer was then added to cells suspended in culture medium.

[0201] The lyophilized aptamer was kept in the dark at RT, then reconstituted to a concentration of 100 μM in PBS supplemented with 1 mM MgCl 2 and stored at −20° C. in the dark.

[0202] B. Experimental Method a. Combinatorial SELEX Protocol Binding SELEX was performed over 11 consecutive rounds using CD3ε-Fc protein bound to protein G magnetic beads (positive selection), IgG1 protein bound to protein G magnetic beads, or beads alone (negative selection, starting from round 3 onwards).

[0203] i. Bead-protein complex preparation The magnetic protein G beads were vortexed and washed once with PBS, then mixed with 100 μL of protein under gentle shaking at RT for 10 min. The beads were then separated using a magnet, the supernatant was discarded, and the beads were resuspended in 350 μL of 1× folding buffer containing 2% BSA.

[0204] To verify bead-protein complex formation, a small amount of sample (before DNA addition) was treated with FC-blocker (Miltenyi), stained with αCD3ε, and analyzed by flow cytometry.

[0205] ii. Initial Library and Enrichment Round Library Preparation and Folding Protocols The library was initially reconstituted to 1 mM. The working concentration in the first round was 14.3 µM, and in rounds 2 to 11, enriched library concentrations of 0.25 to 0.5 µM were used. In each round, the following components were used:

[0206] [Table 15]

[0207] The library, which underwent DNA folding according to the following protocol, was heated to 95°C for 5 minutes, then rapidly cooled on ice for 10 minutes, and kept at 4°C until use.

[0208] iii. SELEX Once the enriched library was folded, 350 μL of the enriched library round was added to 350 μL of CD3ε-FC-beads (positive selection rounds 1–11) or bead-only / IgG1-bead complex (counter selection, rounds 3–11). Incubation times, protein amounts, and washing steps were varied depending on the SELEX round.

[0209] For positive selection, the "positively unbound" fraction of the supernatant was removed and kept at -20°C until NGS preparation. For washing, the beads were precipitated using a magnet, the supernatant was discarded, and the beads were resuspended in 1 mL of 1x folding buffer. After the washing step, the beads were suspended in 300 μL of ultrapure water (UPW) and DNA was eluted at 95°C for 10 minutes. Finally, the beads were precipitated using a magnet, and the "positively bound" supernatant was collected for the PCR step.

[0210] When performing a negative SELEX round, 350 μL of the enriched library round was added to 350 μL of the beads-only / IgG-bead complex, the supernatant was collected, and the fraction was carried forward to the positive selection stage. The fraction binding to the negative sample, designated "bound to negative," was eluted and kept at -20°C until NGS preparation.

[0211] iv. PCR amplification protocol The eluted DNA fractions ("bound" and "unbound") were used as templates for asymmetric PCR (A-PCR) amplification, respectively. The PCR reaction was modified for each round. The PCR components and amplification protocol are shown in Table 16 and Table 17, respectively.

[0212] [Table 16]

[0213] [Table 17]

[0214] v. PCR ssDNA purification The PCR product was concentrated using a 10K Amicon (Millipore, UFC5010BK) and purified using an HLPC ProSEC 300S size exclusion column (Agilent). After purification, the DNA was buffer exchanged using a ssDNA Clean Kit (ZYMO, D7011), the concentration was measured using a NanoDrop, and the DNA was diluted for a new SELEX round.

[0215] b. Evaluation of library pool binding to target protein by real-time PCR The magnetic protein G beads were vortexed, washed once with PBS, and then resuspended with the protein (CD3ε or IgG1) for 10 minutes at room temperature under gentle shaking. The beads were then precipitated under a magnetic field, the supernatant discarded, and the beads resuspended in 125 μL of 1× folding buffer and 2% BSA. Next, the library pools from rounds 3, 6, 9, and 11, as well as the initial random library, were folded (95°C for 5 minutes, on ice for 10 minutes, and maintained at 4°C). 125 μL of each folded DNA library was mixed with the bead-protein complex for 1 hour at 4°C with gentle shaking. After incubation, the beads were precipitated using a magnet and washed three times with 1 mL of folding buffer. Finally, the DNA-binding fraction was eluted with 100 μL UPW at 95°C for 10 minutes and then used as a template for real-time PCR using SYBRGreen Supermix (BIO-RAD).

[0216] c. Evaluation of individual aptamer binding to target proteins: Protein-aptamer binding assay by HPLC 1 μM of prefolded Cy5-labeled aptamer was mixed with 5 μM of protein to a final volume of 60 μL and incubated for 1 h at 4°C or 37°C. Samples were then analyzed at 570 nm absorbance via an HPLC ProSEC 300S size exclusion column (Agilent) to detect the Cy5-labeled aptamer.

[0217] d. Evaluation of individual aptamer binding to cells using flow cytometry 0.5 to 2 × 10 6 Individual cells (isolated pan T cells, B cells, hPBMCs, cynomolgus monkey PBMCs, Jurkat, and Daudi) were washed and resuspended in 0.2–1 mL folding buffer containing 0.1% BSA and 0.01% tRNA.

[0218] 0.25–1.25 μM of a single DNA candidate was fluorescently labeled by mixing with a CpG'-Cy5 tag (1:1 ratio) and allowed to fold (95°C for 5 min, on ice for 10 min, and maintained at 4°C). The labeled DNA aptamer was then incubated with cells in a V-shaped 96-well plate for 1 h at 4°C or 37°C under gentle shaking (αCD8 / αCD4 was added to hPBMCs and Cyno PBMCs for the last 15 min of incubation). After incubation, cells were washed three times with 1x folding buffer and analyzed after each wash using flow cytometry (CytoFlex).

[0219] e. Competitive CD3ε epitope binding assay 0.25×10 6 Jurkat cells were washed once, resuspended in 1x folding buffer containing 0.1% BSA and 0.01% tRNA, and incubated with a 1:20 dilution of αCD3 clone OKT3 (BioLegend, 317302) or αCD3 clone REA613 (Miltenyi, 130-114-519) or with buffer for 15 minutes. Next, 0.25 μM of the folded Cy5-labeled aptamer was incubated with the cells for 1 hour at 37°C under gentle shaking. After incubation, the cells were washed three times with 1x folding buffer and analyzed after each wash using flow cytometry (CytoFlex).

[0220] f. CS6 Effective Concentration 50 (EC50) quantification 5×10 4Jurkat cells were washed and resuspended in 1x folding buffer containing 0.1% BSA and 0.01% tRNA. 0.1–80 nM of CS6 aptamer was labeled with a CpG'-Cy5 tag (1:1 ratio) and allowed to fold (95°C for 5 min, on ice for 10 min, and maintained at 4°C). The DNA aptamer was then mixed with the cells and incubated in a V-shaped 96-well plate at 37°C for 1 h under gentle shaking. After incubation, the cells were washed twice with 1x folding buffer and analyzed via flow cytometry (CytoFlex).

[0221] Example 6: Identification of CD3-targeting aptamers via combinatorial SELEX A significant optimization step of the drug candidate was performed via replacing the T cell engager described above with a novel aptamer that targets the CD3ε ligand on the surface of T cells.

[0222] The selection of CD3 binding aptamer is described herein.T cell targeting aptamer is identified through binding SELEX and hybrid binding Cell-SELEX using recombinant CD3e protein and recombinant protein+T cell, respectively.Final lead is characterized for its binding to target protein and T cell.

[0223] This disclosure describes novel applications of the SELEX methodology. 15 We describe the identification and characterization of a T cell-engaging aptamer from a random library of possible aptamers. This aptamer portion, as part of a bispecific therapeutic entity, was designed to be constant across different patients.

[0224] A total of 11 rounds of combinatorial SELEX were performed using recombinant human CD3ε protein-Fc chimeras. For counter-negative selection, either beads alone (rounds 1-6) or beads conjugated to human IgG1 (rounds 7-11) were used to eliminate all aptamers that nonspecifically bind to the magnetic beads or the Fc component of the recombinant protein (Figure 22). After round 11 of SELEX, the enriched aptamer library was subjected to sequencing and analysis via a specific algorithm. A single candidate was identified and subjected to validation.

[0225] Figure 22B depicts the SELEX steps: counter-selection was initiated using Protein G magnetic beads (1) conjugated to IgG1 (2) and incubated with the DNA aptamer library pool from the previous step (3). Unbound DNA aptamers were then collected for positive selection (4) and incubated with FC-CD3ε-conjugated beads (5), where the bound fraction (6) underwent PCR amplification and HPLC purification for the next round.

[0226] 1. SELEX Round Comparison Assay The original random library "#9.0" and library pools eluted from rounds 3, 6, 9, and 11 were tested for their binding to hCD3ε. Each round was amplified by PCR using a 5' primer labeled with Cy5, followed by incubation with bead-Fc-CD3ε complexes at 4°C for 1 hour. As a negative control, the mutant pool was incubated with bead-IgG1 complexes (Figure 23A). The amount of amplified DNA precipitated with the target protein was found to be much higher for libraries from rounds 6, 9, and 11 than for the random initial library used in binding SELEX. The results showed specific and strong enrichment at round 6 compared to the initial library. Furthermore, a further increase in specific binding was observed in round 11.

[0227] After demonstrating inter-round enrichment using recombinant CD3 protein, we tested whether such enrichment could also be observed in whole cells. Jurkat T cells were incubated with the same Cy5-tagged library pool, washed, and analyzed by flow cytometry. Isolated pan-B cells served as a negative control (Figure 23B). Similar to the protein data, specific and strong inter-round enrichment for target cells was demonstrated.

[0228] 2. NGS results The enriched libraries eluted in rounds 8, 9, 10 and 11 ("bound"), as well as the supernatants from the positive selection rounds ("unbound"), were subjected to sequencing using a high-throughput NGS Illumina NextSeq500.

[0229] After sequencing, the data was analyzed through an algorithm that assigned a single candidate to downstream binding assays. The algorithm utilizes statistical estimators, tests, and metrics.

[0230] The average P-positive and P-negative scores of the top 100 most abundant aptamers in the final round were plotted (Figure 24A), revealing aptamer #6 with a significant bound-unbound ratio (p<0.05; Poisson test, consistent across all rounds) and selected for experimental validation (designated CD3-CS6-9, SEQ ID NOs: 88-91). Nine additional aptamers with high average P-positive values ​​(P>0.5) were assigned identifiers (CD3_Ppos10-18, SEQ ID NOs: 93-101). The identified CD3-binding aptamers are listed in Table 18.

[0231] [Table 18]

[0232] Next, 14 aptamers with high average P values ​​(P > 0.5) (see Table 18) were subjected to multiple sequence alignment to find shared motifs (Figure 24B, top). In comparison, the highlighted candidates (CS6-9) were also aligned to find more robust motifs (Figure 24B, bottom). Additionally, structure prediction analysis was performed using analysis software (mfold, NUPACK) (Figure 24C). This analysis demonstrated that the candidates fold into complex secondary structures, primarily around the motif region. Following this result and in an optimization effort, CD3_CS8 was further edited by trimming the first 9 nucleotides (denoted CD3_CS8cut) that appeared irrelevant to the formation of secondary structures around the motif in CS_CD8. The top five candidates were further confirmed to possess negative ΔG scores and were selected for individual binding assays. In addition to the combinatorial SELEX described above, a hybrid methodology was performed in which the process also included a whole-cell SELEX round.

[0233] [Table 19]

[0234] Example 7: Individual CD3-binding aptamer validation (from Example 6) a. Aptamer candidates demonstrate binding to human CD3ε via HPLC The top five candidates (CS6, CS7, CS8, CS9, and CS8c; SEQ ID NOS: 88–92, respectively) were synthesized with a 5(5′) phosphothioated CpG motif and assayed for human CD3ε (hCD3ε) binding via HPLC size-exclusion column. In this method, the aptamer was labeled with a Cy5 complementary sequence to the CpG site (Cy5-CpG′). The refolded, labeled candidates were then incubated with CD3ε recombinant protein or a negative control IgG1 (at 37°C or 4°C for 1 h) and analyzed by HPLC ProSEC 300S size-exclusion column (Agilent) at 570 nm absorbance. Upon protein binding, the aptamer-protein complex has a higher mass than the free aptamer, and as a result, the retention time (RT) on the column is expected to be shorter. Conversely, for a non-binding aptamer, the RT in the presence of protein will be the same as in the absence of protein. A poly-T sequence was used as a control. All five candidates demonstrated varying levels of binding to the CD3ε target protein (FIG. 25).

[0235] b. Aptamer candidates demonstrate specific binding to Jurkat T cell lines and primary human pan T cells by flow cytometry. After the CS6, CS7, and CS8c candidates demonstrated specific binding to the CD3e recombinant protein, they were assayed for binding to their targets on the surface of T cells, in whole, natural cells, by flow cytometry. For this purpose, a Jurkat T lymphocyte cell line (acute T-cell leukemia, ATCC TIB-152), previously reported to exhibit TCR expression, was used. Initial binding assays with the cells were performed at 4°C for 1 hour. As a negative control, the myeloblast Kasumi-1 cell line (acute myeloblastic leukemia, ATCC CRL-2724) was used. All three candidates were found to bind differentially to the target cells compared to the control cells, with CS6 and CS7 demonstrating better specificity than CS8c (Figure 26A).

[0236] Next, to better mimic physiological conditions, the three candidates were assayed for Jurkat binding at 37°C, where the B lymphoblast Daudi cell line was used as a negative control (lymphoblast, ATCC CCL-213) (Figure 26B). In this experiment, all three candidates bound to the target cells, with CS6 showing the highest level of binding.

[0237] CS6 was selected for further investigation and characterization and was found to bind to normal primary pan T cells but not pan B cells under blocking conditions at 37°C (Figure 26C).

[0238] Next, the effective concentration of CS6 was 50 (EC 50 ) was assessed. Serial dilutions of Cy5-labeled aptamers were incubated with Jurkat cells for 1 hour at 37°C and assessed for binding via flow cytometry (Figure 27). The calculated EC 50 The value was 19.65 nM. Furthermore, the affinity of CS6 for CD3ε was tested by surface plasmon resonance (SPR), and its dissociation constant was K d =31 nM (Figure 28).

[0239] When hybridized to the variable chain exemplary sequence VS20 (SEQ ID NO: 110) to form a bispecific T cell engager construct, CS6 resulted in stimulation of T cells as demonstrated by elevation of the CD69 marker (Figure 29).

[0240] Example 8: TLR9 agonist sequences engineered into bispecific personalized aptamer structures A. CpG motifs in bispecific personalized aptamers alter immune responses TLR9 has recently emerged as a potential therapeutic target due to its ability to promote the presentation of tumorigenic antigens to adaptive immune cells and stimulate the production of mediators with direct antitumor activity. Class C CpG ODN is a potent inducer of IFN-α from plasmacytoid dendritic cells (pDCs) and a potent B cell activator (Marshall (2003), J Leukoc Biol 73(6):781-92), and in vivo studies have demonstrated that C-type ODNs, such as ODN 2395, which combine the effects of type A and type B ODNs, are very potent Th1 adjuvants (Vollmer (2004) Eur. J. Immunol. 34, 251-262).

[0241] A novel CpG sequence was introduced into the bispecific personalized aptamer structure as a dimerization domain linking the two arms together (Figure 30A). The dimerization sequence was 22 nt long and rich in CpG dioligonucleotides (Figure 30C).

[0242] It was first verified that the introduction of the new hybridization domain did not reduce the target lethality associated with the primary mode of action of the bispecific personalized aptamer. During co-culture of healthy donor-derived PBMCs and HCT116 colorectal cancer cell line, the bispecific personalized aptamer was administered once daily for 72 hours, and the Live / Dead TM Dye and flow cytometry analysis followed. No reduction in cytotoxicity was observed using the newly designed bispecific personalized aptamers, and no significant differences were observed among the four tested CpG ODN-loaded bispecific personalized aptamers (Figure 31A).

[0243] Because ODNs containing phosphodiester backbones are degraded by nucleases, nuclease-resistant ODNs with phosphorothioate (PS) backbones have been developed (Eckstein (2014) Nucleic Acid Therapeutics 24:374-387; Pohar et al. (2017) Sci. Rep. 7). Replacement of non-bridging oxygen with sulfur atoms (Figure 30B) is a common chemical modification in the backbones of therapeutic oligonucleotides, and synthetic ODNs can be composed of partial or complete phosphorothioate (PS) backbones for vaccine adjuvants and in cancer therapy (Pohar et al. (2017) Sci. Rep. 7). Next, four different variations of PS modification were tested to exclude interference with the primary mode of action of the bispecific aptamer (sequences in Figure 30C): (i) non-PS: none of the 22 nt comprising the dimerization domain were modified; (ii) 5PS: only the first five 5' nucleotides of the dimerization domain were modified; (iii) 10PS: the first five and last five nucleotides of the dimerization domain were modified; (iv) 22PS: all 22 nt comprising the dimerization domain were modified.

[0244] The CTL3|CpG1|VS12 bispecific personalized aptamer containing different PS variants was examined for HCT116 cytotoxicity. As shown in Figure 31B, full PS (i.e., 22 PS) demonstrated abrogated cytotoxicity. 5PS and 10PS on each monomer produced comparable results, comparable to the original bispecific personalized aptamer without PS, and 10PS caused a slight decrease that was not significantly different. Therefore, the 5PS modification was selected for further study. Two unique variants of the CpG bridge, CpG1 and CpG2, were generated and tested as TLR9 agonists (see Figure 30C for specific sequences). The bispecific personalized aptamer CTL3|CpG1|VS12, in which the first five 5' nucleotides of the dimerization domain were PS-modified, was tested for its immunostimulatory potential and compared with that of the canonical C-type TLR9-activating oligo, ODN2395 (Roda et al. (2005) J. Immunol. 175:1619-1627; Abel et al. (2005) Clin. Diagn Lab Immunol. 12:606-621). Isolated human B cells were cultured with 50 μM CTL3|CpG1|VS12 bispecific aptamer, and the expression of the costimulatory surface marker CD86 was assessed by flow cytometry. To exclude nonspecific effects caused by the presence of either DNA, a dimer of polyT (50 μM) without a CpG motif was used as a control. Similar to the established TLR9 agonist ODN 2395, CTL3|CpG1|VS12 treatment resulted in the upregulation of CD86 on B cells (Fig. 32A). Splenocytes from BALB / c mice were isolated (n=3) and seeded in 96-well plates (500,000 cells / well). Cells were treated for 48 hours with vehicle, ODN negative control (5 μM), ODN 2395 (5 μM) as a positive control, and bispecific aptamer CTL3|CpG1|VS12 (50 μM). After 48 hours of treatment, cells were centrifuged, and the supernatant was collected and analyzed for IL-6 secretion using an IL-6 ELISA kit (Fig. 32B).The CpG2 sequence also demonstrated TLR9 agonism by inducing IFN-α secretion from PBMCs, although this function appeared to be retracted in the context of the bispecific aptamer (FIG. 32C).

[0245] To confirm that the identity of the constant chain did not affect the previously induced CpG function, a bispecific aptamer was formed using the CD3ε targeting moiety CS6 (SEQ ID NO: 116) and VS20 (SEQ ID NO: 110) as the variable moiety. IL-6 secretion was unaffected by the replacement of the aptamer constant and variable arms. Furthermore, the CpG motif was active when two arms were replaced with nonspecific poly-T sequences. Interestingly, the CpG even functioned as single-stranded DNA, although not as potently as in the double-stranded structure (Figure 33A). To corroborate the function of the novel CpG in driving antigen presentation, additional data were generated, demonstrating increased expression of CD86, CD80, and CD58 on human B cells (Figure 33B). Titration plots for these markers were generated to assess the TLR9 agonistic activity EC of the bispecific aptamer. 50 was demonstrated to be approximately 20 μM (FIG. 34).

[0246] Example 9: Materials and Methods for Examples 10-12 A. Material Aptamers Newly identified cancer-targeting tumoricidal aptamer arms were derived from the functional enrichment process as described in PCT Application No. PCT / IB19 / 01082 using the following target cells / organoids: HCT-116 colon cancer cell line (variable chains HCT116-VS6 and -VS12; SEQ ID NOs: 43 and 44, respectively), MCF7 breast cancer cells (MCF7-VS13, -VS16, and -VS19; SEQ ID NOs: 45, 46, and 47, respectively), A5449 adenocarcinoma human alveolar basal epithelial cells (A549-VS3 and VS20; SEQ ID NOs: 107 and 108, respectively), and colorectal cancer (CRC)-derived organoid #13 (CRC-13 VS31, VS48, and VS81; SEQ ID NOs: 113-115, respectively).

[0247] T cell engager sequences (CTL3, CTL5, and CTL6, SEQ ID NOs: 3, 5, and 6, respectively) were derived from the Cell-SELEX binding process as described in Examples 7-9. The CD16 aptamer sequence was obtained from the literature (Boltz et al. (2011) J. Biol. Chem. 286:21896-21905; Li et al. (2019) Molecules doi:10.3390 / molecules24030478). The CD3e-binding aptamer (CS6, SEQ ID NO: 88) was derived from the SELEX binding process using human recombinant CD3e as described in Examples 10-11.

[0248] Aptamers were synthesized by either standard solid-phase synthesis on CPG resin followed by AEX column purification and ultrafiltration or standard desalting. The tumor-targeting immune engager and CpG motif sequences are found in Table 1. Table 20 below lists additional control and supporting sequences used in different experiments.

[0249] [Table 20]

[0250] b. Antibodies and reagents Leaf used for stimulation of human PBMCs TM Purified anti-human CD3 and Leaf TM Purified anti-human CD28 antibody was purchased from Biolegend (ENCO). CD45-FITC antibody used for leukocyte staining was purchased from Miltenyi Biotec (Almog diagnostic). Mitomycin C used as a positive control was purchased from Sigma. Live / Dead for 405 nm excitation. TM Fixative violet dead cell stain kit was purchased from Thermo Fisher (Rhenium).

[0251] c. Cell lines and PBMC isolation HCT-116 human colorectal cell line (ATCC® CCL-247 TM ) were cultured in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (Pen / Strep).

[0252] The MCF10a non-tumorigenic cell line (ATCC® CRL-10317 TM ) were cultured in DMEM / F12 supplemented with 5% horse serum, 1% Pen / Strep, 20 ng / mL EGF, 0.5 mg / mL hydrocortisone, 100 ng / mL cholera toxin, and 10 μg / mL insulin. All cells were cultured at 37°C and 5% CO2.

[0253] PBMCs were cultured in Lymphoprep according to the manufacturer's protocol. TM PBMCs were isolated from peripheral blood from healthy donors (Sheba Hospital, MDA Israel) by Ficoll density gradient centrifugation using Axis-Shield (Axis-Shield). Isolated PBMCs were maintained in RPMI 1640 medium (ATCC) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (Pen / Strep).

[0254] d. Formulation Buffer / Vehicle Phosphate-buffered saline (magnesium- and calcium-free) was supplemented with 1 mM magnesium chloride (MgCl2). The folding buffer was sterilized using a 0.22 μm PVDF membrane filter unit and kept at RT.

[0255] e. animals Female NSG mice, 7-8 weeks old, were purchased from Jackson Labs.

[0256] B. Experimental Method a. Bispecific personalized aptamer formulation The formulation procedure includes the following steps: 1. Reconfiguration Each strand is diluted / reconstituted (if lyophilized) in formulation buffer to the desired concentration. 2. Aptamer folding: a. Heat the strands to 95°C for 5 minutes. b. Rapid cooling on ice for 10 minutes. c. Incubation at RT for 10 min. 3. Bispecific entity formation The two chains (cancer-targeting variable chain and immune engager chain) are then mixed together and incubated at RT for 30 minutes on a rotator.

[0257] b. Cytotoxicity assay HCT116 cells were seeded in 96-well plates for 24 hours, followed by the addition of PBMCs and treatment once daily for 72 hours. After the 72-hour treatment phase, the cell culture medium was removed and maintained while 30 μL trypsin was added to each well for 5 minutes at 37°C, followed by centrifugation at 300 × g for 5 minutes at 4°C. After centrifugation, the cells were resuspended in 100 μL LIVE / DEAD TM The cells were resuspended in Fixative Violet Dead Cell Stain (Thermo Fisher) (1:1,000 in PBS) and incubated on ice in the dark for 30 minutes. The cells were washed once in wash buffer (PBS containing 1% BSA and 2 mM EDTA) and resuspended in 50 μL CD45-FITC antibody solution for 15 minutes on ice in the dark. After analysis by flow cytometry, the cells were washed once.

[0258] c. Gating strategy: To distinguish between immune cells and target HCT116 cells, Dead / Live TM The dye was used in combination with CD45 antibody staining. The mortality of target cells was assessed using Live / Dead TM It was determined by the percentage of cells that stained positive for the dye.

[0259] d.Animals Female NSG TMMice, 7-8 weeks old, were purchased from Jackson Labs, Inc. All animal procedures were performed in the facilities of Tel Aviv Sourasky Medical Center under ethical approval.

[0260] e. Xenograft model induction and intervention (i) HCT116 Early Intervention Model Female NSG TM Mice were treated with 0.5 × 10 PBS at a 1:4 ratio using 0.2 mL / mouse of Cultrex® (basement membrane matrix, type 3). 6 2 x 10 mixed with fresh human PBMCs 6 HCT116 tumor cells were injected subcutaneously (SC) into the right flank of mice. The SC intervention regimen is detailed for each experiment. (ii) MCF7 established tumor model Female NSG TM Mouse, 2 x 10 6 MCF-7 tumor cells were injected SC into the right flank of mice. One week before MCF-7 implantation, the mice were supplemented with water containing estradiol. Established tumors were measured (50–100 mm). 3 ), 15×10 6 Fresh human PBMCs were administered intravenously (IV). Four days after PBMC injection, randomization was performed based on tumor volume, and intratumoral (IT) intervention was initiated three times weekly for a total of eight doses.

[0261] f. Tumor volume assessment method Changes in tumor volume were monitored three times a week with a caliper. Tumor volume was estimated as follows: tumor volume (mm 3 ) = length x width 2 / 2.

[0262] g. Statistical methods All quantitative data are expressed as mean±SEM. Either ANOVA or Student's t-test was used to assess the significance of differences between groups, where appropriate.

[0263] Example 10: Tumoricidal aptamers identified by Aummune's platform were found to be effective in vitro in cancer cell lines and tumor-derived organoids The newly identified cancer-targeting tumoricidal aptamer arms were derived from a functional enrichment process described in PCT Application No. PCT / IB19 / 01082.

[0264] To provide proof-of-concept for the ability of Aummune's platform to identify specific aptamer sequences that function as VSs, the HCT116 colon cancer cell line was used. These targeted cells, along with a negative control of human PBMCs from healthy donors as representative non-tumorigenic cells, were subjected to Aummune's proprietary innovative aptamer selection platform to isolate potent and selective VSs.

[0265] a. Identification of functional aptamer "variable chain 12" via Aummune's SELEX process Aummune's proprietary functional SELEX technology was performed using the human colon cancer cell line HCT116. The enrichment procedure was carried out in 10 15 We started with a random library of aptamers containing a large repertoire of individual sequences. As shown in Figure 35, the aptamer population indeed demonstrated relative enrichment between rounds of enrichment, including the eighth round of functional selection (F3.8), which induced 37.4% apoptotic cells, a 1.5-fold increase over the 25% apoptotic cells (clustered bead population sample) after the first round of functional selection (F3.1).

[0266] The DNA library underwent enrichment for apoptosis-inducing sequences in HCT116 cells during functional Cell-SELEX, resulting in a 1.5-fold increase in caspase 3 / 7 activation in cycle 8 (F3.8) (37%) compared to cycle 1 (F3.1) (25%).

[0267] In the final round of functional selection, the clustered library was incubated with both target ("positive" HCT-116) cells and negatively selected ("negative" PBMCs from healthy donors). Positive and negative events were sorted from each cell population. Finally, libraries from the final round for both target and negative cells were sequenced via NextSeq 500, followed by bioinformatics analysis for each putative aptamer. Each aptamer was assigned two scores: one representing the sequence's propensity to induce apoptosis in target cells (Y-axis, Figure 36), and the second representing the sequence's propensity to induce apoptosis in negatively selected cells (X-axis, Figure 36). The top 44 sequences with the highest Y-axis-X-axis score ratio were individually screened for their apoptosis-inducing potential via high-content fluorescence microscopy.

[0268] Subsequent individual sequence screening was performed using high-content time-lapse fluorescence microscopy: target cells were incubated with candidate aptamers for 24 hours, and time-lapse imaging was applied to identify putative sequences that successfully induced apoptosis in target cells.

[0269] Variable chain 6 (VS6) and VS12 (SEQ ID NOs: 43 and 44, respectively) were selected for further testing for their ability to induce target cell death (Figure 37). VS12 was further evaluated over a range of concentrations, and VS12 exhibited dose-dependent cytotoxicity. The data collectively show that VS12 was able to (i) induce caspase activity, (ii) result in increased target cell death as measured by flow cytometry, and (iii) substantially reduce target cell viability.

[0270] b. Identification of functional aptamer variable chain 13 (VS13), VS16, and VS19 via Aummune's SELEX process Functional Cell-SELEX, designed to obtain functional target-specific cytotoxic aptamers, was performed using the MCF7 human breast cancer cell line (again using the process described herein and in PCT Application No. PCT / IB2019 / 001082).

[0271] During each round of functional enrichment, the aptamer library was incubated with target cells (MCF7) and stained with Annexin V as a cell death marker. PBMCs from healthy donors were used for negative selection.

[0272] As shown in Figure 38A, the aptamer library population demonstrated increasing relative functional enrichment with each round of SELEX iteration. In the final round of functional enrichment, the library was incubated with both target ("positive"; MCF7) cells and counter-selected ("negative"; PBMCs from healthy donors) cells. Positive and negative events were sorted and sequenced. Each aptamer sequence was assigned two scores: (i) the sequence's propensity to induce cell death in target tumor cells (X-axis, Figure 38B) and (ii) the sequence's propensity to induce nonspecific cell death in counter-selected PBMCs (Y-axis, Figure 38B). The top 45 sequences with the highest X-axis to Y-axis score ratios were individually screened for their apoptosis-inducing potential via high-content fluorescence microscopy.

[0273] Subsequent individual sequence screening was performed using high-content time-lapse fluorescence microscopy. MCF7 cells were cultured with candidate aptamers for 24 hours, and time-lapse imaging was applied to identify putative sequences that successfully induced apoptosis in target cells. Vehicle (1x PBS- / - supplemented with 1 mM MgCl2) and random sequences were used as negative controls; staurosporine was used as a positive control. Three aptamer sequences, variable chains (VS13, VS16, and VS19), demonstrated their ability to induce MCF7 cell death as individual aptamers (Figures 39A and 39B).

[0274] The top six candidates (VS4, VS11, VS13, VS16, VS19, and VS43) were further tested for their ability to affect MCF7 cell viability in a dose-dependent manner. VS aptamers were simultaneously added to PBMC cultures to assess the specificity of each candidate. The viability of both MCF7 and PBMC was determined using an XTT assay. Cultures containing either the VS13 or VS16 aptamer resulted in a significant reduction in MCF7 cell viability compared with a nonspecific DNA sequence of the same length composed of polythymidine nucleotides (polyT) (Figure 40A). VS13 and VS16 exhibited desirable characteristics and fulfilled the criteria for promising VS candidates by inducing substantial cell death in the target cell population while having minimal effects on negative healthy PBMCs (Figures 40A and 40B).

[0275] The scatter plot summary shows MCF7 viability (Y-axis) and PBMC viability (X-axis) for the lead aptamers tested (Figure 40B) compared to the positive control (staurosporine) and negative controls (vehicle and untreated). The six lead aptamers and polyT are indicated by hexagons for the 200 μM dose, diamonds for the 100 μM dose, and triangles for the 50 μM dose level. VS13 and VS16 are indicated by "13" and "16" (Figure 40B).

[0276] c. Identification of functional aptamer variable chain 3 (VS3) and VS20 via Aummune's SELEX process Aummune's proprietary technology was then performed using human adenocarcinoma alveolar basal epithelial lung cells A549. Similar to HCT116 and MCF7, functional inter-round enrichment of A549 was demonstrated using the library from round 8 of functional selection (F3.8), which induced 39% apoptotic cell death (Figure 41), a 1.3-fold increase compared to the 30% apoptosis induction by the first-round library pool (F3.1). As detailed in the two examples above, NGS sequencing and subsequent bioinformatics analysis of the final enriched library (F3.8) was performed, and 90 individual aptamer sequences were further evaluated by high-content microscopy.

[0277] Five top candidates (including VS3 and VS20) were assayed for their cytotoxic effects after a single dose administered at 50, 100, and 200 μM concentrations, and the assay concluded with measurement of cell viability ratio via XTT assay (Figure 42).

[0278] d. Aummune's SELEX process applied to colorectal cancer (CRC)-derived organoids The robustness of the platform was demonstrated by providing data generated from the SELEX process performed on organoids derived and grown from primary human tumor tissue.

[0279] Fresh CRC tissue was removed from patients during surgical procedures, collected in dedicated medium, and kept at 4°C until processing. The tissue was then subjected to a first processing step that combined mechanical and enzymatic dispersion using collagenase until fragments of less than 0.1 mm were observed. The tissue fragments were mixed with basement membrane extract (BME) and placed in an incubator to solidify the BME. CRC culture medium was then added to the cells. After two weeks, several organoid structures began to form, and after another three weeks, the number of organoids reached a critical mass for the initiation of the SELEX process (Figure 43).

[0280] As shown in Figure 44A, the aptamer population pool showed a relative functional increase in the seventh round of functional selection (F3.7), which resulted in 31.8% apoptotic cells, a 3.6-fold increase over the 8.7% apoptotic cells observed in the second round pool (F3.2).

[0281] In the final round of functional selection, the enriched library was incubated with both target cells and a counter / negative cell population (PBMCs from healthy donors). Positive and negative events were sorted from each cell population. The enriched library from the final round was then sequenced via NextSeq 500 for both target and negative cells, followed by bioinformatics analysis to identify promising individual aptamer sequences. The sequencing data was analyzed using Aummune's algorithm, which assigned candidates for functional validation of the individual sequences. The algorithm utilized statistical estimators, tests, and metrics. Aummune has successfully performed high-content microscopy screening on organoids in their assembled 3D configuration and within an extracellular support environment (BME) without the need to disperse cells into single-cell suspension. This setup allowed for long screening times (up to 24 hours) and supported tumor cell viability over time. Aummune has tailored the quantification of both active caspases and annexin V using this assembled multicellular organoid method.

[0282] The three variable chains (VS31, VS48 and VS81, respectively, SEQ ID NOs: 113-115) identified by the above-mentioned microscopy screening were individually tested for their ability to induce tumor cell death using CRC13 organoids as targets and luminescence-based viability assays.The variable chains were compared with random sequences with 50% GC content (Figure 44B).

[0283] Example 11: In vitro proof of concept (POC) for novel bispecific personalized aptamer efficacy In some embodiments, the personalized cancer therapeutics described herein are composed of a heterodimeric structure comprising three distinct domains (Figure 1).

[0284] Following functional Cell-SELEX (see Example 10a) designed to obtain functional apoptosis-inducing aptamers targeting the HCT116 cell line, two candidates were selected to generate bispecific leads (i.e., VS6 and VS12).

[0285] The T cell engagers generated and characterized using the process described herein (see Example 3) were used as representative "constant" immune-engaging arms, in addition to a previously characterized CD16-binding natural killer (NK) engager (Boltz et al. (2011) J. Biol. Chem. 286:21896-21905). Other potential immune-modulating aptamers can also be used (Soldevilla et al. (2016) Journal of Immunology Research 2016:1083738; Soldevilla et al. (2017) Immunotherapy - Myths, Reality, Ideas, Future doi:10.5772 / 66964). Five candidate bispecific personalized aptamers were generated (see Figure 45) and are listed in Table 21 below:

[0286] [Table 21]

[0287] NK and CTL bispecific personalized aptamers were evaluated for their cytotoxic effects against the HCT116 target cell line in a coculture setting containing effector PBMCs from healthy donors at an effector:target (E:T) ratio of 80:1. Unless otherwise specified, all treatments were administered once daily at 100 μM for a total duration of 72 hours. Tumor cell viability was subsequently analyzed by flow cytometry using LIVE / DEAD (Thermo Fisher) staining, gating on target cells only. Bispecific aptamers were compared to a vehicle negative control (1x PBS supplemented with 1 mM MgCl2) and a nonspecific DNA dimer, each composed of two polythymidine (polyT) arms of similar oligomeric length as the bispecific strand. The results show high levels of lethality with all five bispecific personalized aptamers targeting HCT116 cells (approximately 55%) and low activity against PBMCs (approximately 17%), similar to the negative control (10-12%) (Figures 46A and 46B). The PBMC lethality data reflect the specificity of the bispecific personalized aptamers compared to mitomycin, a clinically approved chemotherapeutic drug that is highly promiscuous in its cytotoxic activity.

[0288] A. Dose-dependent effects of bispecific personalized aptamers targeting the HCT116 cell line Next, we investigated the ability of bispecific personalized aptamers to target HCT116 cells in a dose-dependent manner. The bispecific personalized aptamers CTL3||VS6, CTL5||VS12, CTL6||VS12, and a control polyT dimer (polyT||polyT) were tested at four concentrations in cocultures of PBMCs with HCT116 cells. Dose-dependence was demonstrated for each of the bispecific personalized aptamers tested, but not with the negative control polyT||polyT dimer (Figure 47).

[0289] B. Bispecific Individualized Aptamers Are Target Cell Specific in Their Cytotoxicity MCF10a is a non-tumorigenic epithelial cell line used as a negative selection along with PBMCs from healthy donors during functional Cell-SELEX to identify the VS12 aptamer and increase the specificity of the aptamer targeting the HCT116 cell line (described in PCT Application No. PCT / IB2019 / 001082, incorporated herein by reference).

[0290] To demonstrate that the bispecific personalized aptamers achieve selectivity while remaining potent against the desired target, their ability to induce cell death was assessed using PBMCs and MCF10a cells from healthy donors (Figures 48A and 48B). CTL3||VS12 exhibited a desirable profile of >60% target cell killing and <30% off-target killing (indicated by squares).

[0291] C. Bispecific Personalized Aptamers Outperform the Cancer-Targeting Aptamer Moiety Alone The target cell cytotoxicity of the bispecific personalized aptamer was compared with that of either monomer alone. Either the CTL6||VS12 bispecific personalized aptamer or one of its monomer chains was tested for its ability to induce HCT116 tumor cell death at an equivalent concentration of 100 μM. The CTL6||VS12 bispecific personalized aptamer was significantly superior to either monomer and the polyT||polyT negative control (Figure 49A). Neither the bispecific personalized aptamer nor the monomer induced PBMC lethality (Figure 49B).

[0292] D. CTL3||VS12 and CTL6||VS12 induced similar cytotoxic effects. An additional promising, previously untested bispecific personalized aptamer lead, CTL3||VS12, was compared in parallel with CTL6||VS12 for its cytotoxic effect against HCT-116 target cells in a co-culture assay with PBMCs. Both bispecific personalized aptamers were found to demonstrate similar cytotoxic effect against target cells (boxes, Figure 50), which was significantly greater than either monomer alone (Figure 50).

[0293] E. POC of CD3-targeting bispecific aptamer conjugates VS12 was hybridized with a T cell engager moiety (CS) to form the bispecific, dual-acting aptamer CS6-VS12. The CS6-VS12 bispecific aptamer was evaluated for its ability to induce target cell cytotoxicity.

[0294] CS6-VS12 was tested for cytotoxicity against the HCT116 colon cancer cell line in a coculture setting containing effector PBMCs from healthy donors at an effector:target (E:T) ratio of 10:1. Tumor cell viability was subsequently analyzed by a luminescence-based cell viability assay. CS6-VS12 was compared to a vehicle negative control (1x PBS supplemented with 1 mM MgCl) and a nonspecific DNA dimer, each composed of two polythymidine (polyT) arms of similar oligomeric length as the bispecific strand (Figure 51).

[0295] F. Bispecific Aptamers Targeting MCF7 Breast Cancer Cells CTL3s containing the T cell engager portion of bispecific aptamers were generated from a multi-donor selection process targeting human CD8 T cells, and their characterization is detailed in Examples 2–4. VS13, VS16, and VS19 were each hybridized to CTL3 to form bispecific aptamers. These VS-CTL3 bispecific aptamers were evaluated for their cytotoxic activity against MCF7 target cells in a coculture setting with PBMCs from healthy donors. Tumor cell lethality was subsequently analyzed by flow cytometry, and viability was analyzed by XTT to obtain complementary information. The bispecific aptamers (CTL3||VS13, CTL3||VS16, and CTL3||VS19) were compared with vehicle and a dimer composed of two poly-T arms. All three bispecific entities were found to have significant cytotoxic activity compared with vehicle and poly-T controls (Figures 52A and 52B).

[0296] Example 12: In vivo POC of bispecific personalized aptamers in HCT116 and MCF7 tumor xenograft models The in vitro validated bispecific personalized aptamers were tested for their ability to destroy target tumor cells in an in vivo setting.

[0297] A. In vivo efficacy of NK cell engager CD16||VS12 Female NSG TM Mice were treated with 0.5 × 10 PBS at a 1:4 ratio using 0.2 mL / mouse of Cultrex® (basement membrane matrix, type 3). 6 2 x 10 mixed with fresh human PBMCs 6 HCT116 tumor cells were injected SC into mice into the right flank and treated with either the NK engager CD16||VS12 or poly-T dimer (poly-T||poly-T) as a control. Figure 53 shows the efficacy of treatment compared to poly-T administration after 12 interventions during the 32-day study. All seven treated mice showed tumor growth inhibition compared to poly-T. Furthermore, CD16||VS12-associated tumor growth attenuation resulted in better survival rates.

[0298] B. In vivo efficacy of the T cell engager CTL6||VS12 As mentioned above, female NSG TM 0.5 × 10 in a 1:4 ratio to mice 6 2 x 10 mixed with fresh human PBMCs 6HCT116 tumor cells were inoculated and treated with either vehicle, CTL6||VS12 from Supplier A, or CTL6||VS12 synthesized by Supplier B. Supplier A provided the aptamer without any modifications and using standard desalting purification methods, while Supplier B provided the aptamer containing inverted dTs at both the 3' and 5' flanking sites and as the product of column purification. Figure 54 shows the efficacy of treatment compared to vehicle and untreated groups after 10 interventions during the initial 27-day study (after day 27, mice began to be sacrificed for ethical endpoint volume). Both groups of CTL6||VS12-treated mice demonstrated significant inhibition of tumor growth. Comparison of tumor volume at day 27 showed significant differences for both bispecific personalized aptamers compared to vehicle (Figure 56). Individual mouse tumor volumes are presented for each bispecific personalized aptamer treatment compared to vehicle up to the end of the study (30 days after the last intervention) (Figures 55A and 55B).

[0299] C. In vivo efficacy of the T cell engager CTL3||VS12 HCT116 colon cancer cells were transformed into immunodeficient female NOD scid gamma (NSG) TM ) Mice were co-transplanted with fresh human PBMCs from healthy donors and then administered vehicle, polyT dimer, or CTL3||VS12 as detailed in Table 22.

[0300] [Table 22]

[0301] Figures 56A and 56B describe HCT116 tumor growth kinetics. Treatment with the bispecific aptamer CTL3||VS12 significantly attenuated HCT116 tumor growth (Figure 57A), resulting in an average tumor size that was approximately 30% smaller in weight than the control group at day 22 (Figure 57B), but not with the nonspecific polyT||polyT dimer. Figure 58 depicts the survival curves of this experiment, suggesting benefit for the treatment groups.

[0302] D. The CS6-VS12 bispecific aptamer attenuates tumor growth in vivo In the xenograft model, HCT116 colon cancer cells were co-implanted into immunodeficient female NSG mice in a mixed fashion (E:T 1:4 ratio) with fresh human PBMCs from healthy donors and administered bispecific personalized aptamer (CS6-VS12, SEQ ID NOs: 116 and 50), polyT duplex, or vehicle.

[0303] Figures 59A and 59B show HCT116 tumor growth kinetics. Treatment with the bispecific aptamer CS6-VS12 significantly attenuated HCT116 tumor growth after a total of 10 interventions, but not with the nonspecific oligonucleotide polyT. At day 30, mice began to be sacrificed for endpoint ethical volume. Individual mouse tumor volumes were presented until day 41 (31 days after the last intervention). Tumor growth inhibition was demonstrated in all CS6-VS12-treated mice (Figure 59B). The reduction in tumor growth translated into a survival benefit for the bispecific treatment group when compared with vehicle (Figure 60).

[0304] E. Efficacy of the MCF7-targeting bispecific aptamer CTL3||VS16 in vivo in an established tumor model The transferability of CTL3||VS16 cytotoxicity from in vitro to in vivo settings was evaluated in an established MCF7 tumor xenograft model.

[0305] [Table 23] A significant inhibition of tumor growth was demonstrated in CTL3-VS16 treated mice compared to vehicle treated mice (FIGS. 61A and 61B).

[0306] F. Efficacy of the murine 4T1-targeting bispecific aptamer CS6-VS32 in vivo in combination with immune checkpoint inhibitors To enable in vivo animal validation in immunocompetent animals (in addition to the xenograft models described above), the murine breast cancer cell line 4T1 was subjected to a functional enrichment platform (as in other examples in Example 10) to identify VS32, which was hybridized with CS6 to form a bispecific aptamer and evaluated in a dual-flank 4T1 tumor model.

[0307] The tendency for inhibited growth of both primary and secondary tumors was demonstrated by intratumoral administration of CS6-VS32 into established primary tumors (Figure 62A). Cyclophosphamide (CTX) chemotherapy was used as a positive control at equivalent doses.

[0308] When administration of CS6-VS32 was combined with the immune checkpoint inhibitor anti-PD1, synergy was demonstrated, resulting in significant tumor growth reduction in both injected and non-injected secondary tumors (Figure 62B).

[0309] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0310] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The present application provides the following: 1. (a) a cancer cell-binding chain that specifically binds to an antigen expressed on cancer cells; (b) a CpG motif sequence; and (c) an immune effector cell-binding chain that specifically binds to an antigen expressed by an immune effector cell; wherein the cancer cell-binding chain is linked to the immune effector cell-binding chain by the CpG motif sequence. 2. The bispecific individualized aptamer described in 1 above, wherein the cancer cell-binding chain induces cell death when contacted with a cancer cell. 3. The bispecific individualized aptamer described in 1 or 2 above, wherein the cell death is apoptosis, necrosis, immunological cell death, autophagy, or necroptosis. 4. The bispecific individualized aptamer according to any one of 1 to 3 above, wherein the cancer cells are patient-derived cancer cells. 5. The bispecific individualized aptamer according to any one of 1 to 4 above, wherein the cancer cells are solid tumor cells. 6. The bispecific individualized aptamer described in 5 above, wherein the cancer cells are carcinoma cells. 7. The bispecific personalized aptamer according to claim 6, wherein the carcinoma cells are breast cancer cells, head and neck cancer cells, bladder cancer cells, or colorectal cancer cells. 8. The bispecific individualized aptamer according to any one of 1 to 4 above, wherein the cancer cells are sarcoma cells. 9. The bispecific individualized aptamer according to any one of 1 to 4 above, wherein the cancer cells are blood cancer cells. 10. The bispecific individualized aptamer according to any one of 1 to 9 above, wherein the cancer cell-binding chain induces cell death when contacted with the cancer cells in vitro. 11. The bispecific individualized aptamer according to any one of 1 to 10 above, wherein the cancer cell-binding chain induces cell death when contacted with the cancer cells in vivo. 12. The bispecific individualized aptamer described in any one of 1 to 11 above, wherein the immune effector cell binding chain mediates lysis of the cancer cells via T cell or NK cell mediated cytotoxicity. 13. The bispecific individualized aptamer described in any one of 1 to 12 above, wherein the cancer cell-binding chain and the immune effector cell-binding chain are linked together by hybridization of the 5' sequence of the cancer cell-binding chain to the 5' sequence of the immune effector cell-binding chain. 14. The bispecific individualized aptamer described in any one of 1 to 13 above, wherein the 5' sequence of the cancer cell-binding chain hybridizes to the 5' sequence of the immune effector cell-binding chain to form a double-stranded CpG motif sequence. 15. The bispecific individualized aptamer according to claim 14, wherein the CpG motif sequence acts as a TLR agonist and induces TLR9-mediated antigen-presenting cell (APC) stimulation and / or increased uptake of tumor antigens. 16. The bispecific individualized aptamer described in any one of 1 to 15 above, wherein the CpG motif sequence induces an anti-tumor immune response. 17. The bispecific individualized aptamer described in any one of 1 to 16 above, wherein the CpG motif sequence induces IL6 secretion, IFNα secretion, and / or B cell activation. 18. A bispecific individualized aptamer described in any one of 1 to 17 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence that is at least 80% identical to any one of SEQ ID NOs: 63 to 66. 19. A bispecific individualized aptamer described in any one of 1 to 18 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 63 to 66. 20. A bispecific individualized aptamer described in any one of 1 to 19 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 63 to 66. 21. A bispecific individualized aptamer described in any one of 1 to 20 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence that is at least 98% identical to any one of SEQ ID NOs: 63 to 66. 22. A bispecific individualized aptamer described in any one of 1 to 21 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising any one of the sequences of SEQ ID NOs: 63 to 66, and optionally the CpG motif sequence is a double-stranded nucleic acid sequence comprising the sequences of SEQ ID NOs: 63 and 64. 23. A bispecific individualized aptamer described in any one of 1 to 22 above, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising at least 15 consecutive nucleotides of any one of SEQ ID NOs: 63 to 66. 24. The bispecific individualized aptamer described in any one of 1 to 23 above, wherein the CpG motif sequence has a length of 30 nucleotides or less. 25. The bispecific personalized aptamer according to any one of 1 to 24 above, wherein the cancer cell-binding chain binds to a cancer antigen selected from prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), tyrosinase, gp100, MART-1 / melan-A, HSP70-2-m, HLA-A2-R17OJ, HPV16-E7, MUC-1, HER-2 / neu, mammaglobin-A, or an MHC-TAA peptide complex. 26. A bispecific individualized aptamer described in any one of 1 to 25 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 27. A bispecific individualized aptamer described in any one of 1 to 26 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 28. A bispecific individualized aptamer described in any one of 1 to 27 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 29. A bispecific individualized aptamer described in any one of 1 to 28 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 98% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 30. The bispecific individualized aptamer described in any one of 1 to 29 above, wherein the cancer cell-binding chain comprises any one of the nucleic acid sequences of SEQ ID NOs: 43 to 62 or 107 to 115. 31. A bispecific individualized aptamer described in any one of 1 to 30 above, wherein the cancer cell-binding strand comprises at least 30 consecutive nucleotides of any one of SEQ ID NOs: 43 to 62 or 107 to 115. 32. A bispecific individualized aptamer described in any one of 1 to 31 above, wherein the cancer cell-binding strand comprises at least 40 consecutive nucleotides of any one of SEQ ID NOs: 43 to 62 or 107 to 115. 33. A bispecific individualized aptamer described in any one of 1 to 32 above, wherein the cancer cell-binding strand comprises at least 50 consecutive nucleotides of any one of SEQ ID NOs: 43 to 62 or 107 to 115. 34. A bispecific individualized aptamer described in any one of 1 to 33 above, wherein the cancer cell-binding strand comprises at least 60 consecutive nucleotides of any one of SEQ ID NOs: 43 to 62 or 107 to 115. 35. The bispecific individualized aptamer according to any one of 1 to 34 above, wherein the cancer cell-binding strand is 120 nucleotides or less in length. 36. The bispecific individualized aptamer according to any one of 1 to 35 above, wherein the cancer cell-binding strand is 90 nucleotides or less in length. 37. The bispecific individualized aptamer according to any one of 1 to 36 above, wherein the cancer cell-binding strand is 80 nucleotides or less in length. 38. The bispecific individualized aptamer described in any one of 1 to 37 above, wherein the cancer cell-binding strand is 63 nucleotides in length or less, and optionally, the cancer cell-binding strand is 63 nucleotides in length. 39. The bispecific individualized aptamer described in any one of 1 to 38 above, wherein the immune effector cell-binding chain binds to an antigen expressed by T cells, NK cells, B cells, macrophages, dendritic cells, neutrophils, basophils, or eosinophils. 40. The bispecific individualized aptamer according to any one of 1 to 39 above, wherein the immune effector cell-binding chain binds to an immune effector cell antigen selected from CD16, Notch-2, other Notch family members, KCNK17, CD3, CD28, 4-1BB, CTLA-4, ICOS, CD40L, PD-1, OX40, LFA-1, CD27, PARP16, IGSF9, SLC15A3, WRB, and GALR2. 41. The bispecific individualized aptamer described in any one of 1 to 40 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 42. The bispecific individualized aptamer described in any one of 1 to 41 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 43. The bispecific individualized aptamer described in any one of 1 to 42 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 44. The bispecific individualized aptamer described in any one of 1 to 43 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 98% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 45. The bispecific individualized aptamer described in any one of 1 to 44 above, wherein the immune effector cell-binding chain comprises any one of the nucleic acid sequences of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 46. ​​The bispecific individualized aptamer described in any one of 1 to 45 above, wherein the immune effector cell-binding strand comprises at least 20 consecutive nucleotides of any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 47. The bispecific individualized aptamer described in any one of 1 to 46 above, wherein the immune effector cell-binding strand comprises at least 30 consecutive nucleotides of any one of SEQ ID NOs: 1 to 42, 88 to 106 or 116. 48. A bispecific individualized aptamer described in any one of 1 to 47 above, wherein the immune effector cell-binding strand comprises at least 40 consecutive nucleotides of any one of SEQ ID NOs: 1 to 42, 88 to 106 or 116. 49. A bispecific individualized aptamer described in any one of 1 to 48 above, wherein the immune effector cell-binding strand comprises at least 50 consecutive nucleotides of any one of SEQ ID NOs: 1 to 42, 88 to 106 or 116. 50. The bispecific individualized aptamer described in any one of 1 to 49 above, wherein the immune effector cell-binding strand is 120 nucleotides or less in length. 51. The bispecific individualized aptamer according to any one of 1 to 50 above, wherein the immune effector cell-binding strand is 90 nucleotides or less in length. 52. The bispecific individualized aptamer according to any one of 1 to 51 above, wherein the immune effector cell-binding strand is 80 nucleotides or less in length. 53. The bispecific individualized aptamer according to any one of 1 to 52 above, wherein the immune effector cell-binding strand is 73 nucleotides or less in length. 54. The bispecific individualized aptamer according to any one of 1 to 53 above, comprising a combination of a cancer cell-binding chain selected from SEQ ID NOs: 43 to 62 or 107 to 115 and an immune effector cell-binding chain selected from SEQ ID NOs: 1 to 42, 88 to 106, or 116. 55. A bispecific individualized aptamer according to any one of 1 to 54 above, which comprises a chemical modification. 56. The bispecific individualized aptamer described in 55 above, which is chemically modified with polyethylene glycol (PEG). 57. The bispecific individualized aptamer described in 56 above, wherein the PEG is linked to the 5' or 3' end of the aptamer. 58. A bispecific individualized aptamer according to any one of 55 to 57 above, which comprises a 5'-end cap. 59. A bispecific individualized aptamer according to any one of 55 to 58 above, which comprises a 3'-end cap. 60. The bispecific individualized aptamer according to claim 59, wherein the 3'-end cap is an inverted thymidine. 61. The bispecific individualized aptamer according to claim 59, wherein the 3'-end cap comprises biotin. 62. The bispecific individualized aptamer according to any one of 55 to 61 above, comprising a 2' sugar substitution. 63. The bispecific individualized aptamer of claim 62, wherein the 2' sugar substitution is a 2'-fluoro, 2'-amino, or 2'-O-methyl substitution. 64. A bispecific individualized aptamer according to any one of claims 55 to 63, comprising locked nucleic acid (LNA), unlocked nucleic acid (UNA) and / or 2'deoxy-2'fluoro-D-arabinonucleic acid (2'-F ANA) sugars in its backbone. 65. A bispecific individualized aptamer according to any one of 55 to 64 above, comprising a methylphosphonate internucleotide bond and / or a phosphorothioate (PS) internucleotide bond. 66. A bispecific individualized aptamer described in any one of 55 to 65 above, wherein the double-stranded CpG motif sequence comprises a partial PS modification. 67. The bispecific individualized aptamer according to any one of the above items 55 to 66, wherein five nucleotides from the 5' end of the double-stranded CpG motif sequence are modified. 68. A bispecific individualized aptamer according to any one of 55 to 67 above, wherein five nucleotides from both the 5' end and the 3' end of the double-stranded CpG motif sequence are modified. 69. A bispecific individualized aptamer described in any one of 55 to 68 above, wherein the double-stranded CpG motif sequence contains a complete PS modification. 70. A bispecific individualized aptamer according to any one of 55 to 69 above, comprising a triazole internucleotide linkage. 71. A bispecific individualized aptamer according to any one of the above items 55 to 70, which is modified with cholesterol or a dialkyl lipid. 72. The bispecific individualized aptamer of claim 71, wherein the cholesterol or dialkyl lipid is linked to the 5' end of the aptamer. 73. The bispecific individualized aptamer according to any one of 55 to 72 above, which comprises modified bases. 74. A bispecific individualized aptamer according to any one of 1 to 73 above, which is a DNA aptamer. 75. The bispecific individualized aptamer according to claim 74, which is a D-DNA aptamer. 76. The bispecific individualized aptamer described in 75 above, which is an R-DNA aptamer. 77. A bispecific individualized aptamer according to any one of 1 to 73 above, which is an RNA aptamer. 78. The bispecific individualized aptamer according to claim 77, which is a D-RNA aptamer. 79. The bispecific individualized aptamer described in 77 above, which is an R-RNA aptamer. 80. A pharmaceutical composition comprising the bispecific individualized aptamer described in any one of 1 to 79 above. 81. The pharmaceutical composition according to claim 80, further comprising a pharmaceutically acceptable carrier. 82. A pharmaceutical composition according to claim 80 or 81, formulated for parenteral administration. 83. A pharmaceutical composition according to any one of items 80 to 82 above, for use in the treatment of cancer. 84. The pharmaceutical composition according to claim 83, wherein the cancer is a solid tumor. 85. The pharmaceutical composition described in 84 above, wherein the cancer is breast cancer. 86. The pharmaceutical composition described in 83 above, wherein the cancer is carcinoma. 87. The pharmaceutical composition according to claim 86, wherein the cancer is colorectal cancer. 88. A method for treating cancer, comprising the step of administering to a subject the bispecific individualized aptamer described in any one of 1 to 87 above. 89. A method for treating cancer, comprising the step of administering to a subject the pharmaceutical composition described in any one of 80 to 88 above. 90. The method according to claim 88 or 89, wherein the administration is parenteral. 91. The method according to claim 90, wherein the administration is subcutaneous administration. 92. The method according to claim 90 or 91, wherein the administration is intratumoral injection. 93. The method according to claim 90 or 91, wherein the administration is peritumoral injection. 94. The method according to any one of claims 88 to 93, wherein two or more doses are administered. 95. A method according to any one of claims 88 to 94, wherein at least 10 to 12 doses are administered. 96. A method according to any one of claims 88 to 95, wherein the administration of two or more doses to a subject is separated by at least one day. 97. A method according to any one of the above items 88 to 96, wherein the cancer is a solid tumor. 98. The method of claim 97, wherein the solid tumor is accessible by intratumoral administration. 99. The method of claim 98, wherein the cancer is breast cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, bladder cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, or colorectal cancer. 100. The method according to any one of claims 88 to 97, wherein the cancer is a sarcoma. 101. The method according to claim 100, wherein the cancer is a blood cancer. 102. A method according to any one of the above items 88 to 101, wherein the subject has undergone chemotherapy. 103. A method according to any one of claims 88 to 102, wherein the subject had a tumor that had been surgically removed. 104. A method according to any one of 88 to 103 above, further comprising the step of administering an additional cancer therapy to the subject. 105. The method according to claim 104, wherein the additional cancer therapy comprises chemotherapy. 106. The method according to claim 104, wherein the additional cancer therapy comprises radiation therapy. 107. The method according to claim 104, wherein the additional cancer therapy comprises surgical removal of the tumor. 108. The method described in claim 104, wherein the additional cancer therapy comprises administering an immune checkpoint inhibitor to the subject. 109. The method according to claim 108, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an anti-CTLA4 antibody. 110. A method for killing cancer cells, comprising the step of contacting cancer cells with an aptamer described in any one of 1 to 87 above. 111. The method according to claim 110, wherein the cancer cells are killed by apoptosis, necrosis, immunological cell death, autophagy, or necroptosis. 112. The method according to claim 110 or 111, wherein the cancer cells are solid tumor cells. 113. The method according to claim 112, wherein the cancer cells are breast cancer cells or colorectal cancer cells. 114. The method according to claim 110 or 111, wherein the cancer cells are sarcoma cells. 115. The method according to claim 110 or 111, wherein the cancer cells are blood cells. 116. A method for producing a bispecific individualized aptamer, comprising the steps of: (1) synthesizing a cancer cell-binding chain; (2) synthesizing an immune effector cell-binding chain; and (3) linking both chains to form a bispecific aptamer, wherein optionally, the two chains are linked by hybridization, covalent bonding, or PEG cross-linking. 117. The cancer cell-binding chain is prepared by the following steps: (a) contacting cancer cells with a plurality of particles ("aptamer cluster particles") having a library of aptamer clusters immobilized on their surfaces, wherein at least a subset of the immobilized aptamer clusters bind to at least a subset of the cancer cells to form cell-aptamer cluster particle complexes; (b) incubating the cell-aptamer cluster particle complexes for a period of time sufficient for at least a portion of the cancer cells in the cell-aptamer cluster particle complexes to perform a cellular function; (c) detecting the cell-aptamer cluster particle complex performing the cellular function; (d) separating the cell-aptamer cluster particle complexes comprising cancer cells performing the cellular function detected in step (c) from other cell-aptamer cluster particle complexes; (e) amplifying the aptamers in the separated cell-aptamer cluster particle complexes to generate a functionally enriched population of aptamers; and (f) identifying the enriched population of aptamers via sequencing, thereby identifying the cancer cell-binding chains. 117. The method according to claim 116, wherein the method is identified through a process comprising: 118. The method according to claim 117, wherein steps (c) and (d) are carried out using a flow cytometer. 119. The method according to claim 117 or 118, further comprising the step of separating the aptamer cluster particles from the target cells in the cell-aptamer cluster particle complexes separated in step (d). 120. The method described in claim 119, further comprising the step of dissociating the aptamers from the particles in the separated aptamer cluster particles. 121. A method described in any of 117 to 120 above, further comprising the step (e') after step (e) and before step (f): (i) forming aptamer cluster particles from the functionally enriched population of aptamers of step (e); and (ii) repeating steps (a) to (e) using the newly formed aptamer cluster particles to generate a further functionally enriched population of aptamers. 122. The method according to claim 121, wherein step (e') is repeated at least twice. 123. The method according to claim 122, wherein step (e') is repeated at least three times. 124. The method according to claim 123, wherein step (e') is repeated at least four times. 125. A method according to any one of claims 121 to 124, wherein step (e') further comprises applying restrictive conditions during successive rounds of enrichment. 126. The method described in claim 125, wherein the restrictive conditions are selected from (i) reducing the total number of particles, (ii) reducing the copy number of the aptamer per particle, (iii) reducing the total number of target cells, (iv) reducing the incubation time, and (v) introducing errors into the aptamer sequence by amplifying the population of aptamers using an error-prone polymerase. 127. A method described in any of 121 to 126 above, wherein the further enriched population of aptamers in step (e') has a two-fold reduction in sequence diversity compared to the library of aptamer clusters in step (a). 128. A method according to any one of 121 to 127 above, wherein each round of step (e') enriches the population of aptamers by at least 1.1 fold for aptamers that alter the cellular function. 129. A method according to any one of the above items 117 to 128, wherein the period is from about 10 minutes to about 5 days. 130. A method according to any one of the above items 117 to 129, wherein the period is from about 1.5 hours to about 72 hours. 131. A method according to any one of the above items 117 to 130, wherein the period is from about 1.5 hours to about 24 hours. 132. A method according to any one of claims 117 to 131, wherein the cancer cells are contacted with a reporter of the cell function before, during, or after the step of contacting the cancer cells with the aptamer cluster particles. 133. A method according to any one of 117 to 131 above, wherein the cancer cells are contacted with the reporter of cell function before, during, or after step (b). 134. The method according to any one of 117 to 133 above, wherein the reporter of cell function is a fluorescent dye. 135. A method according to any one of 117 to 134 above, further comprising the step of isolating the cancer cells from a patient prior to step (a). 136. The method according to claim 135, wherein the cancer cells are isolated from a tumor biopsy or surgical resection. 137. A method according to any one of claims 117 to 134, wherein the cell function is cell viability, cell death, or cell proliferation. 138. The method according to any one of claims 116 to 137, wherein the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain further comprise complementary 5' sequences. 139. The method according to claim 138, wherein step (3) comprises hybridizing the synthesized cancer cell-binding chain and the synthesized immune effector cell-binding chain. 140. The method of claim 138, wherein the complementary 5' sequence comprises a CpG motif. 141. The method according to any one of paragraphs 116 to 140 above, wherein the complementary 5' sequence comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 63 to 66. 142. The method according to any one of paragraphs 116 to 141 above, wherein the complementary 5' sequence comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 63 to 66. 143. The method according to any one of paragraphs 116 to 142 above, wherein the complementary 5' sequence comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 63 to 66. 144. The method according to any one of paragraphs 116 to 143 above, wherein the complementary 5' sequence comprises a nucleic acid sequence that is at least 98% identical to any one of SEQ ID NOs: 63 to 66. 145. A method according to any one of 116 to 144 above, wherein the complementary 5' sequence comprises any one of the nucleic acid sequences of SEQ ID NOs: 63 to 66. 146. The method according to any one of 116 to 145 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 147. The method according to any one of paragraphs 116 to 146 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 148. The method according to any one of 116 to 147 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 149. The method according to any one of paragraphs 116 to 148 above, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 98% identical to any one of SEQ ID NOs: 43 to 62 or 107 to 115. 150. The method described in any one of 116 to 149 above, wherein the cancer cell-binding chain comprises any one of the nucleic acid sequences of SEQ ID NOs: 43 to 62 or 107 to 115. 151. The method of any of paragraphs 116 to 150 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 152. The method of any of paragraphs 116 to 151 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 153. The method of any of paragraphs 116 to 152 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 154. The method of any of paragraphs 116 to 153 above, wherein the immune effector cell-binding chain comprises a nucleic acid sequence that is at least 98% identical to any one of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 155. The method according to any one of 116 to 154 above, wherein the immune effector cell-binding chain comprises any one of the nucleic acid sequences of SEQ ID NOs: 1 to 42, 88 to 106, or 116. 156. A method for treating cancer in a subject, comprising the step of administering to the subject a bispecific personalized aptamer produced using the method described in any one of 116 to 155 above.

Claims

1. (a) a cancer cell-binding chain that specifically binds to an antigen expressed on a cancer cell; (b) a CpG motif sequence; and (c) an immune effector cell-binding chain that specifically binds to an antigen expressed by an immune effector cell; wherein the cancer cell-binding chain is linked to the immune effector cell-binding chain by the CpG motif sequence.

2. 2. The bispecific individualized aptamer of claim 1, wherein the cancer cell-binding chain induces cell death when contacted with a cancer cell.

3. The bispecific personalized aptamer of claim 1 or 2, wherein the cancer cells are patient-derived cancer cells.

4. 4. The bispecific individualized aptamer of any one of claims 1 to 3, wherein said immune effector cell binding chain mediates lysis of said cancer cells via T cell or NK cell mediated cytotoxicity.

5. 5. The bispecific individualized aptamer of any one of claims 1 to 4, wherein the cancer cell-binding chain and the immune effector cell-binding chain are linked together by hybridization of the 5' sequence of the cancer cell-binding chain to the 5' sequence of the immune effector cell-binding chain, and the 5' sequence of the cancer cell-binding chain hybridizes to the 5' sequence of the immune effector cell-binding chain to form a double-stranded CpG motif sequence.

6. The bispecific individualized aptamer of any one of claims 1 to 5, wherein the CpG motif sequence (1) acts as a TLR agonist and induces TLR9-mediated antigen-presenting cell (APC) stimulation and / or increased uptake of tumor antigens, (2) induces an anti-tumor immune response, and / or (3) induces IL6 secretion and / or B cell activation.

7. The bispecific individualized aptamer according to any one of claims 1 to 6, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising a sequence that is at least 80% identical to any one of SEQ ID NOs: 63 to 66.

8. 8. The bispecific individualized aptamer of any one of claims 1 to 7, wherein the CpG motif sequence is a double-stranded nucleic acid sequence comprising any one of the sequences of SEQ ID NOs: 63 to 66, and optionally the CpG motif sequence is a double-stranded nucleic acid sequence comprising the sequences of SEQ ID NOs: 63 and 64.

9. 9. The bispecific personalized aptamer of any one of claims 1 to 8, wherein the cancer cell binding chain binds to a cancer antigen selected from prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), tyrosinase, gp100, MART-1 / melan-A, HSP70-2-m, HLA-A2-R17OJ, HPV16-E7, MUC-1, HER-2 / neu, mammaglobin-A, or an MHC-TAA peptide complex.

10. 10. The bispecific personalized aptamer of any one of claims 1 to 9, wherein the cancer cell-binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 43-62 or 107-115.

11. The bispecific individualized aptamer of any one of claims 1 to 10, wherein the cancer cell-binding chain comprises any one of the nucleic acid sequences of SEQ ID NOs: 43-62 or 107-115.

12. 12. The bispecific personalized aptamer of any one of claims 1 to 11, wherein the immune effector cell binding chain binds to an antigen expressed by T cells, NK cells, B cells, macrophages, dendritic cells, neutrophils, basophils, or eosinophils.

13. 13. The bispecific individualized aptamer of any one of claims 1 to 12, wherein the immune effector cell binding chain binds to an immune effector cell antigen selected from CD16, Notch-2, other Notch family members, KCNK17, CD3, CD28, 4-1BB, CTLA-4, ICOS, CD40L, PD-1, OX40, LFA-1, CD27, PARP16, IGSF9, SLC15A3, WRB, and GALR2.

14. 14. The bispecific individualized aptamer of any one of claims 1 to 13, wherein said immune effector cell binding chain comprises a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-42, 88-106 or 116.

15. 15. The bispecific individualized aptamer of any one of claims 1 to 14, wherein the immune effector cell binding chain comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-42, 88-106 or 116.

16. 16. The bispecific individualized aptamer of any one of claims 1 to 15, comprising a combination of a cancer cell-binding chain selected from SEQ ID NOs: 43-62 or 107-115 and an immune effector cell-binding chain selected from SEQ ID NOs: 1-42, 88-106 or 116.

17. The bispecific individualized aptamer of any one of claims 1 to 16, comprising a chemical modification.

18. A pharmaceutical composition for the treatment of cancer, comprising the bispecific personalized aptamer of any one of claims 1 to 17.

19. 20. Use of a bispecific individualized aptamer according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of cancer.

20. A method of killing cancer cells in vitro, comprising contacting the cancer cells with an aptamer according to any one of claims 1 to 17.

21. A method for producing a bispecific individualized aptamer, comprising the steps of: (1) synthesizing a cancer cell-binding chain; (2) synthesizing an immune effector cell-binding chain; and (3) linking both chains to form a bispecific aptamer, wherein the cancer cell-binding chain is linked to the immune effector cell-binding chain by a CpG motif sequence.

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