T lymphocyte-binding aptamers

Abstract

Provided herein are aptamers that target T lymphocytes and methods for using the same. In certain aspects, provided herein are methods for producing aptamers. In some embodiments, the method includes synthesizing (e.g., chemically synthesizing) a nucleic acid 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: 1-21 (e.g., any one of SEQ ID NOs: 1, 20, and 21).

Description

[Technical Field]

[0001] Related applications This application claims the benefit of priority under U.S. Provisional Patent Application No. 63 / 121,080, filed on 3 December 2020, and U.S. Provisional Patent Application No. 63 / 257,402, filed on 19 October 2021, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] background T cells have established themselves as central effectors in cancer immunotherapy, particularly due to their abundance, killing effect, and proliferative capacity. T cell engagers are bispecific molecules with one end being a constant component of the T cell / CD3 complex and the other end targeting tumor-associated antigens (TAAs). This structure and specificity allows bispecific T cell engagers to physically link T cells to tumor cells, ultimately stimulating T cell activation and subsequent tumor killing. (Huehls AM et al. (2015) Immunol Cell Biol 93(3):290-296; Ellerman D (2019) Methods 154:102-117) Therefore, bispecific molecules that utilize T cells to redirect them to tumor cells are promising therapeutic agents. Over the past 30 years, countless T bispecific antibodies have been developed. To date, only one T bispecific antibody, blinatumomab, has been approved for clinical use in humans, compared to approximately 30 other IgG-based antibody drugs for cancer treatment. This delay is mainly due to the difficulties in protein engineering during the production of these antibodies and the uncertainty regarding the clinical toxicity of these novel constructs (Wu and Cheung (2018) Pharmacol.Ther. 182:161-175).

[0003] Aptamers are single-stranded oligonucleotides that can specifically bind to a variety of targets, including proteins, sugars, and small organic compounds. There is growing interest in using aptamers for the development of both therapeutic and diagnostic agents.

[0004] Aptamers, like antibodies, recognize and bind to their target, but they have many advantages, including shorter production time, lower manufacturing costs, lower batch variability, higher modifiability, better thermal stability, and lower immunogenicity (Zhang, Lai, and Juhas (2019) Molecules 24:pii:E941.doi:10.3390 / molecules24050941).

[0005] Therefore, aptamers that can target T cells have great potential for use as anti-cancer drugs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Huehls AM et al. (2015) Immunol Cell Biol 93(3):290-296 [Non-Patent Document 2] Ellerman D(2019)Methods 154:102-117 [Non-Patent Document 3] Wu and Cheung(2018)Pharmacol.Ther.182:161-175 [Non-Patent Document 4] Zhang, Lai, and Juhas (2019)Molecules 24:pii:E941.doi:10.3390 / molecules24050941 [Overview of the project] [Means for solving the problem]

[0007] Abstract In certain embodiments, aptamers that bind to T cells, and / or aptamers that induce T cell stimulation and / or T cell-mediated cytotoxicity are provided herein. In some embodiments, pharmaceutical compositions comprising such aptamers, methods for treating cancer and / or killing cancer cells and / or stimulating T lymphocytes using such aptamers, and methods for producing such aptamers are provided herein.

[0008] In certain embodiments, aptamers are provided herein that include nucleic acid sequences that are 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 to 21. In certain embodiments, the aptamer includes 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, or at least 50) consecutive nucleotides of any one of SEQ ID NOs: 1 to 21. In certain embodiments, the aptamer comprises at least 40 consecutive nucleotides (e.g., 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, at least 54, 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) of any one of SEQ ID NOs: 1 to 21. In some embodiments, the aptamer comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21). In some embodiments, the aptamer provided herein has a sequence that is essentially comprised of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, or 21). In certain embodiments, the aptamer provided herein has a sequence that is comprised of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, or 21).

[0009] In a particular embodiment, the aptamer provided herein is 100 nucleotides or less in length (for example, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 73 nucleotides or less, 70 nucleotides or less, 63 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 59 nucleotides or less, 58 nucleotides or less, 57 nucleotides or less, 56 nucleotides or less, 55 nucleotides or less, 54 nucleotides or less, 53 nucleotides or less, 52 nucleotides or less, 51 nucleotides or less, or 50 nucleotides or less).

[0010] In some embodiments, the aptamers provided herein can bind to T cells. In some embodiments, the aptamers provided herein bind to the T cell surface protein CD3 (e.g., CD3ε chain, CD3e). In some embodiments, the aptamers can induce T cell-mediated cytotoxicity. In some embodiments, the aptamers can induce cell death of cancer cells (e.g., human cancer cells) via T cell-mediated cytotoxicity. In some embodiments, the cancer cells are patient-derived cancer cells. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are lung cancer cells. In some embodiments, the aptamers induce cell death of cancer cells in vitro. In certain embodiments, the aptamers induce cell death of cancer cells in vivo (e.g., in human and / or animal models). In some embodiments, the aptamers can induce T cell stimulation. In some embodiments, the aptamers can induce T cell proliferation.

[0011] In some embodiments, the aptamers provided herein include one or more chemical modifications. In some embodiments, the aptamers are chemically modified with polyethylene glycol (PEG) (e.g., bonded to the 5' end of the aptamer). In some embodiments, the aptamers include a 5' end cap. In certain embodiments, the aptamers include a 3' end cap (e.g., inverted thymidine, biotin). In some 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, or 54) 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions). In certain embodiments, the aptamer comprises locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'-deoxy-2'fluoro-D-arabinonucleotide (2'-FANA) sugars in its backbone. 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, or 54) methylphosphonate internucleotide bonds and / or phosphorothioate (PS) internucleotide bonds. In certain embodiments, the aptamer contains 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 nucleotide interlinks.In certain embodiments, the aptamer is modified with cholesterol or dialkyl lipids (e.g., at their 5' ends). In some embodiments, the aptamer contains one or more modified bases.

[0012] In certain embodiments, the aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or enantiomerized L-DNA aptamers). In some embodiments, the aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or enantiomerized L-RNA aptamers). In some embodiments, the aptamers comprise a mixture of DNA and RNA.

[0013] In certain embodiments, aptamer conjugates are provided herein, comprising an aptamer provided herein linked to a cancer cell binding moiety (e.g., a small molecule, another aptamer, polypeptide, nucleic acid, protein, and / or antibody). In some embodiments, the aptamer is covalently bound to the cancer cell binding moiety. In some embodiments, the aptamer is acovalently bound to the cancer cell binding moiety. In some embodiments, the aptamer is directly linked to the cancer cell binding moiety. In some embodiments, the aptamer is linked to the cancer cell binding moiety via a linker. In some embodiments, the cancer cell binding moiety binds to an antigen expressed on cancer cells. In some embodiments, the antigens expressed on cancer cells are selected from prostate-specific antigen (PSA), prostatic 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, melan A, B-catenin, CDC27, HSP70-2-m, HLA-A2-R17OJ, AFP, EBV-EBNA, HPV16-E7, MUC-1, HER-2 / neu, mammoglobin A, or MHC-TAA peptide complexes. In some embodiments, the cancer cell binding moiety induces cell death (e.g., apoptosis) upon contact with cancer cells (e.g., human cancer cells). In some embodiments, the cancer cells are patient-derived cancer cells. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are breast cancer cells. In some embodiments, the cancer cell binding moiety induces cell death when it comes into contact with cancer cells in vitro. In certain embodiments, the cancer cell binding moiety induces cell death when it comes into contact with cancer cells in vivo (e.g., in human and / or animal models).

[0014] In certain embodiments, pharmaceutical compositions are provided herein that include an aptamer (e.g., a therapeutically effective amount of an aptamer) provided herein. In certain embodiments, pharmaceutical compositions are provided herein that include an aptamer conjugate (e.g., a therapeutically effective amount of an aptamer conjugate) provided herein. In some embodiments, the pharmaceutical compositions provided herein further include a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions provided herein are formulated for parenteral administration.

[0015] In certain embodiments, the pharmaceutical compositions provided herein are for use in treating cancer. In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer.

[0016] In certain embodiments, methods are provided herein for treating cancer in a subject, the method comprising administering to the subject an aptamer (e.g., a therapeutically effective amount of an aptamer) or a pharmaceutical composition provided herein. In certain embodiments, methods are provided herein for treating cancer in a subject, the method comprising administering to the subject an aptamer conjugate (e.g., a therapeutically effective amount of an aptamer conjugate) or a pharmaceutical composition provided herein. In some embodiments, the administration is parenteral administration (e.g., subcutaneous administration). The administration may be intratumoral injection, subcutaneous injection, or intravesical instillation.

[0017] In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, basal cell carcinoma, bladder cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, or Merkel cell carcinoma. In certain embodiments, the subject is a subject who has received chemotherapy. In certain embodiments, the subject is a subject who has had a tumor surgically removed (e.g., a breast cancer tumor surgically resected).

[0018] In some embodiments, the treatment methods provided herein further comprise administering an additional cancer treatment to a subject. In some embodiments, the additional cancer treatment comprises chemotherapy. In certain embodiments, the additional cancer treatment comprises radiation therapy. In some embodiments, the additional cancer treatment comprises surgical removal of the tumor. In certain embodiments, the additional cancer treatment comprises administration of an immune checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA4 antibody, an anti-TIGIT antibody, an anti-PVR antibody or an anti-nectin antibody, to the subject.

[0019] In certain aspects, a method of killing cancer cells is provided herein, the method comprising contacting the cancer cells with an aptamer or aptamer conjugate provided herein. In some embodiments, the cancer cells die by apoptosis. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are breast cancer cells. In some embodiments, the cancer cells die upon contact with the cancer cells in vitro. In certain embodiments, the cancer cells die upon contact with the cancer cells in vivo (e.g., in a human and / or animal model).

[0020] In certain embodiments, methods for producing aptamers are provided herein. In some embodiments, the method includes synthesizing (e.g., chemically synthesizing) a nucleic acid having a 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 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21). In certain embodiments, the method includes synthesizing a nucleic acid having a sequence having 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, or at least 50) consecutive nucleotides of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21). In certain embodiments, the aptamer comprises at least 40 (e.g., 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, at least 54, 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, at least 70, at least 71, at least 72, or at least 73) consecutive nucleotides of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21). In certain embodiments, the method comprises synthesizing a nucleic acid comprising the sequence of any one of SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21). In some embodiments, the method comprises synthesizing a nucleic acid having a sequence essentially derived from SEQ ID NOs: 1 to 21 (e.g., any one of SEQ ID NOs: 1, 20, and 21).In a particular embodiment, this method involves synthesizing a nucleic acid having a sequence consisting of sequence numbers 1 to 21 (for example, any one of sequence numbers 1, 20, and 21). [Brief explanation of the drawing]

[0021] [Figure 1] Figures 1A and 1B show the scheme of the combined SELEX method. [Figure 2A] Figures 2A and 2B show the SELEX binding comparative assay. The binding assay was performed using the target protein CD3ε bead complex (black) or the control protein IgGl (gray) with the initial random library (Rnd Lib) and library enrichment pools from rounds 3 (R3), 6 (R6), 9 (R9), and 11 (R11). After incubation and washing, library DNA was eluted and the concentration in the supernatant was assessed by real-time PCR. The standard curve was performed using the random library (top). (B) Binding of the Cy5 fluorescently labeled library to Jarcutt T cell lines and Pan B cells was demonstrated by flow cytometry. Dot plots and histogram graphs are shown. Flow data quantification of median Cy5 fluorescence intensity (MFI). [Figure 2B] Figures 2A and 2B show the SELEX binding comparative assay. The binding assay was performed using the target protein CD3ε bead complex (black) or the control protein IgGl (gray) with the initial random library (Rnd Lib) and library enrichment pools from rounds 3 (R3), 6 (R6), 9 (R9), and 11 (R11). After incubation and washing, library DNA was eluted and the concentration in the supernatant was assessed by real-time PCR. The standard curve was performed using the random library (top). (B) Binding of the Cy5 fluorescently labeled library to Jarcutt T cell lines and Pan B cells was demonstrated by flow cytometry. Dot plots and histogram graphs are shown. Flow data quantification of median Cy5 fluorescence intensity (MFI). [Figure 3A-3B]Figures 3A–3C show the results of next-generation sequencing (NGS) analysis. Figure 3A is a dot plot showing the analysis of single aptamer sequences from enriched libraries from the 8th, 9th, 10th, and 11th SELEX rounds, as defined in Example 2. The X-axis represents mean P-negativity, and the Y-axis represents mean P-positivity. The diagonal line represents the threshold between specific binding aptamers and non-specific aptamer sequences with low binding affinity. The top five candidates selected for further investigation are shown with their names. Figure 3B shows a sequence LOGO display of the shared motif (using GLAM2 software) of the top 14 specific binding aptamers (top) and the top 4 selected aptamers (bottom). Figure 3B discloses sequence numbers 59–68, 59, and 69–74, respectively, in order of appearance. Figure 3C shows the secondary structure analysis (mfold) of the five selected candidates. The position of the motif nucleotide is indicated by a red asterisk. Figure 3C discloses sequence numbers 1, 2, 3, 5, and 4 in order of appearance, respectively. [Figure 3C] Figures 3A–3C show the results of next-generation sequencing (NGS) analysis. Figure 3A is a dot plot showing the analysis of single aptamer sequences from enriched libraries from the 8th, 9th, 10th, and 11th SELEX rounds, as defined in Example 2. The X-axis represents mean P-negativity, and the Y-axis represents mean P-positivity. The diagonal line represents the threshold between specific binding aptamers and non-specific aptamer sequences with low binding affinity. The top five candidates selected for further investigation are shown with their names. Figure 3B shows a sequence LOGO display of the shared motif (using GLAM2 software) of the top 14 specific binding aptamers (top) and the top 4 selected aptamers (bottom). Figure 3B discloses sequence numbers 59–68, 59, and 69–74, respectively, in order of appearance. Figure 3C shows the secondary structure analysis (mfold) of the five selected candidates. The position of the motif nucleotide is indicated by a red asterisk. Figure 3C discloses sequence numbers 1, 2, 3, 5, and 4 in order of appearance, respectively. [Figure 4]Figure 4 shows aptamer sequences that bind to the target protein as determined by HPLC. Folded and Cy5-labeled aptamer candidates were assayed for recombinant human CD3ε (hCD3ε) binding. Aptamers were incubated with hCD3e or negative control IIgG1 at 37°C for 1 hour. PolyT was used as the negative control sequence. [Figure 5A] Figures 5A-5C show CS6 binding to T cells as demonstrated by flow cytometry. Jarcut cells and Kami-1 cells were incubated with CpG'-Cy5 labeled CS6, CS7, and CS8c and analyzed by flow cytometry (Figure 5A). Jarcut 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 5B). Isolated pan T cells and pan B cells were incubated with CpG'-Cy5 labeled CS6 and analyzed by flow cytometry. Dot plots of T cells and B cells by Cy5 (X axis) / SSC (Y axis) are shown, along with MFI quantification (Figure 5C). [Figure 5B] Figures 5A-5C show CS6 binding to T cells as demonstrated by flow cytometry. Jarcut cells and Kami-1 cells were incubated with CpG'-Cy5 labeled CS6, CS7, and CS8c and analyzed by flow cytometry (Figure 5A). Jarcut 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 5B). Isolated pan T cells and pan B cells were incubated with CpG'-Cy5 labeled CS6 and analyzed by flow cytometry. Dot plots of T cells and B cells by Cy5 (X axis) / SSC (Y axis) are shown, along with MFI quantification (Figure 5C). [Figure 5C]Figures 5A-5C show CS6 binding to T cells as demonstrated by flow cytometry. Jarcut cells and Kami-1 cells were incubated with CpG'-Cy5 labeled CS6, CS7, and CS8c and analyzed by flow cytometry (Figure 5A). Jarcut 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 5B). Isolated pan T cells and pan B cells were incubated with CpG'-Cy5 labeled CS6 and analyzed by flow cytometry. Dot plots of T cells and B cells by Cy5 (X axis) / SSC (Y axis) are shown, along with MFI quantification (Figure 5C). [Figure 6] Figure 6 shows the effective concentrations of CS6. Jarcut cells were incubated with serially diluted concentrations of CpG'-Cy5-labeled CS6, and the EC50 of the compounds was determined by flow cytometry. [Figure 7] Figure 7 provides a schematic diagram of an exemplary use of a T cell engager aptamer as an aptamer conjugate. In this example, a T cell-binding aptamer is ligated with a second aptamer that targets cancer to obtain a bispecific aptamer entity. Three distinct domains of the therapeutic agent are shown. [Figures 8A-8B] Figures 8A and 8B demonstrate the in vivo efficacy of an exemplary bispecific T cell engager aptamer consisting of a CS6 aptamer hybridized to HCT116 (SEQ ID NO: 21) and a colon cancer cell line-targeted aptamer sequence (named VS12, SEQ ID NO: 22). Female NSG mice were subcutaneously transplanted with HCT-116 tumor cells mixed with human PBMCs, followed by subcutaneous administration of a total of 10 doses of the T cell engager bispecific individualized aptamer. Tumor volume of HCT116 was monitored for the CS6-VS12 treatment group, the poly-T-poly-Tq (non-specific p-DNA aptamer) group, and the vehicle mouse group (A). Individual mouse growth curves are shown in Figure 8B. *** indicates a statistically significant difference (p ≤ 0.001). [Figure 9]Figure 9 shows the Kaplan-Meier survival analysis of treated mice. ** indicates a statistically significant difference (p ≤ 0.01). [Figure 10] Figure 10 shows the three modes of action (MoA) and downstream systemic effects (Panel D) of intratumoral-administered bispecific individualized aptamers (Panels A-C) in solid tumors. [Figure 11] Figure 11 shows the key steps in the individualization process for each patient. [Figure 12] Figure 12 is a schematic diagram of a study design to identify customized variable chains and test bispecific aptamers in two established tumor mouse models. [Figure 13] Figure 13 shows that intratumoral administration of bispecific individualized aptamers significantly attenuates tumor growth. Panel (A) shows the A549 tumor volume of mice treated with CS6-VS20, CS6-VS45, and vehicle for 14 days post-administration. Panel (B) shows the 4T1 tumor volume of mice treated with CS6-VS32 and vehicle for 14 days post-administration. Two-way ANOVA was performed for statistical analysis. * indicates a statistically significant difference (p ≤ 0.05), and *** indicates a statistically significant difference (p ≤ 0.001). [Figure 14] Figure 14 shows the systemic effects of administration of the bispecific individualized aptamer CS6-VS32. In panel (A), lungs were collected at the end of the experiment, cells were plated with 6-thioguanine, and colonies were counted 12–14 days later. In panel (B), serum was collected on the final day and analyzed using a mouse IL-6 ELISA assay. Each circle represents a mouse in the test group (left). Spleens were collected and weighed at the end of the experiment. The bars represent the mean ± SEM (right). [Modes for carrying out the invention]

[0022] Detailed explanation general In certain embodiments, aptamers that selectively bind to T cells, and / or aptamers that selectively induce T cell stimulation and / or T cell-mediated cytotoxicity are provided herein. In some embodiments, pharmaceutical compositions comprising such aptamers, methods for treating cancer and / or killing cancer cells using such aptamers, and methods for producing such aptamers are provided herein.

[0023] definition For convenience, the specific terms used in this specification, the examples, and the appended claims are set forth herein.

[0024] The articles "a" and "an" are used herein to refer to one or more (e.g., at least one) grammatical objects of the articles. For example, "an (an) element" means one element or more elements.

[0025] The terms “oligonucleotide” and “nucleic acid molecule” refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, one or more 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 analogues. If present, modifications to the nucleotide structure may be given before or after the construction of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified, such as by conjugation with labeling components.

[0026] As used herein, the term “aptamer” refers to a short (e.g., less than 200 nucleotides) single-stranded nucleic acid molecule (ssDNA oligonucleotide and / or ssRNA) that can specifically bind to a target molecule, such as a protein or peptide, or to a topographic feature of a target cell.

[0027] The terms “bonding” or “interaction” refer to a stable association between two molecules, for example, an aptamer and a target, due to interactions such as electrostatic interactions, hydrophobic interactions, ionic interactions, π-stacking interactions, coordination interactions, van der Waals interactions, covalent interactions, and / or hydrogen bonding interactions under physiological conditions.

[0028] As used herein, when two nucleic acid sequences form base pairs with each other at each position, they are relative to each other. to " Complementary " mosquito or they are "complementary" to each other.

[0029] The terms “regulation” or “to regulate,” when used in relation to functional properties or biological activity or processes (e.g., enzyme activity or receptor binding), refer to the ability to upregulate (e.g., activate or stimulate), downregulate (e.g., inhibit or suppress), or otherwise alter the quality of such properties, activity, or processes. In certain specific examples, such regulation may be conditional on the occurrence of a particular event, such as activation of a signaling pathway, and / or may only be expressed in certain cell types.

[0030] As used herein, “specific binding” refers to the ability of an aptamer to bind to a given target. Typically, an aptamer binds to approximately 10 targets. -7 M or less, about 10 -8 M or less, or about 10 -9 M or less K D The K molecule binds specifically to its target with an affinity corresponding to that affinity, and its affinity is significantly lower (e.g., less than 2 times, less than 5 times, less than 10 times, less than 50 times, less than 100 times, less than 500 times, less than 500 times, less than 1000 times, or less than 1000 times) than its affinity to nonspecific and unrelated targets (e.g., IgG1 globulin, BSA, casein, or unrelated cells, e.g., HEK293 cells or E. coli cells) or to different aptamer sequences to congeneral targets. D It then binds to the target.

[0031] Aptamer In certain embodiments, aptamers that bind to T cells, and / or induce T cell stimulation and / or T cell-mediated cytotoxicity are provided herein. In some embodiments, pharmaceutical compositions comprising such aptamers, methods for treating cancer and / or killing cancer cells using such aptamers, and methods for producing such aptamers are provided herein.

[0032] In certain embodiments, aptamers are provided herein that include nucleic acid sequences that are 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 to 21. In certain embodiments, the aptamer includes 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) consecutive nucleotides of any one of SEQ ID NOs: 1 to 21. In certain embodiments, the aptamer comprises at least 40 consecutive nucleotides (e.g., 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, at least 54, 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, at least 70, at least 71, at least 72, or at least 73) of any one of SEQ ID NOs: 1 to 21. In some embodiments, the aptamer comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 to 21. In some embodiments, the aptamer provided herein has a sequence that is essentially derived from any one of SEQ ID NOs: 1 to 21. In certain embodiments, the aptamer provided herein has a sequence that is derived from any one of SEQ ID NOs: 1 to 21.

[0033] In the context of two or more nucleic acids, the terms “identical” or “percent identity” refer to two or more sequences or subsequences that are identical or have the same percentage of identical nucleotides (i.e., approximately 60% identity in a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) when compared and aligned to obtain the greatest match in a comparison window or specified region using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, for example, the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / , etc.).

[0034] In a particular embodiment, the aptamer is 100 nucleotides or less in length (for example, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 59 nucleotides or less, 58 nucleotides or less, 57 nucleotides or less, 56 nucleotides or less, 55 nucleotides or less, 54 nucleotides or less, 53 nucleotides or less, 52 nucleotides or less, 51 nucleotides or less, or 50 nucleotides or less).

[0035] In some embodiments, the aptamers provided herein can bind to T cells. In some embodiments, the aptamers provided herein bind to clusters of differentiated 3 (CD3) (e.g., CD3ε chains). In some embodiments, the aptamers can induce T cell stimulation. In some embodiments, the aptamers can induce T cell-mediated cytotoxicity. In some embodiments, the aptamers can induce cell death of cancer cells (e.g., human cancer cells) via T cell-mediated cytotoxicity. In some embodiments, the cancer cells are patient-derived cancer cells. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are lung cancer cells. In some embodiments, the aptamers induce cell death of cancer cells in vitro. In certain embodiments, the aptamers induce cell death of cancer cells in vivo (e.g., in human and / or animal models).

[0036] In some embodiments, the aptamers provided herein include one or more chemical modifications. Exemplary modifications are shown in Table 1.

[0037] [Table 1]

[0038] In certain embodiments, the aptamer includes terminal modifications. In some embodiments, the aptamer is chemically modified with polyethylene glycol (PEG) (e.g., 0.5-40 kDa) (e.g., bonded to the 5' end of the aptamer). In some embodiments, the aptamer includes a 5' end cap (e.g., inverted thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS). In certain embodiments, the aptamer includes a 3' end cap (e.g., inverted thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS).

[0039] In certain embodiments, the aptamers provided herein contain one or more modified sugars (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). In some embodiments, the aptamers contain one or more 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions). In certain embodiments, the aptamer comprises locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'-deoxy-2'fluoro-D-arabinonucleotide (2'-FANA) sugars in its backbone.

[0040] 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, or 54) methylphosphonate internucleotide bonds and / or phosphorothioate (PS) internucleotide bonds (e.g., at its 5' end). In certain embodiments, the aptamer contains 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 nucleotide interbondings. In certain embodiments, the aptamer is modified with cholesterol or dialkyl lipids.

[0041] In some embodiments, the aptamer comprises one or more modified bases (e.g., BzdU, naphthyl, tryptamino, isobutyl, 5-methylcytosine, alkyne (dibenzocyclooctin), azide, maleimide).

[0042] In certain embodiments, the aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or enantiomerized L-DNA aptamers). In some embodiments, the aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or enantiomerized L-RNA aptamers). In some embodiments, the aptamers comprise a mixture of DNA and RNA.

[0043] Aptamers can be synthesized by methods well known to those skilled in the art. For example, aptamers can be synthesized chemically, for example, on a solid support. Solid-phase synthesis can utilize phosphoramidite chemistry. Briefly, the synthesis cycle begins with the removal of the acid-unstable 5'-dimethoxytrityl protecting group (DMT, "trityl") from the hydroxyl functional group of the terminal support-bound nucleoside by UV-controlled treatment with an organic acid. The exposed, highly reactive hydroxyl group is then available to react with the next protected nucleoside phosphoramidite component in the coupling step, forming a phosphite triester skeleton. Next, the acid-unstable phosphite triester skeleton is oxidized to a stable pentavalent phosphate triester. If phosphorothioate modification is desired at a specific skeleton position, the acid-unstable phosphite triester skeleton is sulfurized at this stage instead of the oxidation process to produce a P=S bond instead of a P=O bond. Next, all unreacted 5'-hydroxyl groups are acetylated ("capped") to block these sites during the following coupling step and to avoid internal mismatch sequences. After the capping step, the cycle is restarted by removing the DMT protecting groups and sequentially coupling the next bases according to the desired sequence. Finally, the oligonucleotide is cleaved from the solid support, removing all protecting groups from the backbone and bases.

[0044] Aptamer Conjugate In certain embodiments, aptamer conjugates are provided herein, comprising an aptamer provided herein linked to a cancer cell binding moiety. The cancer cell binding moiety may be, for example, an aptamer, a small molecule, a polypeptide, a nucleic acid, a protein, or an antibody. In some embodiments, the aptamer is covalently bound to the cancer cell binding moiety. In some embodiments, the aptamer is non-covalently bound to the cancer cell binding moiety. In some embodiments, the aptamer is directly linked to the cancer cell binding moiety. In some embodiments, the aptamer is linked to the cancer cell binding moiety via a linker.

[0045] In some embodiments, the cancer cell binding moiety binds to an antigen expressed on cancer cells. In some embodiments, the cancer cell binding moiety binds to a cancer antigen selected from prostate-specific antigen (PSA), prostatic 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, melan A, B-catenin, CDC27, HSP70-2-m, HLA-A2-R17OJ, AFP, EBV-EBNA, HPV16-E7, MUC-1, HER-2 / neu, mammoglobin A, or an MHC-TAA peptide complex.

[0046] In some embodiments, the cancer cell binding moiety induces cell death (e.g., apoptosis) upon contact with cancer cells (e.g., human cancer cells). In some embodiments, the cancer cells are patient-derived cancer cells. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are breast cancer cells. In some embodiments, the cancer cell binding moiety induces cell death upon contact with cancer cells in vitro. In certain embodiments, the cancer cell binding moiety induces cell death upon contact with cancer cells in vivo (e.g., in human and / or animal models).

[0047] Bispecific personalized aptamers In certain embodiments, a bispecific individualized aptamer is provided herein comprising (a) a cancer cell binding chain that specifically binds to an antigen expressed on cancer cells; (b) a CpG motif; and (c) a CD3 binding chain (e.g., a CD3 binding aptamer disclosed herein), wherein the cancer cell binding chain is linked to the CD3 binding chain by a CpG motif.

[0048] In some embodiments, cancer cell binding chains can induce cell death (e.g., apoptosis) in cancer cells (e.g., human cancer cells) upon contact. In some embodiments, the cancer cells are patient-derived cancer cells. In some embodiments, the cancer cells are solid tumor cells (e.g., breast cancer cells). In certain embodiments, the cancer cells are cancer cells (e.g., colorectal cancer cells). In some embodiments, the aptamer induces cell death upon contact with cancer cells in vitro. In certain embodiments, the aptamer induces cell death upon contact with cancer cells in vivo (e.g., in human and / or animal models). In some embodiments, the cancer cell binding chain binds to a cancer antigen selected from prostatic membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), tyrosinase, gp100, MART-l / Melan A, HSP70-2-m, HLA-A2-R17OJ, HPV16-E7, MUC-1, HER-2 / neu, mammoglobin A, or an MHC-TAA peptide complex.

[0049] In a particular embodiment, the cancer cell binding chain is, 28 ~ 51 It includes a nucleic acid sequence that is at least 60% identical to any one of the following (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). In some embodiments, the cancer cell binding chain is the sequence number. 28 ~ 51 It contains any one of the following nucleic acid sequences. In a particular embodiment, the cancer cell binding chain is the sequence number 28 ~ 47It contains at least 30 consecutive nucleotides (for example, 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) of any one of them. In some embodiments, the cancer cell binding chain is the ALCE 28 ~ 51 It has an essential sequence consisting of the following. In a particular embodiment, the cancer cell binding chain is the sequence number. 28 ~ 51 It has an array consisting of [this].

[0050] [Table 2-1] [Table 2-2]

[0051] In the context of two or more nucleic acids, the terms “identical” or “percent identity” refer to two or more sequences or subsequences that are identical or have the same nucleotides at a specified percentage (i.e., approximately 60% identity in a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) when compared and aligned to obtain the greatest match in a specified region using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, for example, the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / , etc.).

[0052] In certain embodiments, the cancer cell binding chain is 120 nucleotides or less in length (for example, 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 chain is approximately 63 nucleotides in length.

[0053] In some embodiments, the CD3-binding chain includes a nucleic acid sequence that is at least 60% identical to any one of sequence numbers 1 to 21 (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). In some embodiments, the CD3-binding chain includes a nucleic acid sequence that is at least 60% identical to any one of sequence numbers 1 to 21.

[0054] In certain embodiments, the CD3-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) consecutive nucleotides from any one of SEQ ID NOs: 1 to 21. In some embodiments, the CD3-binding chain provided herein has a sequence that is essentially comprised of SEQ ID NOs: 1 to 21. In certain embodiments, the CD3-binding chain provided herein has a sequence that is comprised of SEQ ID NOs: 1 to 21. In certain embodiments, the CD3 bond 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 a particular embodiment, the CD3-binding chain is approximately 73 nucleotides long.

[0055] The cancer cell binding chain and the CD3 binding chain can be linked to each other by hybridization of the 5' sequence of the cancer cell binding chain and the 5' sequence of the CD3 binding chain. In certain embodiments, the 5' sequence of the cancer cell binding chain hybridizes to the 5' sequence of the CD3 binding chain to form a CpG-rich motif, a TLR9 agonist sequence. The cancer cell binding chain and the CD3 binding chain can also be linked to each other by direct ligation of the two ends (e.g., the 5' ends) of the double-stranded sequence. In certain embodiments, the double-stranded sequence is a CpG motif, which is a TLR9 agonist sequence.

[0056] In some embodiments, the TLR9 agonist sequence includes a double-stranded region containing at least one CpG motif nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). 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 antitumor response. In some embodiments, the TLR9 agonist sequence induces cytokine production.

[0057] In some embodiments, the CPG motif sequence is the sequence number. 52 ~ 55 A double-stranded nucleic acid sequence containing a sequence that is at least 60% identical to any one of the following (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). In some embodiments, the CPG motif sequence is the sequence number. 52 ~ 55 It is a double-stranded nucleic acid sequence containing one of the following sequences.

[0058] In a particular embodiment, the CpG motif sequence is the sequence number 52 ~ 55 The CpG motif sequence provided herein 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. In some embodiments, the CpG motif sequence provided herein is the sequence number 52 ~ 55 It has an array that is essentially composed of. In certain embodiments, the CpG motif array provided herein is an array sequence 52 ~ 55 It has an array consisting of [this].

[0059] In a particular embodiment, the CpG motif sequence is 35 nucleotides or less in length (for example, 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).

[0060] The bispecific individualized aptamers provided herein may include any combination of the cancer cell binding chain and CD3 binding chain described herein.

[0061] In some embodiments, the bispecific individualized aptamers provided herein include one or more chemical modifications. Exemplary modifications are shown in Table 3.

[0062] [Table 3]

[0063] In certain embodiments, the bispecific individualized aptamer includes terminal modifications. In some embodiments, the bispecific individualized aptamer is chemically modified with polyethylene glycol (PEG) (e.g., 0.5-40 kDa) (e.g., bonded to the 5' end of the aptamer). In some embodiments, the bispecific individualized aptamer includes 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 includes a 3' end cap (e.g., inverted thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS).

[0064] In certain embodiments, the bispecific individualized aptamers provided herein include one or more modified sugars (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). In some embodiments, the bispecific individualized aptamers include one or more 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions). In certain embodiments, the bispecific individualized aptamer contains locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'-deoxy-2'fluoro-D-arabinonucleotide (2'-FANA) sugars in its backbone.

[0065] 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) methylphosphonate internucleotide bonds and / or phosphorothioate (PS) internucleotide bonds.

[0066] In certain embodiments, the bispecific individualized aptamer may contain PS modifications within its double-stranded region (e.g., a CpG motif sequence). For example, the double-stranded region of a bispecific individualized aptamer (e.g., a CpG motif sequence) may contain 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 bonds on one or both strands. In some embodiments, the double-stranded region of a bispecific individualized aptamer (e.g., a CpG motif sequence) may contain partial PS modifications. In certain embodiments, five nucleotides from the 5' end of the double-stranded CpG motif sequence are modified. In other embodiments, five nucleotides from the 5' and 3' ends of the double-stranded CpG motif sequence are modified. In certain embodiments, the double-stranded CpG motif sequence contains complete PS modifications.

[0067] In certain embodiments, the aptamer contains 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 nucleotide interbondings. In certain embodiments, the aptamer is modified with cholesterol or dialkyl lipids (e.g., at their 5' ends).

[0068] In some embodiments, the aptamer comprises one or more modified bases (e.g., BzdU, naphthyl, tryptamino, isobutyl, 5-methylcytosine, alkyne (dibenzocyclooctin), azide, maleimide).

[0069] In certain embodiments, the aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or enantiomerized L-DNA aptamers). In some embodiments, the aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or enantiomerized L-RNA aptamers). In some embodiments, the aptamers comprise a mixture of DNA and RNA.

[0070] Pharmaceutical composition In certain embodiments, pharmaceutical compositions comprising an aptamer provided herein (e.g., a therapeutically effective amount of an aptamer) are provided herein. In certain embodiments, pharmaceutical compositions comprising an aptamer conjugate provided herein (e.g., a therapeutically effective amount of an aptamer conjugate) are provided herein. In some embodiments, the pharmaceutical compositions provided herein further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions provided herein are formulated for parenteral administration (e.g., subcutaneous administration). Administration may be intratumoral injection, subcutaneous injection, or intravesical infusion.

[0071] In certain embodiments, the pharmaceutical composition is for use in the treatment of cancer. In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a carcinoma (e.g., colorectal cancer). In some embodiments, the cancer is lung cancer.

[0072] "Pharmacologically acceptable carriers" means substances that can be included in the compositions described herein without causing serious adverse toxic effects to the patient, and which assist in the administration and absorption of active agents to a subject. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline, phosphate buffer solution, MgCl2, KCl, CaCl2, Ringer's lactate solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylase or starch, fatty acid esters, lipids, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants. Such preparations may be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, and / or aromatics that do not react adversely with the compositions described herein. Those skilled in the art will recognize that other pharmaceutical excipients may be useful.

[0073] Treatment method In some embodiments, methods for treating cancer are provided herein, comprising administering a pharmaceutical composition comprising one or more aptamers provided herein.

[0074] In some embodiments, methods for treating cancer are provided herein, comprising administering a pharmaceutical composition comprising one or more aptamer conjugates provided herein.

[0075] In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. Accordingly, in certain embodiments, methods for delivering the aptamers, aptamer conjugates, and / or pharmaceutical compositions described herein are provided herein.

[0076] In certain embodiments, the pharmaceutical compositions, aptamers, and aptamer conjugates described herein may be administered in conjunction with any other conventional anti-cancer treatment, such as radiotherapy and surgical resection of tumors. These treatments may be applied as needed and / or as indicated, and may be performed before, concurrently with, or after administration of the pharmaceutical compositions, aptamers, aptamer conjugates, dosage forms, and kits described herein.

[0077] In certain embodiments, the method involves administering multiple doses of an aptamer or aptamer conjugate. Separate doses may include any number of two or more doses (e.g., doses), including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21, 22, 23, 24, or 25 doses. Some embodiments include at least 8, 9, 10, 11, 12, 13, 14, or 15 doses. Those skilled in the art can readily determine the number of doses to be administered, or the desirability of administering one or more additional doses, according to methods known in the art for monitoring a therapeutic method and other monitoring methods provided herein. Accordingly, the methods provided herein include methods for providing one or more doses of the aptamers, aptamer conjugates, and / or pharmaceutical compositions described herein, the number of doses can be determined by monitoring the subject and, based on the results of the monitoring, deciding whether to provide one or more additional doses. The decision of whether to provide one or more additional doses may be based on a variety of monitoring results, including, but is not limited to, T cell stimulation, T cell cytotoxic activity, indication or suppression of tumor growth, appearance or suppression of new metastases, the subject's antitumor antibody titer, the subject's overall health status and / or the subject's weight.

[0078] The interval between doses may be any of a variety of periods. In some embodiments, doses may be spaced at intervals of 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 interval between doses may be a function of any of a variety of factors, including monitoring stages, as described in relation to the number of doses, the time until the subject initiates an immune response, and / or the time it takes for the subject to remove the aptamer or aptamer conjugate from normal tissue. In one example, this period may be a function of the time it takes for the subject to initiate an immune response; for example, this period may be longer than the time it takes for the subject to initiate an immune response, for example, more than about one week, more than about ten days, more than about two weeks, or more than about one month; in another example, this period may be shorter than the time it takes for the subject to initiate an immune response, for example, less than about one week, less than about ten days, less than about two weeks, or less than about one month. In yet another example, this period may be a function of the time it takes for the subject to remove the aptamer or aptamer conjugate from normal tissue; for example, this period may be longer than the time it takes for the subject to remove the aptamer or aptamer conjugate from normal tissue, for example, more than about one hour, more than about one day, more than about two days, more than about three days, more than about five days, or more than about one week. In another example, this period may be shorter than the time required for the subject to remove the aptamer or aptamer conjugate from the normal tissue, for example, less than about 1 hour, less than about 1 day, less than about 2 days, less than about 3 days, less than about 5 days, or less than about 1 week.

[0079] The dosage of aptamers or aptamer conjugates described herein is the amount of aptamer or aptamer conjugate effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, and is either the minimum toxicity to the patient or the maximum feasible dose. The effective dose level can be identified using the methods described herein and depends on various pharmacokinetic and pharmacodynamic factors, including the activity of the particular composition administered, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, symptoms, overall health status, and prior medical history of the patient being treated, as well as similar factors well known in the medical field. Generally, the effective dose of an anticancer drug is the amount of the drug that is the minimum effective dose to produce a therapeutic effect. Such an effective dose generally depends on the factors described above.

[0080] Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal), or injection. Injection includes intravenous (IV), intratumoral (IT), intralesional, peritumoral, intramuscular (IM), and subcutaneous (SC) administration. The compositions described herein are not limited to oral, parenteral, enteral, intravenous, intratumoral, intraperitoneal, topical, percutaneous (e.g., using any standard patch), intradermal, ocular, nasal (intranasal), topical, parenteral, e.g., aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, tongue, (trans)buccal, (trans)urethral, ​​vaginal (e.g., transvaginal and perival), implanted, intrabladder, intrapulmonary, intraduodenal, intragastric, and bronchial administration. It can be administered in any form by any effective route, including intravenously. In some embodiments, the aptamers or aptamer conjugates described herein are administered orally, rectally, topically, intravesically, by injection into or near the draining lymph nodes, intravenously, by inhalation or aerosol, or subcutaneously. In some embodiments, administration is parenteral (e.g., subcutaneous). Administration may be intratumoral or peritumoral injection.

[0081] The administration plan may be of any of the various methods and amounts and may be determined by those skilled in the art according to known clinical factors. As is known in the medical field, the dosage for a single patient may depend on many factors, including the species, size, body surface area, age, sex, immune function, tumor size, general health status and specific biomarkers, the specific microorganism being administered, the duration and route of administration, the type and stage of the disease (e.g., tumor size), and other compounds such as drugs administered concurrently.

[0082] The treatment methods described herein may be suitable for the treatment of primary tumors, secondary tumors or metastases, as well as recurrent tumors or cancer. The dosage of the pharmaceutical compositions described herein may be set or adjusted as appropriate depending on the dosage form, route of administration, the severity or stage of the target disease, etc.

[0083] In some embodiments, the dose administered to a subject is sufficient to prevent cancer, delay its onset, slow or halt its progression, prevent cancer recurrence, reduce tumor burden, or contribute to the subject's disease-free survival, tumor cessation time, or overall survival. Those skilled in the art will recognize that the dose depends on the potency of the particular compound used, as well as various factors including the subject's age, species, symptoms, and weight. The size of the dose is also determined by the route, timing, and frequency of administration, as well as the presence, nature, and extent of any adverse side effects that may accompany the administration of the particular compound and the desired physiological effect.

[0084] Appropriate doses and administration plans can be determined by conventional dosing techniques known to those skilled in the art. Generally, treatment is initiated with a small dose less than the optimal dose of the compound. The dose is then gradually increased until the optimal effect is achieved under those circumstances. Effective dosages and treatment protocols can be determined by routine and conventional means, for example, by starting with a low dose in experimental animals and then increasing the dose while monitoring the effect, and similarly systematically changing the administration plan. Animal experiments are commonly used to determine the maximum tolerated dose ("MTD") of a bioactive agent per kilogram of body weight. Those skilled in the art regularly extrapolate doses for efficacy while avoiding toxicity in other species, including humans.

[0085] Accordingly, the dosage of the aptamers or aptamer conjugates provided herein for therapeutic use may vary depending on the specific aptamer or aptamer conjugate, the age, weight, and clinical condition of the recipient patient, as well as the experience and judgment of the clinician or practitioner administering the treatment, among other factors that influence the selected dosage. In general, the dose should be sufficient to slow, preferably regress, tumor growth and most preferably cause complete regression of the cancer.

[0086] Examples of cancers that can be treated by the methods described herein include, but are not limited to, hematological malignancies, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, and leukocythemic leukemia. Leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, leader cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, anaplastic cell leukemia, hairy cell leukemia, hematoblastic leukemia, hematocytic leukemia, histiocytic leukemia, hepatocellular leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia Leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, acinar cell carcinoma, acinar carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma (carcinoma) basocellulare), basaloid cell carcinoma, basal squamous cell carcinoma, bronchoalveolar epithelial carcinoma, bronchiolar carcinoma, bronchogenic lung cancer, cerebrumoid carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, armor-like carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, durum carcinoma durum), embryonic carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spherical cell carcinoma, spindle cell carcinoma, carcinoma spongiosum), squamous cell carcinoma, squamous cell carcinoma, string carcinoma Carcinoma, telangiectaticum (carcinoma telangiectaticum), telangiectodes (carcinoma telangiectodes), transitional cell carcinoma, nodular carcinoma (carcinomaTuberosum, tuberous carcinoma, verrucous carcinoma, choriocarcinoma (carcinoma villosum), giant cell carcinoma (carcinoma gigantocelle), adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, adrenal carcinoma (hypemephroid carcinoma), infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lens carcinoma (lenticular carcinoma, carcinoma lenticulare), lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, medullary carcinoma, black carcinoma, soft carcinoma, mucinous carcinoma, mucinous carcinoma (carcinoma mucosum, mucous carcinoma) Carcinoma, myxoma carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, squamous cell carcinoma, medullary carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fasciosarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma (adipose sarcoma), hydatidiform soft tissue sarcoma, ameloblastic sarcoma, staphyloid sarcoma, green sarcoma, choriosarcomaCarcinoma, fetal sarcoma, Wilms tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jense's sarcoma, Kaposi's sarcoma, Kupfer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, paraosteal sarcoma, reticulocyte sarcoma, rhabdomyosarcoma, serous cystic sarcoma, synovial sarcoma, pyelodiglic osteosarcoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, rhabdomyosarcoma, thrombocytosis, primary macroglobulinemia Examples include small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid tumors, pre-malignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, Harding-Passey melanoma, juvenile melanoma, lentigo malignant melanoma, malignant melanoma, acral lentiginous melanoma, achromatic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subungual melanoma, superficial spreading melanoma, plasmacytoma, colorectal cancer, and rectal cancer.

[0087] In some embodiments, the methods and compositions provided herein relate to the treatment of sarcomas. The term “sarcoma” generally refers to a tumor composed of a substance such as embryonic connective tissue and generally consisting of densely packed cells embedded in a fibrous, heterogeneous or homogeneous substance.

[0088] Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fasciosarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma (adipose sarcoma), hydatidiform soft tissue sarcoma, ameloblastic sarcoma, staphyloid sarcoma, green sarcoma, choriosarcoma This includes carcinoma, 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, Kupfer cell sarcoma, angiosarcoma, leukemosarcoma, malignant mesenchymal sarcoma, paraosteal sarcoma, reticular sarcoma, Rous sarcoma, serosal sarcoma, synovial sarcoma, and telangiectatic osteosarcoma.

[0089] Further exemplary neoplasms that can be treated using the methods and compositions described herein include Hodgkin's disease, non-Hodgkin 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 pancreatic insulinoma, malignant carcinoid, pre-malignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, non-genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[0090] In some embodiments, the cancer being treated is melanoma. The term “melanoma” is interpreted to mean a tumor arising from the melanocyte system of the skin and other organs. Non-limiting examples of melanoma include Harding-Passé melanoma, juvenile melanoma, lentigo malignant melanoma, malignant melanoma, acromian lentigo melanoma, achromatic melanoma, benign juvenile melanoma, Cloudmann melanoma, S91 melanoma, nodular melanoma, subglandular melanoma, and superficial spreading melanoma.

[0091] Certain 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, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, central nervous system cancer, peripheral nervous system cancer, skin cancer, kidney cancer, and all of the above metastases. Certain types of tumors include hepatocellular carcinoma, hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal cancer, thyroid cancer, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma of the lung, basal cell carcinoma, adenocarcinoma (well-differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, Gravitz tumor, and adrenal adenocarcinoma (hypernephroid). This includes adenocarcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, testicular tumors, lung cancer including small cell, non-small cell, and large cell lung cancer, bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon cancer, rectal cancer, and hematopoietic malignancies (including all types of leukemia and lymphoma, including acute myeloid leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma).

[0092] Cancers treated in certain embodiments also include precancerous lesions, such as actinic keratosis (solar keratosis), moles (dysplastic nevi), actinic cheilitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, congenital keratosis, iron deficiency dysphagia, lichen planus, oral submucosal fibrosis, actinic (solar) elastic fibrosis, and cervical dysplasia.

[0093] Cancers treated in some embodiments include, for example, noncancerous or benign tumors of endoderm, ectoderm, or mesenchymal origin, which include, but are not limited to, cholangiomas, colon polyps, adenomas, papillomas, cystadenomas, hepatocellular adenomas, hydatidiform moles, renal tubular adenomas, squamous cell papillomas, gastric polyps, hemangiomas, osteomas, chondromas, 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 available for 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] Treatment of immune disorders In certain embodiments, the aptamers provided herein can inhibit the immune response by blocking T cell activation. In some embodiments, methods for treating autoimmune or inflammatory diseases and / or inhibiting transplant rejection are provided herein, comprising administering a pharmaceutical composition comprising one or more aptamers provided herein to a subject.

[0096] The methods described herein can be used to treat subjects in need of treatment. As used herein, “subjects in need of treatment” includes subjects with inflammatory diseases, immune disorders, and / or who have received organ and / or tissue transplants, as well as subjects who are likely to acquire such diseases or disorders or who are likely to receive such transplants.

[0097] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with autoimmune diseases, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, Macalwells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; allergic diseases, such as food allergies, hay fever, or asthma; infectious diseases, such as infection by Clostridium difficile; inflammatory diseases such as TNF-mediated inflammatory diseases (e.g., gastrointestinal inflammatory diseases such as pouchitis, cardiovascular inflammatory symptoms such as atherosclerosis, or inflammatory lung diseases such as chronic obstructive pulmonary disease) (to partially or completely alleviate these adverse events); as pharmaceutical compositions for suppressing rejection in other situations in which organ transplantation or tissue rejection may occur; as supplements, foods, or beverages for improving immune function; or as reagents for suppressing the proliferation or function of immune cells.

[0098] In some embodiments, the methods provided herein are useful for treating inflammation. In certain embodiments, inflammation of any tissue and organ of the body includes inflammation of the musculoskeletal system, inflammation of the blood vessels, inflammation of the nerves, inflammation of the digestive system, inflammation of the eye, inflammation of the reproductive system, and other inflammations discussed below.

[0099] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with musculoskeletal immune disorders (partially or completely reducing these adverse events). Musculoskeletal immune disorders include, but are not limited to, conditions affecting skeletal joints, including the joints of the hands, wrists, elbows, shoulders, jaw, spine, neck, hips, knees (knews), ankles, and feet, as well as conditions affecting tissues that connect muscles to bones, such as tendons. Examples of such immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendinitis, synovitis, tenosynovitis, synovitis, bursitis, connective tissue inflammation (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, pubic osteitis, and cystic fibrous osteitis).

[0100] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with ocular immunodisorders (partially or completely reducing these adverse events). Ocular immunodisorders refer to immune disorders affecting any structure of the eye, including the eyelids. Examples of ocular immunodisorders that can be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.

[0101] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with neurological immune disorders (partially or completely mitigating these adverse events). Examples of neurological immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, encephalitis, Guillain-Barré syndrome, meningitis, neuromyotonic syndrome, narcolepsy, multiple sclerosis, myelitis, and schizophrenia.

[0102] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation related to the vascular or lymphatic system (partially or completely reducing these adverse events). Examples of inflammation of the vascular or lymphatic system that can be treated with the methods and compositions described herein include, but are not limited to, arthritis, arthritis, phlebitis, vasculitis, and lymphangitis.

[0103] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with gastrointestinal immune disorders (partially or completely mitigating these adverse events). Examples of gastrointestinal immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis. Inflammatory bowel disease includes, for example, certain forms of the associated symptom group recognized in the field. Several major forms of inflammatory bowel disease are known, with Crohn's disease (focal bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) being the most common of these disorders. Furthermore, inflammatory bowel disease includes irritable bowel syndrome, microscopic colitis, lymphocytic-plasmacytic enteritis, celiac disease, collagenous colitis, lymphocytic colitis, and eosinophilic enteritis. Other less common forms of IBD include unclassifiable colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behçet's disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia-associated masses or lesions, and primary sclerosing cholangitis.

[0104] The pharmaceutical compositions described herein can be used, for example, as pharmaceutical compositions for preventing or treating inflammation associated with reproductive system immune disorders (partially or completely reducing these adverse events). Examples of reproductive system immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tuboovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.

[0105] The methods and pharmaceutical compositions described herein can be used to treat autoimmune conditions. Such conditions include, but are not limited to, acute alopecia generalis, Behçet's disease, Chagas disease, chronic fatigue syndrome, autonomic neuropathy, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, type 1 diabetes, type 2 diabetes, giant cell arteritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Henoch-Schönlein purpura, Kawasaki disease, lupus erythematosus, and microscopic purpura. These include enteritis, microscopic polyarteritis, mixed tissue disease, Macalwells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Audrey thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjögren's syndrome, temporal arteritis, Wegner's granulomatosis, warm autoimmune hemolytic anemia, interstitial cystitis, Lyme disease, Morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

[0106] The methods and pharmaceutical compositions described herein can be used to treat T-cell-mediated hypersensitivity disorders. Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including poison ivy), urticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dust mite allergy), and gluten-sensitive enteropathy (celiac disease).

[0107] Other immunodisorders having inflammatory components that can be treated by this method and pharmaceutical compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, connective tissue inflammation, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, interstitial pneumonia, prostatitis, pyelonephritis, and stomatitis, transplant rejection (including organs such as kidneys, liver, heart, lungs, pancreas (e.g., islet cells), bone marrow, cornea, small intestine, skin allografts, skin homografts, and heart valve grafts (xengrafts), serum sickness, and graft-versus-host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, and Sézary's syndrome. These include syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, cancer-related hypercalcemia, pemphigus, herpetic vesicular dermatitis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmitis, iritis and iridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, chemotherapy for fulminant or disseminated pulmonary tuberculosis, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) hemolytic anemia, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, and sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation associated with infectious symptoms (e.g., sepsis). [Examples]

[0108] Materials and methods of Examples 1-2 to 3 A. Material a. Random Library Random Library 9.0 ("Lib9.0") was purchased from IDT. This library contains approximately 10 unique sequences, each consisting of two adjacent 20nt sequences at 3' and 5' that act as primers for PCR amplification during the SELEX procedure.15 It contains a vast repertoire of 40nt-length random sequences. The lyophilized library was reconstituted in ultrapure water (UPW) to a final concentration of 1 mM. The random library sequence is 5'-TCACTATCGGTCCAGACGTA-40N-TATTGCGCCGAGGTTCTTAC-3' (SEQ ID NO: 23), where N represents a random oligonucleotide selected from a mixture of T, A, C, and G nucleotides (in a 1:1:1:1 ratio) that are equally represented. SELEX Pre-Preparation After reconstitution, the library underwent size exclusion QC validation using an HPLC ProSEC 300S column (Agilent). b. Primers and caps for the library A set of 20nt primers and caps was purchased from IDT (Table 4). During incubation with cells, the caps were used to hybridize the primer sites in the library to avoid the possibility of the primer sequences interacting with random 40nt sequence sites. A mixture of 3' and 5' caps (Table 4) was used in each SELEX round at a cap-to-library ratio of 3:1. Forward primers were purchased from IDT with the 5' region labeled with Cy-5 for amplification of sequences detected by fluorescence assay. The lyophilized primers were reconstituted in ultrapure water (UPW) to a final concentration of 100 μM. [Table 4] c. Aptamer folding buffer Phosphate-buffered saline (excluding magnesium and calcium) was supplemented with 1 mM magnesium chloride (MgCl2). The folding buffer was sterilized using a 0.22 μm PVDF membrane filter unit and maintained at 4°C. d.PBMC PBMCs were isolated using Ficol (Lymphoprep, Axis-Shield) density gradient centrifugation according to the manufacturer's protocol. The frozen cynomolgus macaque PBMC (NHP-PC001) was purchased from Creative Biolabs. e. Human Pan T cells and B cells were isolated using the Pan T cell isolation kit (Miltenyi Biotec, 130-096-535) according to the manufacturer's protocol. Human Pan B cells were isolated using the Pan B cell isolation kit (Miltenyi Biotec, 130-101-638) according to the manufacturer's protocol. f. Antibodies, proteins, and enzymes αCD3ε-FITC (catalog number 130-113-690) / APC (catalog number 130-113-687) / VioBlue (catalog number 130-114-519) / APC-Vio770 (catalog number 130-113-688), αCD4-FITC (catalog number 130-114-531), αCD8-FITC (catalog number 130-113-719) / PE-Vio770 (catalog number 130-113-159) and matching isotype controls were purchased from Miltenyi Biotec. The αCD3εOKT3 clone (catalog number 317302) was purchased from BioLegend. Recombinant human CD3 epsilon protein (Fc chimeric His tag) (ab220590), recombinant cynomolgus monkey CD3 epsilon protein (Fc chimeric His tag) (ab220531), and recombinant mouse CD3 epsilon protein (His tag) (ab240841) were purchased from Abeam. Human IgG1 isotype was used as a negative counter selection (InVivoMAb, BE0297). I purchased the Protein G magnetic beads from ThermoFisher (88847). Herculase II Fusion DNA Polymerase (600675), used for asymmetric PCR (A-PCR), was purchased from Agilent, and the real-time PCR iTaq Universal SYBRGreen Supermix (1725124) was purchased from BIO-RAD. g.Cell line The Jarcut, Daudi, and Kasumi-1 cell lines were purchased from ATCC. Jarcut cells (ATCC TIB-152), Daudi cells (ATCC CCL-213), and Kasumi-1 (ATCC CRL-2724) were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FCS) and 1% penicillin and streptomycin (Pen / Strep). All cells were cultured at 37°C and 5% CO2. g. Aptamer Each aptamer was diluted to the desired concentration with folding buffer. The aptamers were heated at 95°C for 5 minutes, then rapidly cooled on ice for 10 minutes, and incubated at room temperature (RT) for 10 minutes. The folded aptamers were then added to cell suspensions in culture medium. The lyophilized aptamers were stored in the dark at RT, then reconstituted in PBS supplemented with 1 mM MgCl2 to a concentration of 100 μM, and stored in the dark at -20°C.

[0109] B. Experimental Method a. Combined SELEX protocol The conjugated SELEX test was performed in 11 consecutive rounds using CD3ε-Fc protein conjugated to Protein G magnetic beads (positive selection), IgG1 protein conjugated to Protein G magnetic beads, or beads alone (negative selection, starting from round 3).

[0110] A. Preparation of bead-protein complexes Magnetic Protein G beads were vortexed, washed once with PBS, and then mixed with 100 μl of protein at RT for 10 minutes under gentle shaking conditions. The beads were then separated with a magnet, the supernatant was discarded, and the beads were resuspended in 350 μl of folding buffer (1x) containing 2% BSA. To verify bead-protein complex formation, small samples (before DNA addition) were treated with the FC blocker (Miltenyi), stained with αCD3ε, and analyzed by flow cytometry.

[0111] B. Preparation and folding protocols for initial and enriched round libraries The library was initially reconstituted to 1 mM. The working concentration for the first round was 14.3 μM, but for rounds 2 through 11, concentrated libraries with concentrations of 0.25–0.5 μM were used. The following components were used in each round: [Table 5] The library was subjected to DNA folding according to the following protocol: heated at 95°C for 5 minutes, then rapidly cooled on ice for 10 minutes, and maintained at 4°C until use.

[0112] C. SELEX Once the concentrated library was folded, 350 µl of concentrated library rounds were added to 350 µl of CD3ε-FC-beads (positive selection rounds 1-11) or to beads only / IgG1-bead complexes (counter selection, rounds 3-11). Incubation time, protein volume, and washing steps differed by SELEX round. For positive selection, the supernatant of the "non-positively bound" fraction was removed and kept at -20°C until NGS preparation. For washing, the beads were precipitated with a magnet, the supernatant was discarded, and the beads were resuspended in 1 ml of folding buffer (1x). After the washing step, the beads were suspended in 300 μl of ultrapure water (UPW) and the DNA was eluted at 95°C for 10 minutes. Finally, the beads were precipitated with a magnet, and the "positively bound" supernatant was collected for the PCR step. If a negative SELEX round was performed, 350 µl of concentrated library round was added to 350 µl of beads only / IgG bead conjugate, and the recovered supernatant fraction was advanced to the positive selection step. The conjugated fraction to the negative sample, referred to as "negatively bound," was eluted and kept at -20°C until NGS preparation.

[0113] D.PCR amplification protocol The eluted DNA fractions ("bound" and "unbound") were used as templates for asymmetric PCR (A-PCR) amplification, respectively. PCR reactions were prepared for each round. The PCR components and amplification protocols are shown in Tables 6 and 7, respectively. [Table 6] [Table 7]

[0114] E.PCR ssDNA purification PCR products were concentrated with 10K Amicon (Millipore, UFC5010BK) and purified using an HLPC ProSEC 300S size exclusion column (Agilent). After purification, the DNA was buffer-exchanged with an ssDNA clean kit (ZYMO, D7011), the concentration was measured using NanoDrop, and the DNA was diluted for a new SELEX round.

[0115] b. Evaluation of library pools that bind to target proteins by real-time PCR. Magnetic Protein G beads were vortexed, washed once with PBS, and then resuspended with protein (CD3ε or IgG1) at RT for 10 minutes under gentle shaking conditions. The beads were then precipitated with a magnet, the supernatant was discarded, and the beads were resuspended in 125 μl of folding buffer (1x) and 2% BSA. Next, the library pools from rounds 3, 6, 9, and 11, and the first random library were folded (95°C for 5 minutes, ice for 10 minutes, maintained at 4°C). 125 μl of each folded DNA library was mixed with the bead-protein complex at 4°C for 1 hour with gentle shaking. After incubation, the beads were precipitated with a magnet and washed three times with 1 ml of folding buffer. Finally, the DNA-bound fraction was eluted at 95°C for 10 minutes using 100 μl UPW and then used as a template for real-time PCR using SYBRGreen Supermix (BIO-RAD).

[0116] c. Evaluation of individual aptamers that bind to target proteins: Protein-aptamer binding assay by HPLC A 1 μM folded Cy5-labeled aptamer was mixed with a 5 μM protein to a final volume of 60 μl and incubated at 4°C or 37°C for 1 hour. Next, to detect the Cy-labeled aptamer, the sample was analyzed for absorption at 570 nm via HPLC ProSEC 300S size exclusion column (Agilent).

[0117] d. Evaluation of individual aptamers that bind to cells using flow cytometry. 0.5~2×10 6 Individual cells (isolated Pan T cells, B cells, hPBMCs, cynomolgus monkey PBMCs, Jarcutt, and Daudi) were washed and resuspended in 0.2–1 ml of folding buffer containing 0.1% BSA and 0.01% tRNA. Single DNA candidates of 0.25–1.25 μM were fluorescently labeled by mixing with a CpG'-Cy5 tag (1:1 ratio) and folded (95°C for 5 minutes, ice for 10 minutes, maintained at 4°C). The labeled DNA aptamers were then incubated with cells in a V-shaped 96-well plate at 4°C or 37°C for 1 hour under gentle shaking conditions (hPBMCs and Cyno PBMCSs were supplemented with αCD8 / αCD4 during the last 15 minutes of incubation). After incubation, cells were washed three times with folding buffer (1x), and analyzed using flow cytometry (CytoFlex) after each wash.

[0118] e. Competitive CD3 epsilon epitope binding assay 0.25 × 10 6The Jurkat cells were washed once and resuspended in folding buffer (1X) containing 0.1% BSA and 0.01% tRNA, and incubated for 15 minutes with αCD3 clone OKT3 (BioLegend, 317302) or αCD3 clone REA613 (Miltenyi, 130-114-519) at a 1:20 dilution or with buffer. Next, 0.25 uM of folded Cy5-labeled aptamer was incubated with the cells for 1 hour at 37 °C under gentle shaking conditions. After incubation, the cells were washed three times with folding buffer (1X) and analyzed using flow cytometry (CytoFlex) after each wash.

[0119] f. Quantification of CS6 effective concentration 50 (EC50) 5×10 4 The Jurkat cells were washed and resuspended in 1X folding buffer containing 0.1% BSA and 0.01% tRNA. The CS6 aptamer at 0.1 - 80 nM was labeled with CpG’-Cy5 tag (1:1 ratio) and folded (95 °C for 5 minutes, ice for 10 minutes, maintained at 40 °C). Next, the DNA aptamer was mixed with the cells and incubated for 1 hour at 37 °C in a V-bottom 96-well plate under gentle shaking conditions. After incubation, the cells were washed twice with folding buffer (1X) and analyzed by flow cytometry (CytoFlex).

[0120] Example 2 - Identification of CD3-targeted aptamers by binding SELEX The selection of CD3-binding aptamers is described herein. Aptamers targeting T cells were identified by binding SELEX and hybrid binding cell-SELEX using recombinant CD3e protein and recombinant protein + T cells, respectively. The final leads were characterized for their binding to the target protein and T cells.

[0121] The present disclosure uses the SELEX methodology in a novel application to 15We describe the identification and characterization of T cell-engaging aptamers from a random library of potential aptamers. This aptamer portion was designed to be constant across different patients as part of a bispecific therapeutic entity.

[0122] SELEX binding was performed in a total of 11 rounds using recombinant human CD3 epsilon protein Fc chimeras. For counter-negative selection, either beads alone (rounds 1-6) or beads conjugated with human IgG1 (rounds 7-11) were used to remove all aptamers that nonspecifically bind to the magnetic beads or the Fc component of the recombinant protein (Figure 1). After 11 rounds of SELEX, the enriched aptamer library was subjected to sequencing and analysis using specific algorithms. A single candidate was identified and validated.

[0123] Figure 1B shows the steps of SELEX: Counter selection begins with (1) Protein G magnetic beads, (2) which are conjugated with IgG1, and (3) incubated with a pool of DNA aptamers from the previous step. Next, (4) unbound DNA aptamers are collected for positive selection, (5) which are incubated with beads conjugated with FC-CD3ε, and the bound fraction (6) undergoes PCR amplification and HPLC purification for the next round.

[0124] 1. SELEX Round Comparative Assay The original random library "No. 9.0" and library pools eluted from rounds 3, 6, 9, and 11 were tested for binding to hCD3a. After incubation with the Bcads-Fc-CD3ε complex at 4°C for 1 hour, each round was amplified by PCR using Cy-5 labeled 5' primers. As a negative control, the mutant pool was incubated with the beads-IgG1 complex (Figure 2A). The amount of amplified DNA precipitated with the target protein was found to be significantly higher in the libraries from rounds 6, 9, and 11 than in the random initial library used for binding SELEX. The results showed specific and strong enrichment at round 6 compared to the initial library. Furthermore, there was another increase in specific binding observed in round 11. After demonstrating inter-round enrichment using recombinant CD3 protein, the inventors tested whether such enrichment could be observed in whole-cell conditions. Jarcut T cells were incubated with the same Cy5-tagged library pool, washed, and analyzed by flow cytometry. Isolated Pan B cells were used as a negative control (Figure 2B). Similar to protein data, specific and potent inter-round enrichment of target cells was demonstrated.

[0125] 2.NGS results The concentrated libraries eluted in rounds 8, 9, 10, and 11 ("bound"), as well as the supernatant from the positive selection round ("unbound"), were subjected to sequencing using the high-throughput NGS Illumina NextSeq500. After sequencing, the data were analyzed using an algorithm that assigned a single candidate to a downstream binding assay. This algorithm utilizes statistical estimators, tests, and metrics. The mean P-positive and P-negative scores of the top 100 aptamers that were most abundant in the final round were plotted (Figure 3A), and aptamers with a significant binding-to-non-binding ratio (p<0.05; Poisson test, agreed across all rounds) were highlighted as described above in #6 and selected for experimental validation (referred to as CD3-CS6-9, SEQ ID NOs. 1-5). Nine additional aptamers with high mean P-positive values ​​(P-positive >0.5) were assigned identifiers (CD3_Ppos10-18, SEQ ID NOs. 6-14). The identified CD3-binding aptamers are listed in Table 8. [Table 8] Next, multiplex sequence alignment was performed on 14 aptamers with high average P-positive values ​​(P-positive > 0.5) (see Table 8), and a common motif was identified (Figure 3B, upper). Comparison revealed that the highlighted candidates (CS6-CS9) were also aligned, and a more robust motif was discovered (Figure 3B, lower). Furthermore, structural prediction analysis was performed using analysis software (mfold, NUPACK) (Figure 3C). This analysis demonstrated that the candidates primarily folded into complex secondary structures around the motif region. Based on these results, as an attempt at optimization, CD3 CS8 was further edited by trimming the first 9 nucleotides (denoted as CD3_CS8cut) that appeared irrelevant to the formation of the secondary structure around the motif presented in CS_CD8. The top five candidates were further confirmed to have negative ΔG scores and were selected for individual binding assays. In addition to the conjugated SELEX mentioned above, a hybrid methodology was implemented that also included a whole-cell SELEX round in the process. [Table 9]

[0126] Example 3 - Verification of individual CD3-binding aptamers a. The aptamer candidate demonstrates binding to human CD3a via HPLC. The top five candidates (CS6, CS7, CS8, CS8c, and CS9, SEQ ID NOs. 1-5) were synthesized using a 5(5') phosphorothioate-modified CpG motif and assayed for human CD3ε (hCD3ε) binding via an HPLC size exclusion column. In this method, the aptamers were labeled with a Cy5 sequence complementary to the CpG site (Cy5-CpG'). The folded and labeled candidates were then incubated with CD3ε-recombinant protein or with negative control IgG1 (1 hour at 37°C and 4°C) and analyzed for absorption at 570 nm using an HPLC ProSEC 300S size exclusion column (Agilent). When protein is bound, the aptamer-protein complex is expected to have a larger mass than the free aptamer, resulting in a shorter retention time (RT) on the column. Conversely, for unbound aptamers, the RT in the presence of protein is expected to be the same as the RT in the absence of protein. For control, polyT sequences were used. All five candidates demonstrated binding to the CD3 epsilon target protein at varying levels (Figure 4).

[0127] b. The aptamer candidates demonstrate specific binding to Jurcut 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, their binding to their target on the T cell surface in the context of undenatured whole cells was assayed by flow cytometry. For this purpose, we used the Jarcutt T lymphocyte cell line (acute T-cell leukemia, ATCC TIB-152), which has been previously reported to express TCR. The initial binding assay using cells was performed at 4°C for 1 hour. As a negative control, the myeloblast cell line Kasumi-1 (acute myeloblastic leukemia, ATCC CRL-2724) was used. All three candidates were found to bind differentially to target cells compared to control cells, but CS6 and CS7 showed better specificity than CS8c. (Figure 5A) Next, to better mimic physiological conditions, the three candidates were assayed for binding to jarcuts at 37°C. Here, the B lymphoblast Daudi cell line (lymphoblasts, ATCC CCL-213) was used as a negative control (Figure 5B). In this experiment, the three candidates bound to the target cells when CS6 showed the highest binding level. CS6 was selected for further investigation and characterization. It was found to bind to normal primary Pan T cells at 37°C under blocking conditions but not to Pan B cells (Figure 5C). Next, the effective concentration of CS6 is 50 (EC 50 The following was evaluated: Serial dilutions of the -Cy5-labeled aptamer were incubated with Jurkat cells at 37°C for 1 hour, and binding was evaluated by flow cytometry (Figure 6). The calculated EC5 50 The value was 19.65 nM.

[0128] Materials and methods of Examples 4-5 A. Material a. Animals I purchased 7-8 week old female NSG mice from Jackson Labs. b. Cell lines and peripheral mononuclear cells (PBMCs) The HCT-116 human colorectal cell line (ATCC® CCL-247®) was cultured according to the instructions for ATCC. PBMCs were isolated from healthy donor peripheral blood by Ficol density gradient centrifugation using Lymphoprep® (Axis-Shield) according to the manufacturer's protocol. The isolated PBMCs were frozen in FBS + 20% DMSO medium. Cell viability tests and immunophenotyping of lymphocyte subpopulations (DuraClone) and activation markers were performed after isolation and upon thawing on the day of injection. c. Aptamer The cancer-targeting aptamer arm, variable chain 12 (VS12, SEQ ID NO: 22), was derived from a functional enrichment process as described in PCT application number PCT / IB19 / 01082, using the HCT-116 colon cancer cell line as the target cell. The CS6 T cell engager sequence (SEQ ID NO: 21) was derived from a SELEX binding process as described in Examples 2-3. The aptamers were synthesized as a single oligonucleotide chain and column-purified. Complementary CpG motif sequences were added to both the cancer-targeting aptamer and the immune-engager aptamer to enable hybridization and the generation of bispecific aptamer conjugates. Non-specific poly-T oligonucleotides were used as controls. Although the two poly-T chains also hybridized to form a double helix, in this case, the hybridization domain was not a functional domain. The full-length sequences are shown in Table 10. [Table 10] c. Pharmaceutical buffer / vehicle Phosphate-buffered saline (excluding magnesium and calcium) supplemented with 1 mM magnesium chloride (MgCl2). The folding buffer is sterilized by filtration and used immediately.

[0129] B. Experimental Method a. Bispecific individualized aptamer formulations The formulation procedure includes the following steps: 1. Reconfiguration Dilute / reconstitute each chain to the desired concentration in the formulation buffer (if lyophilizing). 2. Folding the aptamer: a. Heat the chain at 95°C for 5 minutes. b. Rapidly cool on ice for 10 minutes. Incubate in c.RT for 10 minutes. 3. Formation of dual specific entities b. Next, the two strands (cancer-targeting variable strand and immune-engager strand) are mixed together and incubated at RT for 30 minutes in a rotating apparatus.

[0130] Introduction and intervention of xenograft models Female NSG mice, 2 x 10 6 Individual HCT-116 tumor cells and 0.5 × 10⁶ 6 Thawed human PBMCs were mixed with Cultrex® in a 1:4 ratio and subcutaneously (SC) injected into the right flank of mice. On day 7, IV re-boost of PBMCs from the same donor was performed, resulting in 8 × 10⁶ mice. 5 The cells were injected into each mouse. Table 11 details the SC intervention regimens. [Table 11]

[0131] a. Method for evaluating tumor volume Changes in tumor volume were monitored three times a week using calipers. Tumor volume was estimated as follows: Tumor volume (mm³) = (length × width) 2 ) / 2

[0132] b.Statistical methods All quantitative data are expressed as mean ± SEM. To assess the significance of differences between groups, analysis of variance or Student's t-test was used as needed.

[0133] Example 5 - Proof of Concept (POC) regarding the effects of a novel bispecific T cell engager. 1. Representative structure of a bispecific conjugate aptamer In some embodiments, the personalized cancer therapeutics described herein are composed of a heterodimer structure having three distinct domains (Figure 7). In some embodiments, bispecific personalized conjugate aptamers are designed to target specific neoantigens and surface molecules presented by a patient's cancer cells, promoting both direct cancer cell lethality and immune-associated responses. In some embodiments, efficacy is achieved through three distinct modes of action (MoAs) incorporated into a single therapeutic entity, as described below.

[0134] A. Variable chains: Direct killing of cancer cells by individualized aptamers In some embodiments, this part is 10 15 The process begins with a random pool of potential leads and is described in detail in PCT application number PCT / IB19 / 01082. In short, the personalized process is designed to identify the aptamer that best promotes targeted killing of cancer cells without harming healthy cells. Patient-specific chains are identified by performing a Cell and Functional SELEX process, screening candidates with high-throughput microscopy, and confirming the activity and specificity of top candidates while attempting to eliminate off-target effects, including selectivity testing.

[0135] B. Steady chain: T cell engager aptamers In some embodiments, the aptamer arm is designed to target T cells and mediate the lysis of target cancer cells by engaging with immune cells.

[0136] CpG motif with C.TLR9 agonist activity The two aptamer arms of the bispecific structure are cross-linked to each other by nucleic acid-base hybridization of complementary single-stranded overhangs. This hybridization domain is rich in CpGs and designed to induce stimulation of TLR9-mediated antigen-presenting cells (APCs) and increased uptake of tumor antigens. The stimulated APCs then migrate to tumor-discharge lymph nodes, where they cross-present phagocytotoxic T lymphocytes with the phagocytotoxic tumor antigens, resulting in an adaptive systemic anti-tumor immune response.

[0137] 2. In vivo POC of CD3-targeting bispecific aptamer conjugates in an HCT116 tumor xenograft model HCT-116 cells and a human PBMC mix (E:T 1:4 ratio) were co-injected in a mixed manner, followed by administration of bispecific individualized aptamers (CS6-VS12, SEQ ID NOs. 21 and 22), polyT double helix, or vehicle. Figure 8A illustrates the HCT116 tumor growth dynamics. Treatment with the bispecific aptamer CS6-VS12 significantly attenuated HCT116 tumor growth after a total of 10 interventions, whereas treatment with the nonspecific oligonucleotide polyT did not. At day 30, mice began to be sacrificed due to ethical limits on the endpoint. Individual mouse tumor volumes are displayed up to day 41 (31 days after the last intervention). Inhibition of tumor growth was demonstrated in all CS6-VS12 treated mice (Figure 8B). The reduction in tumor growth translated into a survival benefit in the bispecific treatment group compared to the vehicle-based treatment group. Figure 9 shows that, up to day 41 of the experiment, all 8 mice in the CS6-VS12 group survived, compared to 2 out of 9 mice (22.2%) and 3 out of 8 mice (37.5%) in the vehicle group and polyT group, respectively. In summary, the in vivo results of this model demonstrate the effectiveness and efficacy of the CS6-VS12 treatment.

[0138] Example 6 - Dual-specific individualized aptamer F. Representative structure of bispecific individualized aptamers In some embodiments, the personalized cancer therapeutics described herein are composed of a heterodimer structure having three distinct domains (Figure 7). In certain embodiments, the platform described herein is designed to provide a personalized cancer therapy that treats the patient with a personalized solution optimized for the unique set of symptoms and potential drug targets presented by each patient, as reflected in fresh tissue samples of the patient's tumor. In some embodiments, the bispecific personalized aptamer is designed to target specific neoantigens and surface molecules presented by the patient's cancer cells, promoting both direct lethality and immune-associated responses to the cancer cells. In some embodiments, efficacy is achieved by three distinct modes of action (MoAs) incorporated into a single therapeutic entity, as described below.

[0139] 1. Individualized chains: Direct killing of cancer cells by individualized aptamers In some embodiments, this part is 10 15 The process begins with a random pool of potential leads and is described in detail in PCT application number PCT / IB19 / 01082. In short, the personalized process is designed to identify the aptamer that best promotes targeted killing of cancer cells without harming healthy cells. Patient-specific chains are identified by performing a Cell and Functional SELEX process, screening candidates with high-throughput microscopy, and confirming the activity and specificity of top candidates, including attempting to eliminate off-target effects through selectivity testing (Figure 10A).

[0140] 2. Immunomodulatory chain: Lysis of cancer cells by T or NK cell-mediated cytotoxicity In some embodiments, the aptamer arm is a CD3-binding aptamer disclosed herein (e.g., containing any one of the sequences of SEQ ID NOs: 1-21) (Figure 10B). This immunomodulatory arm may be designed to be shared among different patients.

[0141] 3. CpG motif with TLR9-agonist activity In some embodiments, the two aptamer arms of the bispecific structure are crosslinked with each other by nucleic acid-base hybridization of single-stranded overhangs of complementary sequences. This hybridization domain is rich in CpG and designed to induce stimulation of TLR9-mediated antigen-presenting cells (APCs) and increased uptake of tumor antigens (Figure 10C). The stimulated APCs then migrate to tumor-discharge lymph nodes, where they cross-present phagocytotoxic T lymphocytes with the phagocytotoxic tumor antigens, resulting in an adaptive systemic anti-tumor immune response (Figure 10D).

[0142] G. Individualized processes for each patient In some embodiments, the personalized process as a cancer treatment platform includes several key steps (Figure 11): 1. Obtain two types of primary suitable samples from the target. a. Tumor biopsy b. Healthy tissue used as a negative control, consisting of either normal tissue from the biopsy site or peripheral blood mononuclear cells (PBMCs). 2. Performing the selection process described herein to identify personalized aptamers that induce tumor cell death while leaving healthy cells intact; 3. Preparation and hybridization of both strands to obtain bispecific individualized aptamers; 4. Bispecific individualized aptamers are administered to each individual target.

[0143] Example 7 - In vivo efficacy of bispecific aptamers Intratumoral administration of bispecific aptamers was tested. Using two tumor cell lines, tumor-specific aptamers functioning as variable chains were identified using the aptamer identification process disclosed herein. Specifically, tumor-killing aptamers VS20 and VS45 were identified using human alveolar adenocarcinoma A549 cells, and tumor-killing aptamer VS32 was identified using mouse triple-negative breast cancer 4T1 cells. Subsequently, CS6 CD3e-targeted aptamers were hybridized to each tumor-killing aptamer. The sequences of each of these aptamers are shown in Table 12. Each of these three bispecific compounds was tested for functionality in vivo using the process illustrated in Figure 12. [Table 12] As illustrated in Figure 13, each of the tested bispecific entities (CS6-VS20, CS6-VS45, and CS6-VS32) was found to be an established tumor (50-100 mm). 3 When administered intratumor at a total dose of 2 mg, it effectively resulted in a significant reduction in corresponding tumor growth in vivo. In a 4T1 syngeneic immune mouse model, intratumoral administration of CS6-VS32 (2 mg) further demonstrated effects beyond local inhibition of tumor growth in the injected lesions. 4T1 tumors have the ability to spontaneously metastasize to various organs, including the liver and lungs. Because 4T1 cells are resistant to 6-thioguanine, a clonal assay was used to measure the number of lung metastases in treated mice. Figure 14A As can be seen, a reduction in lung metastases was observed. Furthermore, the indications for CpG motif / TLR9 activation were evaluated, and slight increases in circulating IL-6 and spleen weight were also reported in the treated mouse group (Figure 14B).

[0144] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In case of any conflict, the application containing the definitions herein shall prevail.

[0145] Equivalents Those skilled in the art will recognize, or can verify by routine experimentation alone, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the following claims. In certain embodiments, for example, the following are provided: (Item 1) An aptamer containing a nucleic acid sequence that is at least 80% identical to one of sequence numbers 1 through 21. (Item 2) The aptamer according to item 1, wherein the aptamer contains a nucleic acid sequence that is at least 90% identical to any one of sequence numbers 1 to 21. (Item 3) The aptamer according to item 1 or 2, wherein the aptamer contains a nucleic acid sequence that is at least 95% identical to any one of sequence numbers 1 to 21. (Item 4) The aptamer according to any one of items 1 to 3, wherein the aptamer contains a nucleic acid sequence that is at least 98% identical to any one of sequence numbers 1 to 21. (Item 5) The aptamer described in any one of items 1 to 4, wherein the aptamer contains the nucleic acid sequence of any one of sequence numbers 1 to 21. (Item 6) The aptamer described in any one of items 1 to 4, wherein the aptamer contains the nucleic acid sequence of any one of sequence numbers 1 to 21. (Item 7) An aptamer containing at least 20 consecutive nucleotides from one of sequence numbers 1 through 21. (Item 8) The aptamer according to item 7, wherein the aptamer comprises at least 30 consecutive nucleotides of any one of sequence numbers 1 to 21. (Item 9) The aptamer according to item 7 or 8, wherein the aptamer comprises at least 40 consecutive nucleotides of any one of sequence numbers 1 to 21. (Item 10) The aptamer according to any one of items 7 to 9, wherein the aptamer comprises at least 50 consecutive nucleotides of any one of sequence numbers 1 to 21. (Item 11) The aptamer according to any one of items 7 to 10, wherein the aptamer contains at least 50 consecutive nucleotides of any one of sequence numbers 1 to 21, or at least 64 consecutive nucleotides of sequence numbers 1 to 21. (Item 12) The aptamer described in any one of items 1 to 11, wherein the aptamer is 100 nucleotides or less in length. (Item 13) The aptamer described in any one of items 1 to 12, wherein the aptamer is 90 nucleotides or less in length. (Item 14) The aptamer described in any one of items 1 to 13, wherein the aptamer is 80 nucleotides or less in length. (Item 15) The aptamer described in any one of items 1 to 14, wherein the aptamer is 73 nucleotides or less in length. (Item 16) The aptamer described in any one of items 1 to 15, wherein the aptamer binds to T cells. (Item 17) The aptamer described in any one of items 1 to 16, wherein the aptamer binds to T cells. (Item 18) The aptamer described in any one of items 1 to 17, wherein the aptamer binds to the T cell antigen CD3. (Item 19) The aforementioned aptamer, (a) T cell-mediated cytotoxicity; and / or (b) Cancer cell death by T cell-mediated cytotoxicity An aptamer described in any one of items 1 through 18 that induces the following: (Item 20) The aforementioned aptamer, (a) cytokine secretion; and / or (b) T cell activation An aptamer described in any one of items 1 through 19 that induces the following: (Item 21) The method according to item 20, wherein the aptamer induces cell death in cancer cells in vitro. (Item 22) The method according to item 20, wherein the aptamer induces cell death in cancer cells in vivo. (Item 23) The aptamer described in any one of items 20 to 22, wherein the aforementioned cell death is apoptosis. (Item 24) The aptamer according to any one of items 20 to 23, wherein the cancer cells are cancer cells derived from the patient. (Item 25) The aptamer according to any one of items 20 to 24, wherein the cancer cells are solid tumor cells. (Item 26) The aptamer according to item 25, wherein the cancer cells are breast cancer cells or colorectal cancer cells. (Item 27) The aptamer according to any one of items 1 to 26, wherein the aptamer includes chemical modification. (Item 28) The aptamer according to item 27, wherein the aptamer is chemically modified with polyethylene glycol (PEG). (Item 29) The aptamer according to item 28, wherein the PEG is bound to the 5' end of the aptamer. (Item 30) The aptamer according to any one of items 27 to 29, wherein the aptamer includes a 5' terminal cap. (Item 31) The aptamer according to any one of items 27 to 30, wherein the aptamer includes a 3' terminal cap. (Item 32) The aptamer according to item 31, wherein the 3' terminal cap is an inverted thymidine. (Item 33) The aptamer according to item 31, wherein the 3' terminal cap contains biotin. (Item 34) The aptamer according to any one of items 27 to 33, wherein the aptamer includes a 2' sugar substitution. (Item 35) The aptamer according to item 34, wherein the 2' sugar substitution is a 2'-fluoro, 2'-amino, or 2'-O-methyl substitution. (Item 36) The aptamer according to any one of items 27 to 35, wherein the aptamer comprises locked nucleic acid (LNA), unlocked nucleic acid (UNA), and / or 2'-deoxy-2'fluoro-D-arabinonucleotide (2'-FANA) sugar in its backbone. (Item 37) An aptamer according to any one of items 27 to 36, comprising methylphosphonate internucleotide bonds and / or phosphorothioate (PS) internucleotide bonds. (Item 38) The aptamer described in any one of items 27 to 37, wherein the aptamer includes a triazole nucleotide bond. (Item 39) An aptamer as described in any one of items 27 to 38, which is modified with cholesterol or a dialkyl lipid. (Item 40) The aptamer according to item 39, wherein the cholesterol or dialkyl lipid is linked to the 5' end of the aptamer. (Item 41) The aptamer according to item 27, wherein the aptamer comprises a 5' phosphorothioate (PS) internucleotide bond and a 3' terminal cap-inverted thymidine. (Item 42) The aptamer according to any one of items 27 to 41, comprising the aptamer modified with a base. (Item 43) The aptamer described in any one of items 1 to 42, wherein the aptamer is a DNA aptamer. (Item 44) The aptamer described in item 43, wherein the aptamer is a D-DNA aptamer. (Item 45) The aptamer described in item 43, wherein the aptamer is an enantiomerized L-DNA aptamer. (Item 46) The aptamer described in any one of items 1 to 42, wherein the aptamer is an RNA aptamer. (Item 47) The aptamer described in item 46, wherein the aptamer is a D-RNA aptamer. (Item 48) The aptamer described in item 46, wherein the aptamer is an enantiomerized L-RNA aptamer. (Item 49) An aptamer conjugate containing an aptamer described in any one of items 1 to 48, which is linked to a cancer cell binding site. (Item 50) The aptamer conjugate described in item 48, wherein the aptamer is covalently bound to the cancer cell binding portion. (Item 51) The aptamer conjugate according to item 49, wherein the aptamer is non-covalently bound to the cancer cell binding portion. (Item 52) The aptamer conjugate according to any one of items 49 to 51, wherein the aptamer is directly linked to the cancer cell binding portion. (Item 53) The aptamer conjugate according to any one of items 49 to 51, wherein the aptamer is linked to the cancer cell binding portion via a linker. (Item 54) The aptamer conjugate according to any one of items 49 to 53, wherein the cancer cell binding portion binds to an antigen expressed on cancer cells. (Item 55) An aptamer conjugate according to any one of items 49 to 54, wherein the cancer cell binding portion induces cell death upon contact with cancer cells. (Item 56) An aptamer conjugate as described in any one of items 49 to 55, wherein the aforementioned cell death is apoptosis. (Item 57) The aptamer conjugate described in any one of items 49 to 56, wherein the cancer cells are solid tumor cells. (Item 58) The aptamer conjugate described in item 57, wherein the cancer cells are breast cancer cells or colorectal cancer cells. (Item 59) An aptamer conjugate according to any one of items 49 to 58, wherein the cancer cell binding portion induces cell death when it comes into contact with the cancer cells in vitro. (Item 60) The aptamer conjugate according to any one of items 49 to 59, wherein the cancer cell binding portion induces cell death when it comes into contact with the cancer cells in vivo. (Item 61) The aptamer conjugate according to any one of items 49 to 60, wherein the cancer cell binding portion is an aptamer, small molecule, polypeptide, nucleic acid, protein, or antibody. (Item 62) A pharmaceutical composition comprising an aptamer as described in any one of items 1 through 48. (Item 63) A pharmaceutical composition comprising an aptamer conjugate as described in any one of items 49 to 62. (Item 64) The pharmaceutical composition according to item 62 or 63, further comprising a pharmaceutically acceptable carrier. (Item 65) The pharmaceutical composition according to any one of items 62 to 64, wherein the pharmaceutical composition is formulated for parenteral administration. (Item 66) A pharmaceutical composition according to any one of items 62 to 65, for use in the treatment of cancer. (Item 67) The pharmaceutical composition according to item 66, wherein the cancer is a solid tumor. (Item 68) The pharmaceutical composition according to item 67, 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. (Item 69) A method for treating cancer, the method comprising administering an aptamer described in any one of items 1 to 48. (Item 70) A method for treating cancer, the method comprising administering an aptamer conjugate as described in any one of items 49 to 62. (Item 71) A method for treating cancer, the method comprising administering a pharmaceutical composition described in any one of items 62 to 68 to a subject. (Item 72) The method according to any one of items 69 to 71, wherein the administration is parenteral. (Item 73) The method according to item 72, wherein the administration is by intratumor injection. (Item 74) The method according to any one of items 69 to 73, wherein the cancer is a solid tumor. (Item 75) The method according to item 74, 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. (Item 76) The method described in any one of items 69 to 75, wherein the subject has previously received chemotherapy. (Item 77) The method described in any one of items 69 to 76, wherein the subject has previously undergone surgical removal of a tumor. (Item 78) The method according to any one of items 69 to 77, further comprising administering additional cancer treatment to the subject. (Item 79) The method according to item 78, wherein the additional cancer treatment includes chemotherapy. (Item 80) The method according to item 78, wherein the additional cancer treatment includes radiation therapy. (Item 81) The method according to item 78, wherein the additional cancer treatment includes surgical removal of the tumor. (Item 82) The method according to item 78, wherein the additional cancer treatment includes the administration of an immune checkpoint inhibitor to the subject. (Item 83) The method according to item 82, 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. (Item 84) A method for killing cancer cells, the method comprising inducing CTL activity by contacting a CTL with an aptamer described in any one of items 1 to 48. (Item 85) A method for killing cancer cells, the method comprising contacting the cancer cells with an aptamer conjugate described in any one of items 49 to 62. (Item 86) The method according to item 84 or 85, wherein the cancer cells are killed by apoptosis. (Item 87) The method according to any one of items 84 to 86, wherein the cancer cells are solid tumor cells. (Item 88) The method according to item 87, wherein the cancer cells are colorectal cancer cells. (Item 89) The method according to item 87, wherein the cancer cells are breast cancer cells. (Item 90) A method for producing an aptamer, the method comprising synthesizing a nucleic acid molecule having a sequence that is at least 80% identical to any one of sequence numbers 1 to 21. (Item 91) The method according to item 90, wherein the nucleic acid molecule contains a nucleic acid sequence that is at least 90% identical to any one of sequence numbers 1 to 21. (Item 92) The method according to item 90 or 91, wherein the nucleic acid molecule contains a nucleic acid sequence that is at least 95% identical to any one of sequence numbers 1 to 21. (Item 93) The method according to any one of items 90 to 92, wherein the nucleic acid molecule contains a nucleic acid sequence that is at least 98% identical to any one of sequence numbers 1 to 21. (Item 94) The method according to any one of items 90 to 93, wherein the nucleic acid molecule comprises one nucleic acid sequence from sequence numbers 1 to 21. (Item 95) The method according to any one of items 90 to 94, wherein the nucleic acid molecule comprises one nucleic acid sequence from sequence numbers 1 to 21. (Item 96) A method for treating an autoimmune disorder in the subject, comprising administering an aptamer described in any one of items 1-18 or 21-48. (Item 97) A method for treating the inflammatory disease described above, comprising administering an aptamer described in any one of items 1-18 or 21-48. (Item 98) A method for treating transplant rejection of the subject, comprising administering an aptamer described in any one of items 1-18 or 21-48 to the subject. (Item 99) A method for treating an autoimmune disorder in a subject, comprising administering the pharmaceutical composition described in item 62 to the subject. (Item 100) A method for treating the inflammatory disease of the subject, comprising administering the aptamer described in item 62 to the subject. (Item 101) A method for treating transplant rejection of the subject, comprising administering the aptamer described in item 62 to the subject.

Claims

1. An aptamer comprising one of the nucleic acid sequences of sequence numbers 1, 20, and 21, which binds to the T cell antigen CD3.

2. The aptamer according to claim 1, wherein the aptamer binds to T cells.

3. The aforementioned aptamer, (a) T cell-mediated cytotoxicity; (b) T cell-mediated cell death of cancer cells; (c) cytokine secretion; and / or (d) T cell activation The aptamer according to claim 1 or 2, which induces in vitro or in vivo.

4. The aptamer according to claim 3, wherein the aptamer induces cell death in cancer cells, and the cancer cells are patient-derived cancer cells and / or solid tumor cells.

5. The aptamer according to claim 4, wherein the cancer cells are breast cancer cells or colorectal cancer cells.

6. The aptamer according to any one of claims 1 to 5, wherein the aptamer includes chemical modification.

7. An aptamer conjugate comprising the aptamer according to any one of claims 1 to 6, which is linked to a cancer cell binding portion.

8. The aptamer conjugate according to claim 7, wherein the aptamer is directly linked to the cancer cell binding portion or linked to the cancer cell binding portion via a linker.

9. The aptamer conjugate according to claim 8, wherein the cancer cell binding portion binds to an antigen expressed on cancer cells and / or induces cell death upon contact with cancer cells.

10. The aptamer conjugate according to any one of claims 7 to 9, wherein the cancer cells are solid tumor cells.

11. The aptamer conjugate according to claim 10, wherein the cancer cells are breast cancer cells or colorectal cancer cells.

12. The aptamer conjugate according to any one of claims 7 to 11, wherein the cancer cell binding portion is an aptamer, a small molecule, a polypeptide, a nucleic acid, a protein, or an antibody.

13. A pharmaceutical composition comprising the aptamer according to any one of claims 1 to 6.

14. A pharmaceutical composition comprising the aptamer conjugate according to any one of claims 7 to 12.

15. The pharmaceutical composition according to claim 13 or 14, further comprising a pharmaceutically acceptable carrier.

16. A composition for use in treating cancer, comprising an aptamer according to any one of claims 1 to 6.

17. A composition for use in treating cancer, comprising an aptamer conjugate according to any one of claims 7 to 12.

18. A pharmaceutical composition according to any one of claims 13 to 15 for use in treating cancer.

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