Method and composition for treating cancer

Abstract

To provide an improved chimeric molecule for treating cancer.SOLUTION: A chimeric molecule includes an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain which inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47, which is an aptamer-siRNA chimera (AsiC) as one aspect, and the inhibitory nucleic acid specifically binds to a gene product of a selected gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 864,726, filed June 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Government support This invention was made with government support under Grant No. CA184718 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on June 18, 2020, is named 701039-095310WOPT_SL.txt and is 53,904 bytes in size.

[0004] Technical Field The technology described herein relates to chimeric molecules comprising an EpCAM-binding molecule and an inhibitory nucleic acid, and methods of using such compositions for the treatment of cancer, eg, epithelial cancer. [Background technology]

[0005] background RNA interference (RNAi) has been explored for therapeutic use in liver to reduce gene expression.However, liver is unique in that it is easy to transfect RNAi molecules.The delivery of small RNA and the resulting gene knockdown in other tissues continues to be inefficient and ultimately ineffective.In particular, the obstacle of delivery is a major obstacle to the use of RNAi to treat cancer. Summary of the Invention

[0006] overview As described herein, the present inventors have developed novel chimeric aptamer-siRNA molecules (AsiCs) that demonstrate improved efficacy over existing AsiCs and can successfully synergize in the treatment of cancer. These AsiCs target cancer cell markers to specifically direct therapeutic siRNA molecules to cancer cells, thereby increasing delivery efficacy and therapeutic effectiveness while reducing the potential for side effects.

[0007] In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; MCL1; and CD47.

[0008] In some embodiments of any of the aspects, the molecule is an aptamer-siRNA chimera (AsiC). In some embodiments of any of the aspects, the inhibitory nucleic acid specifically binds to a gene product of a selected gene.

[0009] In some embodiments of any of the aspects, the EpCam-binding aptamer domain comprises the sequence of any of SEQ ID NOs: 63-68. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises a sequence selected from SEQ ID NOs: 1-62 and 69-126, or a reverse complement thereof.

[0010] In some embodiments of any of the aspects, the chimeric molecule comprises a first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain. In some embodiments of any of the aspects, the first inhibitory nucleic acid domain and the at least one additional inhibitory nucleic acid domain comprise different sequences but each inhibit expression of the same gene. In some embodiments of any of the aspects, the first inhibitory nucleic acid domain and the at least one additional inhibitory nucleic acid domain each inhibit expression of different genes. In some embodiments of any of the aspects, the at least second inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of PLK1 and MCL1.

[0011] In some embodiments of any of the aspects, the molecule comprises the sequence of one of SEQ ID NOs: 127-137. In some embodiments of any of the aspects, the 3' end of the chimeric molecule comprises dTdT. In some embodiments of any of the aspects, the chimeric molecule comprises at least one 2'-F pyrimidine. In some embodiments of any of the aspects, the chimeric molecule further comprises a chemotherapeutic agent.

[0012] In one aspect of any of the embodiments, described herein is a pharmaceutical composition, kit, or combination comprising a chimeric molecule described herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments of any of the aspects, the composition, kit, or combination comprises at least two different chimeric molecules, the chimeric molecules having different aptamer domains or inhibitory nucleic acid domains. In some embodiments of any of the aspects, the different inhibitory nucleic acid domains recognize different targets. In some embodiments of any of the aspects, the different inhibitory nucleic acid domains have different sequences and recognize the same target.

[0013] In one aspect of any of the embodiments, a. a first chimeric molecule described herein; b. a second chimeric molecule, i. comprising a chimeric molecule as described herein, wherein the inhibitory nucleic acid domain of the second chimeric molecule inhibits expression of a different gene than the first chimeric molecule; or ii. an EpCAM-binding aptamer domain; and an inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of PLK1 and MCL1; and a chimeric molecule comprising a second chimeric molecule; and c. optionally, a pharmaceutically acceptable carrier Described herein is a pharmaceutical composition, kit, or combination comprising:

[0014] In one aspect of any of the embodiments, described herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject a chimeric molecule, composition, kit, or combination described herein. In some embodiments of any of the aspects, the cancer is epithelial cancer, breast cancer, colon cancer, or triple-negative breast cancer. In some embodiments of any of the aspects, the administration is subcutaneous. In some embodiments of any of the aspects, the subject is further administered an additional cancer treatment. In some embodiments of any of the aspects, the cancer treatment is paclitaxel. [Brief explanation of the drawings]

[0015] [Figure 1] This shows that EpCAM is highly expressed in epithelial cancers. [Figure 2] Graphs of gene knockdown using the indicated AsiC in the indicated cell types are depicted. [Figure 3] Figure 1 shows that human TNBC tumors take up Cy3-EpCAM-AsiC at a higher rate than normal breast tissue. [Figure 4] Figure 1 shows that EpCAM-AsiC inhibits EpCAM+ breast cancer cell lines in an in vitro cancer stem cell assay. Rows 1-3 are basal A TNBC, row 4 is luminal, and rows 5-6 are basal B TNBC. [Figure 5-1] 1 shows that ex vivo treatment of EpCAM+ TNBC cells blocks tumor initiation. [Figure 5-2] See the description of Figure 5-1. [Figure 6-1] 1 shows selective uptake of Alexa750-EpCAM-AsiC into EpCAM+ tumors. [Figure 6-2] See the description of Figure 6-1. [Figure 7] We show that EpCAM-AsiC, which targets PLK1, inhibits EpCAM+ TNBC tumor growth. [Figure 8]This is an illustration of anti-tumor immunity. (Excerpt from Sahin and Tureci Science, 2018) [Figure 9A] Figures 9A-9E show that knockdown of RNA quality control pathways enhances antitumor immunity: Figure 9A shows tumor suppression, Figure 9B shows an increase in CD8+ TILs, Figure 9C shows a reduction in inhibitory receptors on CD8+ TILs (first series is EpCAM aptamer, second series is UPF2 AsiC), Figure 9D shows greater degranulation and tumor killing by CD8+ TILs, and Figure 9E shows greater cytokine production by CD8+ TILs. [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 9D] See the legend to Figure 9A. [Figure 9E] See the legend to Figure 9A. [Figure 10] 10 depicts a graph showing an increase in CD8 TILs in 4T1 tumors treated with Upf2 EpCAM-AsiC. [Figure 11-1] We show that disruption of DNA repair by knockdown of PARP1 and APE1 improves tumor immunity. [Figure 11-2] See the description of Figure 11-1. [Figure 12A] Figures 12A-12E show that knockdown of PTPNT2 enhances anti-tumor immunity: Figure 12A shows tumor suppression, Figure 12B shows an increase in CD8+ TILs, Figure 12C shows an increase in tumor antigen presentation, Figure 12D shows greater cytokine production by CD8+ TILs, and Figure 12E shows greater cytokine production by CD4+ TILs. [Figure 12B] See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A. [Figure 12D] See the legend to Figure 12A. [Figure 12E] See the legend to Figure 12A. [Figure 13] 1 shows that knockdown of CD47 by AsiC inhibits tumor growth. The series in the bar graph are, in order, EpCAM aptamer, 3 μM CD47 AsiC, and 4 μM CD47 AsiC. [Figure 14] 10 depicts a graph showing that CD47-AsiC increases TAM in vivo phagocytosis of 4TE-eGFP tumors. [Figure 15] Figures 15A-15B show that CD47 knockdown induces anti-tumor responses. Figure 15A shows an increase in CD8+ T cells and a decrease in suppressive Tregs in tumors. Figure 15B shows an increase in th T cell activity in tumors. In both Figures 15A and 15B, the sequence is, in order, EpCAM aptamer and CD47 AsiC. [Figure 16-1] This shows that CD47-AsiC treatment causes TILs to express fewer inhibitory receptors. In the bar graph, the series is EpCAM aptamer and CD47 AsiC in order. In the pie chart, the series is 5, 4, 3, 2, and 1 inhibitory receptor in order, starting from the blunt end of the arrow. [Figure 16-2] See the description of Figure 16-1. [Figure 17] We show that CD47-AsiC controls tumors better than anti-CD47 antibodies (currently in phase II clinical trials). [Figure 18A]Figures 18A-18E illustrate the synergistic effects of combining AsiCs. Figure 18A illustrates UPF2+CD47, Figure 18B illustrates UPF2+CD47+MCL1, Figure 18C illustrates UPF2+CD47+MCL1+PLK1, and Figure 18D illustrates UPF2+CD47+MCL1+PLK1+PARP1+APE1+PD-L1. Figure 18E illustrates the effects of PARP1-AsiC, PARP1+PD-L1 AsiC, and mixed AsiC (UPF2+CD47+MCL1+PLK1+PARP1+APE1+PD-L1 AsiC) on CD8+ TILs. The sequences in Figure 18E are, in order, control, PARP1 AsiC, PARP1+PD-L1 AsiC, and mixed AsiC. [Figure 18B] See the description of Figure 18A. [Figure 18C] See the description of Figure 18A. [Figure 18D-1] See the description of Figure 18A. [Figure 18D-2] See the description of Figure 18A. [Figure 18E] See the description of Figure 18A. [Figure 19] Mice were treated with a cocktail of CD47, UPF2, PLK1, and MCL1 AsiC, showing that treated tumor cells do not downregulate EpCAM. [Figure 20] Results are compared for PD-L1 AsiC and CD47 AsiC. The series in the bar graph are, in order, EpCAM aptamer, CD47 AsiC, and PD-L1 AsiC. [Figure 21-1] Illustrates a comparison of single EpCAM-AsiC versus AsiC combinations. [Figure 21-2] See the description of Figure 21-1. [Figure 22] Illustrates seven EpCAM-AsiC combinations for treating 4T1E tumor-bearing mice. [Figure 23-1] Illustrates treatment of ErbB2ΔEx16+ mice with combination AsiC. [Figure 23-2]See the description of Figure 23-1. [Figure 24] Illustrates treatment of ErbB2ΔEx16+ mice with combination AsiC. [Figure 25-1]Figures 25A-25G show the tumor-inhibitory and immunomodulatory capabilities of UPF2 AsiC. Figure 25A: Comparison of tumor growth kinetics in mice burdened with 4T1E tumors and treated with either EpCAM Apt or UPF2 AsiC (5 mg / kg, every 3 days, indicated by red arrows). Figure 25B: Ratio of CD8+ TILs to tumor-infiltrating CD4+Foxp3+ Tregs in each group of tumors. a-b, n=5 mice / group. Figure 25C: H&E staining (top) and IHC staining for CD8+ TILs (bottom) using orthotopically implanted 4T1E tumors from mice treated with either EPCAM Apt or UPF2 AsiC. Black arrows indicate mammary glands. Tailless arrows indicate necrotic areas. White arrows indicate CD8+ TILs. Two mice per group were used for imaging, with similar results. Scale bar: 100 mM. Figure 25D: Comparison of CD8+ TIL numbers per selected area. For each group, six fields / sections were selected for counting. Two tumors / groups were examined, with similar results. Figure 25E: Percentage of CD8+ TILs producing IFN-g and TNF induced by PMA and ionomycin. Figure 25F: Degranulation of CD8+ TILs measured by their CD107a / CD107b surface expression, as well as expression of the cytotoxic molecules granzyme B and perforin, after 6 hours of co-incubation with 4T1E tumor cells in which UPF2 was knocked down by UPF2 siRNA. Representative flow images of CD8+ TIL degranulation are also shown (right). (Figure 25G) Targeted 51Cr-labeled UPF2 siRNA-treated 4T1E tumor cell lysis by CD8+ TILs enriched from 4T1E tumors treated with either EpCAM Apt or UPF2 AsiC. The effector:target ratio was 5:1. Figures 25E-25F, n=5 samples / group. Figure 26G, n=3 samples / group. For each sample, CD8+ TILs were pooled from two mice. Data show mean + sem and represent at least two experiments. For all figures: *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 25-2] See the description of Figure 25-1. [Figure 25-3] See the description of Figure 25-1. [Figure 26] Figures 26A-26D illustrate the comparison of tumor-inhibiting and immunomodulatory capabilities between PARP1 AsiC and the PARP1 inhibitor olaparib. Figure 26A: Comparison of tumor growth in mice bearing 4T1E tumors treated with EpCAM Apt, PARP1 AsiC (5 mg / kg, every 3 days), or olaparib (50 mg / kg, daily). Figure 26B: Comparison of the ratio of CD8+ TILs to tumor-infiltrating CD4+ Tregs in each tumor group. Figure 26C: Percentage of CD8+ TILs producing cytokines IFN-g and TNF induced by PMA and ionomycin. Figure 26D: Percentage of CD4+ TILs producing cytokines IFN-g and TNF induced by PMA and ionomycin. n=4 mice / group. Data shown are mean + sem. [Figure 27-1]Figures 27A-27M show the tumor-inhibitory and immunomodulatory capabilities of CD47 AsiC. Figure 27A: Comparison of tumor growth in mice bearing 4T1E tumors treated with either EpCAM Apt or CD47 AsiC. Arrows indicate each treatment. Figure 27B: Ratio of CD8+ TILs to tumor-infiltrating CD4+ Tregs in each group of tumors. Figures 27C-27D: Percentage of CD8+ TILs (Figure 27C) and CD4+ TILs (Figure 27D) producing IFN-g and TNF, induced by PMA and ionomycin. Figure 27E: Production of cytotoxic granules, granzyme B and perforin, by CD8+ TILs. Figure 27F: Ratio of M1-like TAMs to M2-like TAMs in mice treated with either EpCAM Apt or CD47 AsiC. Figure 27G, Percentage of CD11c+DEC205+ DCs relative to CD45+ cells in each group of tumors. Figure 27H, MFI levels of CD40, CD86, and MHCII on CD11c+DEC205+ DCs. Figures 27A-27H, n=5 mice / group. Figure 27I, Percentage of eGFP+ TAMs from 4T1E-eGFP tumors that phagocytosed tumor cells. n=5 mice / group. Figure 27J, In vitro phagocytosis by TAMs of control or CD47 siRNA-treated 4T1E-eGFP tumor cells. n=3 samples / group. Figure 27K, Comparison of tumor growth in 4T1E tumor-bearing mice treated with EpCAM Apt or CD47 AsiC and injected with isotype control Ab or depleted of CD8+ T cells, CD4+ T cells, or macrophages. Figure 27L: CD8+ TILs isolated from tumors in mice treated with EpCAM Apt, CD47 AsiC, or CD47 AsiC with Mac depletion were stimulated with 4T1E tumor cells for 6 hours. Cytokine production (left) and degranulation of CD8+ TILs were compared. The series, from left to right, are EpCAM Apt, CD47 AsiC, and CD47 AsiC + Mac depletion. Figures 27K-27L, n=5 samples / group. Figure 27M: Comparison of 4T1E tumor growth in mice treated with either EpCAM Apt, CD47 AsiC, or anti-CD47 antibody (Ab). n=5 mice / group.a–m, Data shown are mean + sem and represent at least two experiments. [Figure 27-2] See the description of Figure 27-1. [Figure 28-1] Figures 28A-28I illustrate the synergistic antitumor effects of immunomodulatory EpCAM-AsiC and EpCAM-AsiC in combination with anti-PD-1. Figure 28A: Comparison of tumor growth in mice bearing 4T1E tumors treated with EpCAM aptamer, or eGFP AsiC as a control, UPF2 AsiC, CD47 AsiC, MCL1 AsiC, Parp1 AsiC, or a combination of four immunomodulatory EpCAM-AsiCs targeting UPF2, CD47, MCL1, and Parp1. Arrows indicate the days on which mice received AsiC subcutaneously. Figure 28B: Individual tumor growth curves in mice treated with either EpCAM aptamer or the four EpCAM-AsiC combinations. Figure 28C: Comparison of the number of CD8+ TILs per mg of tumor between mice treated with EpCAM Apt or the combined AsiCs. Figure 28D: Ratio of CD8+ TILs to tumor-infiltrating CD4+Foxp3+ Tregs in each group of tumors. Figure 28E: Percentage of CD8+ TILs producing IFN-g and TNF induced by PMA and ionomycin. Right: Representative flow plot. Figure 28F: Percentage of CD4+ TILs producing IFN-g and TNF induced by PMA and ionomycin. Figure 28G: Production of cytotoxic granules, granzyme B and perforin, by CD8+ TILs. Figures 28A-28G, n=4 mice / group. Figure 28H: Comparison of tumor growth in mice bearing 4T1E-eGFP tumors treated with EpCAM aptamer or four immunomodulatory EpCAM-AsiC combinations. n=5 mice / group. Figure 28I. Comparison of tumor growth in mice bearing 4T1E tumors treated with EpCAM aptamer or AsiC in combination with isotype control or anti-PD-1 Ab. n=4 mice / group. Figures 28A, 28H, 28I. Arrows indicate the time for each treatment. Data shown are mean + sem. [Figure 28-2]See the description of Figure 28-1. [Figure 29-1] Figures 29A-29F illustrate the effect of immunomodulatory EpCAM-AsiC on tumor-infiltrating immune cells analyzed by single-cell RNA-Seq. Enriched CD45+ cells (n = 2 mice / group) from mice bearing orthotopic 4T1E tumors treated with either the EpCAM aptamer or the EpCAM-AsiC cocktail were pooled for single-cell RNA-Seq. Figure 29A: Immune cells from the combination of all four samples were projected onto a uniform manifold approximation and projection (UMAP) plot. Each dot represents a cell colored by its inferred cluster identity. Cont: Contaminating cells. Figure 29B: Heatmap showing the Z-score-normalized expression of differentially expressed genes (DEGs) as canonical and cell-type markers across different clusters. Each lane represents a biological sample. Figures 29C-29D, Gene Ontology (GO) enrichment of DEGs upregulated in the EpCAM-AsiC cocktail-treated group for proliferating T cells (Figure 29C) and monocytes / macrophages (Figure 29D). Dashed lines indicate p-values ​​of 0.05. Figures 29E-29F, Heatmaps showing the averaged per cluster expression of genes known to be involved in T cell activation, effector function, and memory formation, and exhaustion in the T cell cluster (Figure 29E), or genes involved in defining monocyte / macrophage phenotype and function in the monocyte / macrophage cluster (Figure 29F), and the normalized Z-scores across clusters. [Figure 29-2] See the description of Figure 29-1. [Figure 29-3] See the description of Figure 29-1. [Figure 29-4] See the description of Figure 29-1. [Figure 30-1]Figures 30A-30K illustrate the antitumor efficacy of combined immunomodulatory EpCAM-AsiC in ErbB2ΔEx16 transgenic mice. Figure 30A: Experimental scheme of tumor induction and treatment in ErbB2ΔEx16 transgenic mice. Figure 30B: Left: Quantitative comparison of EpCAM expression on GFP+ 4T1E-eGFP tumor cells and GFP+ ErbB2ΔEx16 transgenic tumor cells. Right: Representative histogram of EpCAM expression on GFP+ tumor cells. Figure 30C: Comparison of the percentage of CD8+ TILs (left) and CD4+ TILs (right) relative to live cells in 4T1E-eGFP tumors and ErbB2ΔEx16 transgenic tumors. Figures 30B-30C: 4T1E-eGFP, n=5 mice / group; ErbB2ΔEx16, n=7 mice / group. Figure 30D: Comparison of tumor growth in doxycycline-fed ErbB2ΔEx16 transgenic mice treated with the EpCAM aptamer or a combination of four immunomodulatory EpCAM-AsiCs. Figure 30E: Left: Histogram showing a comparison of EpCAM expression on tumor cells from mice treated with the EpCAM aptamer or the combined EpCAM-AsiCs. Right: Quantitative comparison of EpCAM expression on tumor cells in the two groups. Figure 30F: In vivo TAM phagocytosis measured by the percentage of GFP+ TAMs in each group of ErbB2ΔEx16 transgenic mice. Figures 30G-30H: Percentage of IFN-g and TNF-producing CD8+ TILs (Figure 30G) and CD4+ TILs (Figure 30H) induced by PMA and ionomycin. Figures 30I-30K, Production of cytotoxic granules, granzyme B and perforin, by CD8+ TILs (Figure 30I), CD4+ TILs (Figure 30J), and NK cells (Figure 30K) in each group of tumors. Figures 30D-30K, n=6 mice / group. Figures 30B-30K, Data shown are mean + sem. [Figure 30-2] See the description of Figure 30-1. [Figure 31-1]Figures 31A-31D show the antitumor efficacy of combined immunomodulatory EpCAM-AsiC in a lung metastatic 4T1E-Luc tumor model. Figure 31A: Representative luminescence images of mouse lung regions at different time points after tail vein injection of 4T1E-Luc tumor cells. Upper lane: group treated with EpCAM aptamer. Lower lane: group treated with combined EpCAM-AsiC. Figure 31B: Total luminescence photon flux of lung metastases in each treatment group at different time points after tumor cell implantation. Data represent mean + / - sem. Figure 31C: Percentage of CD8+ TILs producing IFN-g and TNF induced by PMA and ionomycin. Figure 31D: Percentage of CD8+ TILs producing IFN-g and TNF induced by PMA and ionomycin. Figures 31A-31D, EpCAM aptamer, n=5 mice / group, combined AsiC, n=6 mice / group. Figures 31C-31D, data represent mean + sem. [Figure 31-2] See the description of Figure 31-1. [Figure 32A] Figures 32A-32B show EpCAM expression and EpCAM aptamer uptake efficiency by mouse and human BC cells. Figure 32A: EpCAM protein expression on mouse (top) and human (bottom) BC cells. The mouse L929 cell line was used as a negative control. The numbers on each graph indicate the MFI value of EpCAM. Figure 32B: Fitting curve analysis to measure the binding / internalization affinity of Cy3-labeled EpCAM aptamer by different mouse (left) and human (right) BC cell lines. The binding / internalization affinity values ​​(Kd) for each cell line are shown in the table. Each data point is collected from pooled samples from three replicate wells. Each experiment was repeated at least twice with similar results. [Figure 32B] See the description of Figure 32A. [Figure 33-1]Figures 33A-33G illustrate the titration of siRNA in BC cells. Figure 33A: Comparison of UPF2 mRNA levels in MDA-MB-231 cells transfected with different concentrations of human-specific or mouse and human cross-reactive (CR) UPF2 siRNA. Figure 33B: UPF2 gene knockdown efficiency of 100 nM UPF2 CR siRNA in different mouse and human BC cell lines. Figures 33C-33G: Comparison of CD47 (Figure 33C), PARP1 (Figure 33D), APE1 (Figure 33E), MCL1 (Figure 33F), and PD-L1 (Figure 33G) mRNA levels in 4T1E cells transfected with different concentrations of mouse-specific and / or mouse and human CR siRNA. Arrows in each graph indicate the siRNA selected for EpCAM AsiC design. Data represent the mean + sem performed in duplicate (Figures 33A, 33C-33G, n=2 / condition) or triplicate (Figure 33B, n=3). [Figure 33-2] See the description of Figure 33-1. [Figure 34-1]Figures 34A-34I illustrate EpCAM AsiC knockdown gene expression in EpCAM+ 4T1E cells in vitro and in vivo. Figure 34A: Gene knockdown efficiency of 100 nM siRNA targeting each gene in 4T1E tumor cells (n=3 / group). Control was cells treated with negative control siRNA. Figure 34B: Design of CD47 AsiC. The folding structure of the EpCAM aptamer is predicted by the mfold web server. Figure 34B discloses SEQ ID NOs: 128 and 78, respectively, in order of appearance. Figure 34C: Gene knockdown efficiency of 4 mM EpCAM-AsiC targeting each gene in 4T1E tumor cells (n=3 / group). Control was cells treated with EpCAM aptamer (Apt). Mock: medium control. Figure 34D: Comparison of UPF2 mRNA levels in EpCAM+ 4T1E tumor cells and EpCAM-CD45- cells isolated from 4T1E tumors treated with EpCAM aptamer, eGFP AsiC, or UPF2 AsiC. Figure 34E: Comparison of %UPF2+ cells in EpCAM+ 4T1E tumor cells from mice treated with EpCAM aptamer, eGFP AsiC, or UPF2 AsiC. Right: Representative flow images. Figure 34F: Ratio of mRNA to pre-mRNA for well-established NMD substrates in EpCAM+ tumor cells from mice treated with UPF2 AsiC, normalized to that from mice treated with EpCAM aptamer. Figures 34D-34F, n=3 mice / group. Figures 34G-34I, Comparison of CD47 mRNA (Figure 34GF), PARP1 mRNA (Figure 34H), and MCL1 mRNA levels (Figure 34I) in EpCAM+ 4T1E tumor cells and EpCAM-CD45- cells isolated from 4T1E tumors treated with EpCAM aptamer, CD47 AsiC, PARP1 AsiC, or MCL1 AsiC. n=3 mice / group. Data are mean + sem and represent at least two independent experiments. [Figure 34-2] See the description of Figure 34-1. [Figure 34-3] See the description of Figure 34-1. [Figure 35]Figures 35A-35C illustrate the effects of siRNA gene knockdown and EpCAM-AsiC on BC cell viability. Figure 35A: Viability of 4T1E tumor cells treated for 72 hours with negative control siRNA or siRNA knocking down UPF2, CD47, PARP1, APEX1, PD-L1, or MCL1. Cell viability was assessed by CellTiter-Glo. n=5 samples / group. Figures 35B-35C: Viability (Figure 35B) and rate of cell proliferation over 3 days (Figure 35C) of 4T1E tumor cells treated for 72 hours with medium (mock), 4 mM EpCAM aptamer, or 4 mM EpCAM AsiC targeting UPF2 or CD47. n=4 / group. Data shown are mean + sem. [Figure 36] Figures 36A-36B show the long-term tumor inhibition and immunomodulatory capabilities of UPF2 AsiC. Figure 36A: Eight-day-old 4T1E tumor-bearing mice were treated with either EpCAM Apt or UPF2 AsiC (5 mg / kg, every 3 days; arrows indicate loaded mice). Tumor growth kinetics is shown. Figure 36B: Percentage of IFN-g and TNF-producing CD8+ TILs induced by PMA and ionomycin. EpCAM Apt group, n=7; UPF2 AsiC group, n=8. Data represent mean + sem. [Figure 37]Figures 37-37B show that UPF2 knockdown in EpCAMhi MDA-MB-231 BC cells generated novel mRNA isoforms and increased the usage of NMD-sensitive isoforms. Figure 37A, left: Various mRNA isoforms (including NMD-insensitive and NMD-sensitive transcripts) for the DNAJC2 gene (top) and the LAT2 gene (bottom). Right: Comparison of isoform fractions (IF) between cells treated with negative siRNA (light gray) and UPF2 siRNA (black). Figure 37B, left: Two mRNA isoforms (one protein-coding transcript and one NMD-sensitive transcript) for the CENPH gene. Right: Comparison of isoform fractions (IF) between cells treated with negative siRNA (light gray) and UPF2 siRNA (black). Figures 37A-37B, left graphs, longer black bars indicate protein-coding exons; shorter black bars indicate non-coding exons; intervening lines represent introns. [Figure 38] Figures 38A-38C show the tumor-suppressing and immunomodulatory capabilities of other EpCAM AsiCs. Figure 38A: Comparison of tumor growth in 4T1E tumor-bearing mice treated with EpCAM Apt or APE1 AsiC. Figure 38B: PD-L1 expression on EpCAM+ 4T1E tumor cells from 2-week-old 4T1E tumor-bearing mice. Cells were gated on live+ CD45- EpCAM+ cells. Figure 38C: Comparison of 4T1E tumor growth in mice treated with EpCAM Apt or PD-L1 AsiC. Figures 38A and 38C, n=5 mice / group. Arrows indicate each treatment. Data represent mean + sem. [Figure 39]Figures 39A-39C illustrate the gating strategy for MDSC subsets and M1- and M2-like TAMs in 4T1E mouse breast tumors. In Figure 39A, mononuclear, singlet, and viable tumor-infiltrating immune cells were first gated on CD45+ cells while gating out CD3+ / CD19+ / TCRb+ / Ter119+ cells. The remaining CD45+ myeloid cells were gated on Gr-1hiCD11b+ PMN-MDSCs, Gr-1intCD11b+ MO-MDSCs, and Gr-1-CD11b+ cells. Gr-1-CD11b+ cells were then gated on F4 / 80+ TAMs and CD206-MHCII+ M1-like TAMs and CD206+MHCII+ M2-like TAMs. Figure 39B: Comparison of the ratio of M1 TAM to M2 TAM in 4T1E tumors treated with EpCAM aptamer or CD47 AsiC. Cells were gated on CD45+CD11b+F4 / 80+MCHII+ TAM. Numbers indicate the percentage of each subset relative to TAM. Figure 39C: Phagocytosis by TAM of 4T1E-eGFP tumor cells treated with negative siRNA or CD47 siRNA in vitro. Numbers indicate the percentage of GFP+ TAM. [Figure 40] Figures 40A-40C illustrate the depletion of CD8+ T cells, CD4+ T cells, and macrophages in 4T1E tumor-bearing mice. Figure 40A: Experimental scheme of CD47 AsiC treatment and immune cell depletion in 4T1E tumor-bearing mice. Figure 40B: Representative flow plots of CD4+ T cells and CD8+ T cells in the peripheral blood of mice treated with isotype control Ab or anti-CD8 Ab and / or anti-CD4 Ab. Figure 40C: Effect of TAM depletion in mice treated with isotype control or anti-CSF1R Ab (0.3 mg / treatment). Data shown are the percentage of CD11b+F4 / 80+MHCII+ TAMs relative to CD45+ tumor-infiltrating immune cells. n=5 mice / group. Data shown are mean + sem. [Figure 41]Figures 41A-41C illustrate a comparison of anti-tumor efficacy between CD47 AsiC and anti-CD47 Ab. Figures 41A-41B, Percentage of IFN-g and TNF-producing CD8+ TIL (Figure 41A) and CD4+ TIL (Figure 41B) cells induced by PMA and ionomycin in mice treated with either EpCAM Apt, CD47 AsiC, or anti-CD47 Ab. Figure 41C, Percentage of PMN-MDSC and MO-MDSC relative to viable cells. Right: Representative flow plots of these two cell subsets in three groups of mice. a-c, n=5 mice / group. Data shown are mean + sem. [Figure 42] Figures 42A-42F illustrate the tumor-inhibitory and immunomodulatory capabilities of MCL1 AsiC. Figure 42A: Viability of 4T1E tumor cells treated with 4 mM EpCAM aptamer or EpCAM AsiC targeting MCL1 for 48-96 hours. n=3 samples / group. Figure 42B: Comparison of tumor growth in mice bearing 4T1E tumors treated with either EpCAM Apt or MCL1 AsiC. Arrows indicate each treatment. Figure 42C: Ratio of CD8+ TILs to tumor-infiltrating CD4+Foxp3+ Tregs in each tumor group. Figures 42D-42E: Percentage of CD8+ TILs (Figure 42Dd) and CD4+ TILs (Figure 42E) producing IFN-g and TNF induced by PMA and ionomycin. Figure 42F: Production of the cytotoxic molecules granzyme B and perforin by CD8+ TILs. Figures 42B-42F, n=5 mice / group. Figures 42A-42F, data shown are mean+sem and represent two experiments. [Figure 43]Figures 43A-43G illustrate the synergistic effect of immunomodulatory EpCAM-AsiC in the 4T1E-eGFP tumor model. Figure 43A: Number of CD8+ TILs per mg of tumor in mice bearing 4T1E-eGFP tumors treated with the EpCAM aptamer or a combination of four EpCAM-AsiCs targeting UPF2, CD47, MCL1, and Parp1. Figure 43B: Ratio of CD8+ TILs to tumor-infiltrating CD4+Foxp3+ Tregs in each group of tumors. c-d: Percentage of CD8+ TILs (Figure 43C) and CD4+ TILs (Figure 43D) producing IFN-g and TNF, induced by PMA and ionomycin. Figures 43E-43F: Production of cytotoxic granules, granzyme B and perforin, by CD8+ TILs (Figure 43E) and CD4+ TILs (Figure 43F). Figure 43G, Mean fluorescence intensity (MFI) of EpCAM expression for eGFP+ tumor cells from each group of tumors. Figures 43A-43G, n=5 mice / group. Data shown are mean + sem. [Figure 44] Figures 44A-44E illustrate the synergistic effect of immunomodulatory EpCAM-AsiC and anti-PD-1. Figure 44A: MFI levels of expression of the co-inhibitors PD-1, CTLA-4, 2B4, TIM-3, and LAG-3 on CD44+CD8+ TILs in mice bearing 4T1E tumors treated with EpCAM aptamer or combined AsiC with isotype or anti-PD-1 antibodies. Figure 44B: Number of CD8+ TILs per mg of tumor in each group of mice. Figure 44C: Number of NK cells per mg of tumor. Figures 44D-44E: Percentage of IFN-g and TNF-producing CD8+ TILs (Figure 44D) and CD4+ TILs (Figure 44E) induced by PMA and ionomycin. Figures 44A-44E, n=4 mice / group. Data shown are mean + sem. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description Targeting therapeutic molecules to diseased cells can improve the effectiveness of treatment and reduce side effects.The technology described herein relates to a chimeric molecule that binds to EpCAM, a common marker of epithelial cancer cells, and an inhibitory nucleic acid designed to target a specific gene that the inventors have demonstrated is essential for the growth and survival of cancer cells.The specific chimeric molecule described herein has been demonstrated to have surprisingly improved efficacy over earlier generations of such chimeric molecules, and can also exhibit synergistic effects when used in combination.Therefore, the present invention describes improved compositions and methods for treating cancer, such as epithelial cancer.

[0017] In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; PTPN2; MCL1; SMG1; TREX1; CMAS; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of UPF2; PARP1; MCL1; and CD47.

[0018] As used herein, a "chimeric molecule" refers to a molecule, e.g., a nucleic acid molecule, that contains two or more distinct regions, each composed of at least one monomer unit, i.e., in the case of AsiC compounds, a nucleotide. Chimeras are not naturally occurring molecules and are by definition engineered. The regions can be distinct in function or structure.

[0019] The EpCAM-binding aptamer domain specifically binds to EpCAM, thereby targeting the chimeric molecule to the cells that express EpCAM, such as cancer cells.This reduces both the size of therapeutic dose and the chance of off-target effects.As used herein, the term "aptamer" refers to the single-stranded nucleic acid that can bind to cells and target molecules (such as polypeptides).Nucleic acid aptamers include RNA, DNA, and / or synthetic nucleic acid analogs (such as PNA) that can specifically bind to target molecules.The generation and therapeutic use of aptamers have been well established in the art.See, for example, U.S. Patent No. 5,475,096.

[0020] As used herein, "EpCAM" or "epithelial cell adhesion molecule" refers to a transmembrane glycoprotein that mediates Ca2+-independent homotypic cell-cell adhesion in epithelial cells. The sequence of EpCAM is known for various species, for example, human EpCAM (see, for example, NCBI Gene ID: 4072; protein sequence: NCBI Ref Seq: NP_002345.2).

[0021] As a non-limiting example, an exemplary EpCAM-binding aptamer is provided herein. In some embodiments of any of the aspects, the EpCAM aptamer can comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 67 or 68. This aptamer has the particular advantage of working with similar potency against human and mouse EpCAM, allowing it to be tested in vivo in immunocompetent mice to determine whether it is immunostimulatory.

[0022] Table 1: Exemplary EpCAM aptamer sequences. [f] indicates a 2' fluro-pyrimidine modification. TIFF2025169317000001.tif27140

[0023] Additional EpCAM aptamers are known in the art. For example, The aptamer illustrated in TIFF2025169317000002.tif11160 and below is a known EpCAM aptamer. TIFF2025169317000003.tif154153

[0024] Further exemplary EpCAM aptamers are illustrated below and are commercially available from Aptagen (see, e.g., Kim et al. Identification of DNA Aptamers toward Epithelial Cell Adhesion Molecule via Cell-SELEX. Molecules and Cells, 2014, 37(10), 742-746 ISSN: 0219-1032, which is incorporated herein by reference in its entirety). TIFF2025169317000004.tif127128

[0025] In some embodiments of any of the aspects, the EpCAM aptamer can comprise, consist of, or consist essentially of the sequence of any of SEQ ID NOs: 63-68.

[0026] In some embodiments of any of the aspects, the EpCAM aptamer can comprise, consist of, or consist essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to the sequence of any of SEQ ID NOs: 63-68. In some embodiments of any of the aspects, the EpCAM aptamer can comprise, consist of, or consist essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to the sequence of any of SEQ ID NOs: 63-68 and retaining wild-type EpCAM binding activity.

[0027] As mentioned above, the chimeric molecule further comprises an inhibitory nucleic acid domain. As used herein, "inhibitory nucleic acid domain" refers to a domain that contains an inhibitory nucleic acid. As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can inhibit the expression of a target, such as double-stranded RNA (dsRNA), siRNA, miRNA, antisense oligonucleotide, etc. The use of inhibitory nucleic acid (iNA) allows targeted degradation of mRNA transcripts, resulting in a decrease in the expression and / or activity of the target, or changes in mRNA processing (for example, changes in splicing).

[0028] An inhibitory nucleic acid domain comprises one inhibitory nucleic acid, although the chimeric molecules described herein can comprise multiple inhibitory nucleic acid domains, e.g., repeats of a single inhibitory nucleic acid domain, or a collection of multiple different inhibitory nucleic acid domains.

[0029] In some embodiments of any of the aspects, the inhibitory nucleic acid can be an siRNA. In some embodiments of any of the aspects, the compositions described herein can include an EpCAM-binding domain that includes an aptamer and an inhibitory nucleic acid domain that includes an siRNA, for example, the composition can include an aptamer-siRNA chimera (AsiC).

[0030] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can comprise an RNA strand (antisense strand) having a region of 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of a targeted mRNA transcript.

[0031] As used herein, the terms "inhibitory oligonucleotide," "inhibitory nucleic acid," or "iNA" refer to an agent containing an oligonucleotide, e.g., a DNA or RNA molecule, that mediates targeted cleavage of an RNA transcript. In some embodiments of any of the aspects, the inhibitory oligonucleotides described herein result in inhibition of the expression and / or activity of a target gene. Inhibitory nucleic acids useful in the methods and compositions of the invention include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases / locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of a target nucleic acid and modulate its function. In some embodiments of any of the aspects, the inhibitory nucleic acid includes antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified linkages, interfering RNA (RNAi), short interfering RNA (siRNA); micro-interfering RNA (miRNA); small, temporal RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA), or a combination thereof. For further disclosure regarding inhibitory nucleic acids, see US2010 / 0317718 (antisense oligos); US2010 / 0249052 (double-stranded ribonucleic acid (dsRNA)); US2009 / 0181914 and US2010 / 0234451 (LNAs); US2007 / 0191294 (siRNA analogs); US2008 / 0249039 (modified siRNAs); and WO2010 / 129746 and WO2010 / 040112 (inhibitory nucleic acids).

[0032] In some embodiments of any of the aspects, the iNAs described herein result in the inhibition of the expression and / or activity of a target, e.g., one or more of the genes described herein. In some embodiments of any of the aspects, contacting a cell with an inhibitor (e.g., an iNA) results in a reduction in target mRNA levels in the cell of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to, and including, 100% of the target mRNA levels found in a cell without the presence of the iNA. In some embodiments of any of the aspects, administering an inhibitor (e.g., an iNA) to a subject results in a reduction in target mRNA levels in the subject of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA levels found in the subject without the presence of the iNA.

[0033] In some embodiments of any of the aspects, the iNA can be a dsRNA. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize and form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, generally perfectly complementary, to the target sequence. The target sequence can be derived from the sequence of an mRNA formed during expression of the target and can span, for example, one or more intron boundaries. The other strand (the sense strand) comprises a region that is complementary to the antisense strand, such that the two strands hybridize to form a duplex structure when combined under appropriate conditions. Typically, the duplex structure is 15 to 30 base pairs in length (inclusive), more typically 18 to 25 base pairs in length (inclusive), even more typically 19 to 24 base pairs in length (inclusive), and most typically 19 to 22 base pairs in length (inclusive). Similarly, the region of complementarity to the target sequence is 15 to 30 base pairs in length (inclusive), more commonly 18 to 25 base pairs in length (inclusive), even more commonly 19 to 24 base pairs in length (inclusive), and most commonly 19 to 21 base pairs in length (inclusive). In some embodiments of any of the aspects, the dsRNA is 15 to 20 nucleotides in length (inclusive), and in other embodiments, the dsRNA is 25 to 30 nucleotides in length (inclusive). As those skilled in the art will recognize, the target region of an RNA that is targeted for cleavage is most often a portion of a larger RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNA with duplexes as short as 9 base pairs can mediate RNAi-directed RNA cleavage under some circumstances. In most cases, the target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0034] Exemplary embodiments of types of inhibitory nucleic acids can include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art.

[0035] In some embodiments of any of the aspects, the nucleic acid of the iNA, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanded repertoire of partners, removal of a base (abasic nucleotide), or a conjugated base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, the RNA that contains modified backbone or non-natural internucleoside bond.The RNA with modified backbone includes, among others, that does not have phosphorus atom in backbone.For the purpose of this specification and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments of any aspect, the modified RNA has phosphorus atom in its internucleoside backbone.

[0036] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates, including 3'-alkylenephosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified RNA backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; those with amide backbones; and others with mixed N, O, S, and CH constituent moieties. and oligonucleosides having heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the native phosphodiester backbone is represented as --O--P--O--CH2--).

[0037] In other RNA mimics suitable for or intended for use in iNA, both the sugar and internucleoside linkage, i.e., backbone, of the nucleotide unit are replaced with novel groups. The basic unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are held to the aza nitrogen atom of the amide portion of the backbone and are bound directly or indirectly.

[0038] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0039] The RNA of an iRNA can also be modified to contain another unlocked nucleic acid (UNA). A UNA is an acyclic derivative of RNA that lacks the C2'-C3' bond of the ribose ring. See, for example, Langkjaer et al. Bioorganic & Medicinal Chemistry 2009 17:5420-5. A UNA at the 5' end of an RNA molecule can improve iRNA targeting. See, for example, Snead et al. Molecular Therapy Nucleic Acids 2013 2:E103. In some embodiments, the 5' position of the chimeric molecule and / or inhibitory nucleic acid is a UNA.

[0040] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In some embodiments of any of the aspects, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3; also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group.Another exemplary modification is the O(CH)ON(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, group, described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH--O--CH--N(CH), also described herein below in the Examples.

[0041] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked dsRNA, and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0042] Inhibitory nucleic acids can also include modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-methyluracil (pseudouracil), 5-hydroxymethyluracil (5-methyl-C ... Other synthetic and natural nucleobases include uracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyanal, other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids described in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications, have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution.

[0043] The preparation of the above-described modified nucleic acids, backbones, and nucleobases is well known in the art.

[0044] Another modification of the inhibitory nucleic acids featured in the invention involves chemically linking the inhibitory nucleic acid to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 4:1053-1060), and the like. 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0045] The double-stranded inhibitory nucleic acids described herein can further comprise one or more single-stranded nucleotide overhangs. Double-stranded inhibitory nucleic acids can be synthesized by standard methods known in the art, as discussed further below, for example, by use of an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc. In some embodiments of any of the aspects, the antisense strand of the double-stranded inhibitory nucleic acid has an overhang of 1 to 10 nucleotides at the 3' and / or 5' end. In some embodiments of any of the aspects, the sense strand of the double-stranded inhibitory nucleic acid has an overhang of 1 to 10 nucleotides at the 3' and / or 5' end. In some embodiments of any of the aspects, at least one end of the double-stranded inhibitory nucleic acid has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2, nucleotides. Double-stranded inhibitory nucleic acids with at least one nucleotide overhang have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. In some embodiments of any of the aspects, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0046] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an inhibitory nucleic acid, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a double-stranded inhibitory nucleic acid extends beyond the 5'-end of the other strand, or vice versa. A double-stranded inhibitory nucleic acid can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang can be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a double-stranded inhibitory nucleic acid.

[0047] The term "blunt" or "blunt-ended" used herein in relation to double-stranded inhibitory nucleic acid means that there are no unpaired nucleotides or nucleotide analogs at a given end of dsRNA, i.e., there are no nucleotide overhangs.One or both ends of double-stranded inhibitory nucleic acid can be blunt.When both ends of double-stranded inhibitory nucleic acid are blunt, double-stranded inhibitory nucleic acid is said to be blunt-ended.For clarity, a "blunt-ended" double-stranded inhibitory nucleic acid is a double-stranded inhibitory nucleic acid that has both ends blunt, i.e., there are no nucleotide overhangs at either end of the molecule.In most cases, such a molecule will be double-stranded throughout its entire length.

[0048] In this aspect, one of the two strands is complementary to the other of the two strands, and one of the strands is substantially complementary to the sequence of target gene precursor or mature miRNA.Therefore, in this aspect, double-stranded inhibitory nucleic acid comprises two oligonucleotides, one oligonucleotide is described as sense strand, and the second oligonucleotide is described as the corresponding antisense strand of sense strand.As described elsewhere herein and known in the art, the complementary sequence of double-stranded inhibitory nucleic acid can also be contained as the self-complementary region of a single nucleic acid molecule, rather than being on separate oligonucleotides.In some embodiments, only a part of the molecule, for example, only inhibitory nucleic acid domain, is a double-stranded molecule.

[0049] Those skilled in the art are well aware that inhibitory nucleic acids having a duplex structure of 20-23, but particularly 21, base pairs have been found to be particularly effective in inducing antisense-mediated inhibition (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer inhibitory nucleic acids can be similarly effective.

[0050] Furthermore, it is contemplated that further optimization can be achieved for any identified sequence by systematically adding or removing nucleotides to generate longer or shorter sequences, and testing the sequences generated by walking up or down the target RNA from that point through longer or shorter size windows.Again, this approach to generating new candidate targets, combined with testing the effectiveness of inhibitory nucleic acids based on their target sequences in inhibition assays known in the art or described herein, can result in further improvement of inhibition efficiency.Furthermore, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or changing in overhangs, or other modifications known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0051] The inhibitory nucleic acids described herein can contain one or more mismatches to the target sequence. In some embodiments of any of the aspects, the inhibitory nucleic acids described herein contain three or fewer mismatches. When the antisense strand of an inhibitory nucleic acid contains mismatches to the target sequence, the area of ​​mismatch is preferably not located in the center of the region of complementarity. When the antisense strand of an inhibitory nucleic acid contains mismatches to the target sequence, the mismatch is preferably limited to within the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23-nucleotide inhibitory nucleic acid strand complementary to a region of a target gene or its precursor, the strand generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an inhibitory nucleic acid containing mismatches to the target sequence is effective in inhibiting the expression of a target gene. In particular, when a specific region of complementarity in a target gene is known to have polymorphic sequence variation within the population, it is important to consider the efficacy of the inhibitory nucleic acid with a mismatch in inhibiting the expression of the target gene.

[0052] In some embodiments of any of the aspects, the ligand alters the distribution, targeting, or lifespan of the inhibitory nucleic acid agent into which it is incorporated. In some embodiments of any of the aspects, the ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cellular or organ compartment, a tissue, an organ, or a region of the body, e.g., compared to a species in which such a ligand is not present. Preferred ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0053] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0054] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a hepatocyte or macrophage, among others. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0055] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)isothiazolinone, and the like.

[0039] Included are compounds such as (amino) cholenoic acid, dimethoxytrityl, or phenoxazine, and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0056] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types such as hepatocytes or macrophages. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.

[0057] The ligand can be, for example, a substance, e.g., a drug, that can increase the uptake of an inhibitory nucleic acid agent into a cell by, for example, disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0058] In some embodiments of any of the aspects, the ligand attached to the inhibitory nucleic acid described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, containing multiple phosphorothioate linkages in the backbone, are also acceptable as ligands (e.g., as PK-modulating ligands) in the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0059] For macromolecular and hydrophilic drug molecules that cannot easily cross bilayer membranes, entrapment in the endosomal / lysosomal compartments of cells is considered to be the greatest obstacle to their effective delivery to the site of action. Numerous approaches and strategies have been devised to address this problem. For liposomal formulations, the use of fusogenic lipids in the formulation is the most common approach (Singh, RS, Goncalves, C. et al. (2004). On the Gene Delivery Efficacies of pH-Sensitive Cationic Lipids via Endosomal Protonation. A Chemical Biology Investigation. Chem. Biol. 11, 713-723.). Other components that exhibit pH-sensitive endosomolytic activity through protonation and / or pH-induced conformational changes include charged polymers and peptides.Examples include Hoffman, AS, Stayton, PS et al. (2002). Design of "smart" polymers that can direct intracellular drug delivery. Polymers Adv. Technol. 13, 992-999;Kakudo, Chaki, T., S. et al. (2004). Transferrin-Modified Liposomes Equipped with a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery System. Biochemistry 436, 5618-5628;Yessine, MA and Leroux, JC (2004). Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug Deliv. Rev. 56, 999-1021;Oliveira, S., van Rooy, I. et al. (2007). Fusogenic peptides enhance endosomal escape improving Inhibitory nucleic acid-induced silencing of oncogenes. Int. J. Pharm. 331, 211-4. They are generally used in the context of drug delivery systems, such as liposomes or lipoplexes.For example, for folate receptor-mediated delivery using liposomal formulations, pH-sensitive fusogenic peptides have been incorporated into liposomes to enhance activity by improving drug unloading during the uptake process (Turk, MJ, Reddy, JA et al. (2002). Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs is described in Biochim. Biophys. Acta 1559, 56-68).

[0060] The chimeric molecules described herein can be conjugated or attached to a polymer to extend their half-life. Suitable polymers include cholesterol, PEG, liposomes, or Fc.

[0061] In certain embodiments, the endosomolytic component can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and / or fusogenicity. Peptidomimetics can be small protein-like chains designed to mimic peptides. Peptidomimetics can result from the modification of existing peptides to alter the molecular properties or from the synthesis of peptide-like molecules using unnatural amino acids or their analogs. In certain embodiments, they have improved stability and / or biological activity compared to peptides. In certain embodiments, the endosomolytic component exhibits its active conformation at endosomal pH (e.g., pH 5-6). An "active" conformation is one in which the endosomolytic component promotes endosome lysis and / or transport of the modular composition of the present invention or any of its components (e.g., nucleic acids) from the endosomes to the cytoplasm of a cell.

[0062] Exemplary endosomolytic components include GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments, the endosomolytic component can contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component can be linear or branched. Exemplary primary sequences of endosomolytic components include: Contains TIFF2025169317000005.tif17156.

[0063] In certain embodiments, more than one endosomolytic component can be incorporated into the inhibitory nucleic acid agent of the present invention.In some embodiments of any of the aspects, this will involve incorporating more than one of the same endosomolytic component into the inhibitory nucleic acid agent.In other embodiments, this will involve incorporating two or more different endosomolytic components into the inhibitory nucleic acid agent.

[0064] These endosomolytic components can mediate endosomal escape, for example, by changing their conformation at endosomal pH. In certain embodiments, endosomolytic components exist in a random coil conformation at neutral pH and can rearrange into an amphipathic helix at endosomal pH. As a result of this conformational transition, these peptides may insert into the lipid membrane of the endosome, causing leakage of endosomal contents into the cytoplasm. Because the conformational transition is pH-dependent, endosomolytic components may exhibit little or no fusogenic activity while circulating in the blood (pH approximately 7.4). As used herein, "fusogenic activity" is defined as the activity that results in the disruption of the lipid membrane by the endosomolytic component. One example of fusogenic activity is the disruption of the endosomal membrane by the endosomolytic component, which results in endosomal lysis or leakage and the transport of one or more components (e.g., nucleic acids) of the modular composition of the present invention from the endosome to the cytoplasm.

[0065] Those skilled in the art can test and identify suitable endosomolytic components.For example, the ability of a compound to respond to pH environment, for example, change charge, can be tested by routine methods, for example, in cell assays.In certain embodiments, test compound is combined with or contacted with cells, and the cells are allowed to internalize the test compound, for example, by endocytosis.Then, endosome preparations can be made from the contacted cells, and can be compared with the endosome preparations from control cells.A change, for example, a decrease, in the endosomal fraction from the contacted cells compared to the control cells indicates that the test compound can function as a fusogenic agent.Alternatively, the contacted cells and the control cells can be evaluated, for example, by microscopic observation, for example, by optical microscopy or electron microscopy, to determine the difference in the endosomal population in cells.Test compound and / or endosomes can be labeled, for example, to quantify endosomal leakage.

[0066] In another type of assay, the inhibitory nucleic acid agent described herein is constructed with one or more test fusion agents or putative fusion agents.The inhibitory nucleic acid agent can be labeled for easy visualization.Once the inhibitory nucleic acid agent is taken up by cells, the ability of endosomolytic components to promote endosomal escape can be evaluated, for example, by preparing endosome preparations or by microscopic observation techniques that allow the visualization of labeled inhibitory nucleic acid agent in the cytoplasm of cells.In certain other embodiments, the inhibition of gene expression or any other physiological parameter can be used as a surrogate marker for endosomal escape.

[0067] In other embodiments, circular dichroism spectroscopy can be used to identify compounds that exhibit pH-dependent structural transitions.Two-stage assays can also be performed, where the first assay evaluates the ability of the test compound alone to respond to changes in pH, and the second assay evaluates the ability of the modular composition that includes the test compound to respond to changes in pH.

[0068] In some embodiments of the aspects described herein, the ligand or conjugate is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue, for example, non-renal target tissue of the body.Other molecules that can bind to HSA can also be used as ligand.For example, neproxin or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) be used to adjust the binding to serum protein, for example, HAS.

[0069] In another aspect, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent.Preferably, such an agent is amphipathic.An exemplary agent is a peptide such as tat or antennopedia.When the agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids.The helical agent is preferably an α-helical agent, and preferably has a lipophilic phase and a lipophobic phase.

[0070] Peptides suitable for use in the present invention can be natural peptides, such as tat or antenopedia peptides, synthetic peptides, or peptidomimetics. Furthermore, peptides can be modified, for example, peptides can contain non-peptide or pseudopeptide bonds and D-amino acids. Peptidomimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to inhibitory nucleic acid agents can affect the pharmacokinetic distribution of the inhibitory nucleic acid, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0071] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can comprise a hydrophobic membrane translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide has the amino acid sequence RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence TIFF2025169317000007.tif4128) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of carrying large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence from the HIV Tat protein TIFF2025169317000008.tif4128 and sequences derived from Drosophila Antennapedia protein TIFF2025169317000009.tif4128 has been found to function as a delivery peptide. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic tethered to the dsRNA agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the following structural modifications can be utilized:

[0072] A "cell-penetrating peptide" can penetrate cells, such as microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bisected amphipathic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0073] In some embodiments of any of the aspects, the inhibitory nucleic acid oligonucleotides described herein further comprise a carbohydrate conjugate. Carbohydrate conjugates are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound that has as part of its carbohydrate moiety one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or higher (preferably C5-C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-C8). In some embodiments of any of the aspects, the carbohydrate conjugate further comprises other ligands, such as, but not limited to, PK modulators, endosomolytic ligands, and cell-penetrating peptides.

[0074] In some embodiments of any of the aspects, the inhibitory nucleic acid domain specifically binds to the gene product of one of the genes listed herein (e.g., UPF2; PARP1; APE1; PD-L1; PTPN2; SMG1; TREX1; CMAS; CD47; PLK1; and / or MCL1). Those skilled in the art know how to design and create inhibitory nucleic acids that inhibit one or more of the genes listed herein. Illustrative, non-limiting examples of inhibitory nucleic acid domain sequences are provided herein below.

[0075] In some embodiments of any of the aspects, the inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; PTPN2; SMG1; MCL1; TREX1; CMAS; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. In one aspect of any of the embodiments, described herein is a chimeric molecule comprising an EpCAM-binding aptamer domain and at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; MCL1; and CD47.

[0076] As used herein, "UPF2" or "regulator of nonsense transcripts 2" refers to a gene encoding a protein that is part of an exon junction complex that regulates mRNA surveillance. The sequence of UPF2 is known in the art for many species, for example, human UPF2 (NCBI Gene ID: 26019) mRNA (NCBI Ref Seq: NM_015542.4 and NM_0805992.2).

[0077] As used herein, "PARP1" or "poly[ADP-ribose] polymerase 1" refers to a gene encoding poly ADP-ribosylase, which targets nuclear proteins on the single strand of DNA. PARP1 sequences are known in the art for many species, for example, human PARP1 (NCBI Gene ID: 142) mRNA (NCBI Ref Seq: NM_001618.4).

[0078] As used herein, "APE1," "APEX1," or "Apurinic / Apyrimidinic (AP) Endonuclease 1" refers to a gene encoding an endonuclease involved in base excision repair. The sequence of APE1 is known in the art for many species, for example, human APE1 (NCBI Gene ID: 328) mRNA (NCBI Ref Seq: NM_001244249.2, NM_001641.4, NM_080648.3, and NM_080649.3).

[0079] As used herein, "PD-L1" or "programmed death-ligand 1" refers to a gene encoding a transmembrane protein that regulates immune activity. Sequences of PD-L1 are known in the art for many species, for example, human PD-L1 (NCBI Gene ID: 29126) mRNA (NCBI Ref Seq: NM_001267706.1, NM_001314029.2, and NM_014143.4).

[0080] As used herein, "PTPN2" or "tyrosine protein phosphatase non-receptor type 2" refers to a gene encoding a tyrosine phosphatase that acts on EGFR and Shc. The sequence of PTPN2 is known in the art for many species, for example, human PTPN2 (NCBI Gene ID: 5771) mRNA (NCBI Ref Seq: NM_001207013.1, NM_001308287.1, NM_002828.4, NM_080422.2, and NM_080423.2).

[0081] As used herein, "SMG1" or "serine / threonine protein kinase 1" refers to a gene encoding a member of the phosphatidylinositol 3-kinase-related kinase protein family involved in the nonsense-mediated mRNA decay (NMD) pathway. The sequence of PTPN2 is known in the art for many species, e.g., human SMG1 (NCBI Gene ID: 23049) mRNA (NCBI Ref Seq: NM_015092.4).

[0082] As used herein, "TREX1" or "3 prime repair exonuclease" refers to the gene encoding the 5'-3' exonuclease that forms part of the SET complex. The sequence of TREX1 is known in the art for many species, for example, human TREX1 (NCBI Gene ID: 11277) mRNA (NCBI Ref Seq: NM_007248.5 and NM_033629.6).

[0083] As used herein, "CMAS" or "cytidine monophosphate N-acetylneuraminic acid synthase" refers to a gene encoding an enzyme that converts N-acetylneuraminic acid (NeuNAc) to cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuNAc). This process is important in the formation of sialylated glycoproteins and glycolipids. This modification plays a role in cell-to-cell communication and immune response. CMAS sequences are known in the art for many species, for example, human CMAS (NCBI Gene ID: 55907) mRNA (NCBI Ref Seq: NM_018686.6).

[0084] As used herein, "CD47" refers to a gene encoding a membrane protein involved in the increase in intracellular calcium concentration that occurs during cell adhesion to the extracellular matrix. The encoded protein is also a receptor for the C-terminal cell-binding domain of thrombospondin, and may play a role in membrane trafficking and signal transduction. The sequence of CD47 is known in the art for many species, for example, human CD47 (NCBI Gene ID: 961) mRNA (NCBI Ref Seq: NM_001777.3 and NM_198793.2).

[0085] The inhibitory nucleic acid domain of the chimeric molecules described herein can comprise one or more siRNA sequences. Exemplary siRNA sequences are provided in Tables 2 and 3, and in Tables 5 and 6, below.

[0086] In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 1-62, 69-126, and 149-162. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-62, 69-126, and 149-162. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-62, 69-126, and 149-162, and which retains the wild-type activity of the reference sequence (e.g., the ability to specifically bind to a target gene product and / or inhibit expression of a target gene).

[0087] In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 1-30, 38-56, 63-97, and 103-122. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-30, 38-56, 63-97, and 103-122. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-30, 38-56, 63-97, and 103-122, and which retains the wild-type activity of the reference sequence (e.g., the ability to specifically bind to a target gene product and / or inhibit expression of a target gene).

[0088] In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 1-25, 38-56, 63-92, and 103-122. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-25, 38-56, 63-92, and 103-122. In some embodiments of any of the aspects, the inhibitory nucleic acid domain comprises, consists of, or consists essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence selected from SEQ ID NOs: 1-25, 38-56, 63-92, and 103-122, and which retains the wild-type activity of the reference sequence (e.g., the ability to specifically bind to a target gene product and / or inhibit expression of a target gene).

[0089] Table 2: Exemplary siRNA sequences TIFF2025169317000010.tif47159TIFF2025169317000011.tif238159TIFF2025169317000012.tif151159

[0090] Table 3 provides pairs of sense and antisense sequences.It is contemplated herein that for the function of siRNA, either sense or antisense sequence can be incorporated into chimeric molecules, for example, in the same continuous nucleic acid strand as aptamer.Therefore, in some embodiments of any aspect, any chimeric molecule described herein can comprise one of the inhibitory nucleic acid sequences provided herein or its reverse complementary strand.

[0091] (Table 3) Exemplary inhibitory nucleic acid domains. [f] indicates a 2' fluoro-pyrimidine modification; {Phos(H).} indicates a 5' phosphate; d indicates a 2' deoxy base. When antisense sequences are provided, they are antisense to the direct sense sequence. TIFF2025169317000013.tif245165TIFF2025169317000014.tif222165

[0092] The chimeric molecules described herein can include more than one inhibitory nucleic acid domain, for example, in tandem or adjacent to an aptamer. Such a structure allows the chimeric molecule to inhibit the expression of multiple genes, provide increased doses of an inhibitory nucleic acid domain, and / or provide multiple different inhibitory nucleic acid domains targeting the same gene, thereby allowing for greater inhibition.

[0093] In some embodiments of any of the aspects, the chimeric molecule comprises a first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain, e.g., a second, and optionally a third, fourth, fifth, or more inhibitory nucleic acid domains.

[0094] In some embodiments of any of the aspects, the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise the same sequence. In some embodiments of any of the aspects, the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise different sequences but each inhibit expression of the same gene. In some embodiments of any of the aspects, the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise different sequences and each inhibit expression of a different gene. Any of the above combinations of inhibitory nucleic acid domains can reflect any of the possible pairwise combinations when three or more inhibitory nucleic acid domains are used. For example, in a chimeric molecule having three inhibitory nucleic acid domains in which the first and second domains comprise the same sequence, the third domain can comprise i) the same sequence, ii) a different sequence that inhibits expression of the same gene, or iii) a different sequence that inhibits expression of a different gene.

[0095] In some embodiments of any of the aspects, the at least second inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of PLK1 and MCL1.

[0096] As used herein, "PLK1" or "polo-like kinase 1" refers to a gene encoding a Ser / Thr protein kinase belonging to the CDC5 / Polo subfamily. It is highly expressed during mitosis. PLK1 sequences are known in the art for many species, for example, human PLK1 (NCBI Gene ID: 5347) mRNA (NCBI Ref Seq: NM_005030.6).

[0097] As used herein, "MCL1" or "myeloid leukemia cell differentiation protein 1" refers to a gene encoding a member of the Bcl-2 family that regulates apoptosis. Sequences of MCL1 are known in the art for many species, for example, human MCL1 (NCBI Gene ID: 4170) mRNA (NCBI Ref Seq: NM_001197320.1 and NM_021960.5).

[0098] In some embodiments of any of the aspects, described herein is a first chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47; and a second chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47. In some embodiments of any of the aspects, described herein is a first chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47; and a second chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. In some embodiments of any of the aspects, described herein is a first chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; and CD47; and a second chimeric molecule comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; and CD47.

[0099] In some embodiments of any of the aspects, described herein are at least four chimeric molecules that collectively comprise inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PARP1; and CD47. In some embodiments of any of the aspects, described herein are at least six chimeric molecules that collectively comprise inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PARP1; MCL1; PD-L1; and CD47.

[0100] In some embodiments of any of the aspects, the chimeric molecules described herein can include one or more linkers, for example, between the EpCAM-binding domain and the inhibitory nucleic acid, or between one or both of those domains and the additional ligand or moiety. The linkers can be cleavable or non-cleavable.

[0101] The term "linker" or "linking group" refers to a moiety (e.g., an organic moiety) that connects two portions of a compound. In some embodiments of any of the aspects, the linker can be a polypeptide or a nucleic acid that functions to attach two domains or portions. In some embodiments of any of the aspects, the linker connects the 5' EpCAM-binding domain to at least one 3' inhibitory nucleic acid domain. In some embodiments of any of the aspects, the linker connects the 3' EpCAM-binding domain to at least one 5' inhibitory nucleic acid domain.

[0102] The linker can comprise, for example, 1 to 1000 nucleotides or more. In some embodiments of any of the aspects, the linker comprises 1 to 100, 10 to 100, 100 to 900, 200 to 800, 300 to 700, 500 to 1000, or 700 to 1000 nucleotides. In some embodiments of any of the aspects, the linker can be 11 to 10 nucleotides in length, e.g., 1 to 3 nucleotides or 3 nucleotides in length.

[0103] The length of the linker can be optimized for one or more desired properties (eg, separation of domains, prevention of self-hybridization, etc.).

[0104] In some embodiments of any of the aspects, the linker can comprise a direct bond or an atom such as carbon, oxygen, or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkyl, alkylheteroaryl Arylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclyl acrylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkylheteroaryl; wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic;R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In some embodiments of any of the aspects, the linker is 1 to 24 atoms, preferably 4 to 24 atoms, preferably 6 to 18 atoms, more preferably 8 to 18 atoms, and most preferably 8 to 16 atoms;

[0105] A cleavable linking group is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties held together by the linker. In some embodiments of any of the aspects, the cleavable linking group is cleaved at least 10 times faster, and preferably at least 100 times faster, in the target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in blood or serum).

[0106] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include: oxidizing enzymes or reductases or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and the redox agents that are selected for specific substrates or do not have substrate specificity; esterase; endosomes or agents that can create an acidic environment, for example, those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (can be substrate specific), and phosphatases.

[0107] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0108] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the target cell. Further examples of cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., disulfide linking groups (-SS-)), phosphate-based cleavable linking groups, ester-based cleavable linking groups, and peptide-based cleavable linking groups.Representative United States patents that teach the preparation of RNA conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,60 No. 3; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824 ,941;No.4,835,263;No.4,876,335;No.4,904,582;No.4,958,013;No.5,082,830;No.5,112,963;No.5,214,136;No.5,082,830;No.5,1 No. 12,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810 ; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is incorporated herein by reference.

[0109] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradation agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition and a second condition, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluation in a cell-free or cultured condition and confirm with further evaluation in a whole animal. In some embodiments of any of the aspects, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0110] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single compound or even at a single nucleoside within an inhibitory nucleic acid.

[0111] In some embodiments of any of the aspects, the chimeric molecules described herein can include at least one region in which the nucleic acid has been modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid to the inhibitory nucleic acid. Additional regions of the inhibitory nucleic acid can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of the inhibitory nucleic acid in inhibiting gene expression. Consequently, when chimeric inhibitory nucleic acids are used, comparable results can often be obtained with shorter inhibitory nucleic acids compared to, for example, phosphorothioate deoxydsRNA hybridized to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0112] In certain instances, the nucleic acid of an inhibitory nucleic acid can be modified with a non-ligand group. Numerous non-ligand molecules have been conjugated to inhibitory nucleic acids to enhance their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), and the like. 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such nucleic acid conjugates are listed above. A typical conjugation protocol involves the synthesis of a nucleic acid bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either while the nucleic acid is still attached to the solid support or in solution phase after cleavage of the nucleic acid. Purification of the nucleic acid conjugate by HPLC typically results in a pure conjugate.

[0113] In some embodiments of any of the aspects, the chimeric molecule can further comprise a second strand, e.g., a nucleic acid strand capable of hybridizing to at least a portion of the inhibitory nucleic acid domain. Exemplary second or complementary (antisense) strands are provided elsewhere herein.

[0114] In some embodiments of any of the aspects, the chimeric molecule can further include a domain that is complementary to at least a portion of the inhibitory nucleic acid domain, e.g., a nucleic acid sequence that can hybridize to at least a portion of the inhibitory nucleic acid domain. Exemplary sequences are those provided elsewhere herein as the second or complementary (antisense) strand.

[0115] In some embodiments of any of the aspects, a chimeric molecule described herein can comprise, consist of, or consist essentially of the sequence of one of SEQ ID NOs: 127-137 and 163-168. In some embodiments of any of the aspects, a chimeric molecule described herein can comprise, consist of, or consist essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to the sequence of one of SEQ ID NOs: 127-137 and 163-168. In some embodiments of any of the aspects, the chimeric molecules described herein can comprise, consist of, or consist essentially of a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to one of SEQ ID NOs: 127-137 and 163-168, and that retains the wild-type activity (e.g., binding ability or cancer-inhibiting activity) of the reference sequence.

[0116] In some embodiments of any of the aspects, a chimeric molecule described herein can comprise, consist of, or consist essentially of a sequence of one of SEQ ID NOs: 127-137 and 163-168 to which an antisense sequence as shown in Table 4, 5, or 6 hybridizes. In some embodiments of any of the aspects, a chimeric molecule described herein can comprise, consist of, or consist essentially of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to a sequence of one of SEQ ID NOs: 127-137 and 163-168 to which an antisense sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to an antisense sequence as shown in Table 4, 5, or 6 hybridizes. In some embodiments of any of the aspects, a chimeric molecule described herein can comprise, consist of, or consist essentially of a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to one of SEQ ID NOs: 127-137 and 163-168, to which an antisense sequence hybridizes that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to the antisense sequence as set forth in Table 4, 5, or 6, and that retains the wild-type activity (e.g., binding ability or cancer-inhibiting activity) of the reference sequence.

[0117] (Table 4) Exemplary AsiCs. Note: The bolded portion indicates the EpCAM aptamer sequence; the italicized portion (UUU) is the linker region; the regular text region is the siRNA region. [f] indicates a 2' fluoro-pyrimidine modification; {Phos(H).} indicates a 5' phosphate; and d indicates a 2' deoxy base. TIFF2025169317000015.tif239161

[0118] In some embodiments of any of the aspects, the chimeric molecules described herein can further comprise, for example, a chemotherapeutic agent conjugated to the chimeric molecule. Exemplary, non-limiting chemotherapeutic agents include paclitaxel and other chemotherapeutic agents described herein.

[0119] In one aspect of any of the embodiments, described herein is a pharmaceutical composition, kit, or combination comprising at least one chimeric molecule described herein and, optionally, a pharmaceutically acceptable carrier. The composition, kit, or combination can comprise a plurality of sequence-distinct chimeric molecules or a population of single chimeric molecules.

[0120] In some embodiments of any of the aspects, the composition, kit, or combination comprises at least two chimeric molecules, the chimeric molecules having different aptamer domains or inhibitory nucleic acid domains. In some embodiments of any of the aspects, the different inhibitory nucleic acid domains recognize different targets. In some embodiments of any of the aspects, the different inhibitory nucleic acid domains have different sequences and recognize the same target.

[0121] In one aspect of any of the embodiments, i) a first chimeric molecule described herein, comprising at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; MCL1; PD-L1; PTPN2; SMG1; TREX1; CMAS; and CD47; and ii) a second chimeric molecule, comprising a chimeric molecule described herein, wherein at least one inhibitory nucleic acid domain of the second chimeric molecule inhibits expression of a different gene than the first chimeric molecule and / or inhibits expression of a gene selected from the group consisting of PLK1 and MCL1; a second chimeric molecule, Described herein are pharmaceutical compositions, kits, or combinations comprising:

[0122] A kit is a collection of materials or components, including at least one of the chimeric molecules described herein. The exact nature of the components configured in the kit depends on its intended purpose. In some embodiments of any of the aspects, the kit is specifically configured for human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0123] In some embodiments of any of the aspects, the kit includes instructions for use. "Instructions for use" typically include specific language describing techniques to be used in using the components of the kit to affect a desired outcome in a subject. Still in accordance with the present invention, "instructions for use" may include specific language describing the preparation of the chimeric molecule and / or at least one method parameter, such as dosage requirements and administration instructions, typically for an intended purpose. Optionally, the kit also contains other useful components, such as measuring tools, diluents, buffers, pharmaceutically acceptable carriers, syringes, or other useful implements, as would be readily recognized by one of ordinary skill in the art.

[0124] The materials or components assembled in the kit can be provided to the practitioner and stored in any convenient and suitable manner that preserves their operability and usefulness. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room temperature, refrigerated, or frozen. The components are typically contained in suitable packaging. As used herein, the phrase "packaging" refers to one or more physical structures used to contain the contents of the kit, such as the composition of the present invention. The packaging material is preferably constructed by well-known methods to provide a sterile, contaminant-free environment. The package also preferably provides an environment that protects from light, humidity, and oxygen. As used herein, the term "package" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, polyester (e.g., polyethylene terephthalate, or Mylar), capable of holding individual kit components. Thus, for example, the package can be a glass vial used to contain a suitable amount of a composition containing a certain volume of the chimeric molecule described herein. The packaging material generally has exterior labeling indicating the contents and / or purpose of the kit and / or its components.

[0125] In a combination of multiple chimeric molecules, the different chimeric molecules can be provided in a mixture or a single formulation, or they can be provided in separate formulations that are packaged or provided as a set or kit.

[0126] In some embodiments of any of the aspects, the technology described herein relates to a pharmaceutical composition comprising at least one chimeric molecule described herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments of any of the aspects, the active ingredient of the pharmaceutical composition comprises at least one chimeric molecule described herein. In some embodiments of any of the aspects, the active ingredient of the pharmaceutical composition consists essentially of at least one chimeric molecule described herein. In some embodiments of any of the aspects, the active ingredient of the pharmaceutical composition consists of at least one chimeric molecule described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C. 12 Alcohols, e.g., ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably herein. In some embodiments of any of the aspects, the carrier inhibits degradation of the active agent, e.g., at least one chimeric molecule described herein.

[0127] In some embodiments of any of the aspects, a pharmaceutical composition comprising at least one chimeric molecule described herein can be in a parenteral dosage form. Because parenteral dosage forms typically bypass a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized before administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for patient administration, including, but not limited to, DUROS®-type dosage forms and dose dumping.

[0128] Suitable vehicles that can be used to provide parenteral dosage forms of at least one chimeric molecule disclosed herein are well known to those skilled in the art. Examples include, but are not limited to: water for injection USP; saline; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of pharmaceutically acceptable salts of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the present disclosure, including conventional and controlled-release parenteral dosage forms.

[0129] Pharmaceutical compositions comprising at least one chimeric molecule can also be formulated to be suitable for oral administration, for example, as discrete dosage forms such as, but not limited to, tablets (including, but not limited to, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as, but not limited to, syrups, elixirs, solutions, or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of a disclosed compound and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia, PA. (2005).

[0130] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms can result in large variations in the concentration of the drug in the patient's blood and other tissues. These variations can affect numerous parameters, such as administration frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, etc. Advantageously, controlled-release formulations can be used to control the drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled-release or extended-release dosage forms or formulations can be used to ensure that maximum drug efficacy is achieved while minimizing potential adverse effects and safety concerns that can arise from both administering an underdose of the drug (i.e., below the minimum therapeutic level) and exceeding the drug's toxic level. In some embodiments of any of the aspects, at least one chimeric molecule can be administered in a sustained-release formulation.

[0131] Controlled-release pharmaceutical products share a common goal: improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled-release preparations in medical treatments is characterized by the use of a minimum amount of active pharmaceutical ingredient to cure or control a condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended drug activity; 2) reduced dosing frequency; 3) improved patient compliance; 4) less total drug use; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved treatment efficacy; 9) enhanced or reduced loss of drug activity; and 10) improved rate of disease or condition control. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0132] Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) that quickly produces the desired therapeutic effect, and then gradually and continuously release other amounts of drug to maintain this level of therapeutic or prophylactic effect over a long period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug being metabolized and excreted from the body. The controlled release of the active ingredient can be stimulated by various conditions, including but not limited to pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0133] A variety of known controlled release or long-term release dosage forms, formulations and devices can be adapted to be used with the salt and composition of the present disclosure.Examples include but are not limited to those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (e.g., OROS® (Alza Corporation, Mountain View, Calif. USA)), or combinations thereof, which provide the desired release profile in various ratios.

[0134] In some embodiments of any of the aspects, the chimeric molecule described herein is provided in a kit or combination with, or in a composition further comprising, or administered together with, an immune checkpoint inhibitor, e.g., an immune checkpoint inhibitor antibody reagent.

[0135] Immune checkpoint inhibitors inhibit one or more immune checkpoint proteins. The immune system has multiple inhibitory pathways, which are crucial for maintaining self-tolerance and regulating immune response. For example, in T cells, the amplitude and quality of response are initiated through antigen recognition by T cell receptors and regulated by immune checkpoint proteins, which balance costimulatory and inhibitory signals. In some embodiments of any of the aspects, subjects or patients are treated with at least one inhibitor of immune checkpoint proteins. As used herein, "immune checkpoint protein" refers to a protein that, when active, exhibits an inhibitory effect on immune activity, for example, T cell activity.Exemplary immune checkpoint proteins may include: PD-1 (e.g., NCBI Gene ID: 5133); PD-L1 (e.g., NCBI Gene ID: 29126); PD-L2 (e.g., NCBI Gene ID: 80380); TIM-3 (e.g., NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g., NCBI Gene ID: 201633); KIR (e.g., NCBI Gene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DD1-α (e.g., NCBI Gene ID: 64115); A2AR (e.g., NCBI Gene ID: 135); B7-H3 (e.g., NCBI Gene ID: 80381); B7-H4 (e.g., NCBI Gene ID: 79679); BTLA (e.g., NCBI Gene ID: 151888); IDO (e.g., NCBI Gene ID: 3620); TDO (e.g., NCBI Gene ID: 6999); HVEM (e.g., NCBI Gene ID: 8764); GAL9 (e.g., NCBI Gene ID: 3965); 2B4 (a member of the CD2 family of molecules, expressed in all NK, gamma delta, and memory CD8+ (alpha beta) T cells) (e.g., NCBI Gene ID: 51744); CD160 (also known as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7.

[0136] Non-limiting examples of immune checkpoint inhibitors (checkpoint target and manufacturer in parentheses) may include: MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb); atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (Ox40; Medimmune); CP-870,893 (CD40; Genentech); tremelimumab (CTLA-4; Mediimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Mediimmune); and IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211); anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834); TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med.207:2187-94); anti-CTLA-4 antibodies described in U.S. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238; tremelimumab (ticilimumab, CP-675,206); ipilimumab (10D1, also known as MDX-D010); U.S. Patent No. 7,488,802; PD-1 and PD-L1 blockers, as described in PCT published patent applications WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; nivolumab (MDX 1106, BMS 936558, ONO 4538); lambrolizumab (MK-3475 or SCH 900475); CT-011; AMP-224; and BMS-936559 (MDX-1105-01). The foregoing references are incorporated herein by reference in their entireties.

[0137] In some embodiments of any of the aspects, the immune checkpoint protein is PD-1 or PD-L1. In some embodiments of any of the aspects, the immune checkpoint protein is PD-1.

[0138] In some embodiments of any of the aspects, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb); atezolizumab (PD-L1; Genentech); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Mediimmune); or PD-1 and PD-L1 blockers described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT published patent application numbers: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. In some embodiments of any of the aspects, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb); pidilizumab (PD-1; Medivation); AUNP12 (PD-1; Aurigene); or a PD-1 blocker described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT published patent application numbers: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.

[0139] As used herein, the term "antibody reagent" refers to a polypeptide that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. An antibody reagent can include an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent can include a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies, such as single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments, as well as complete antibodies.

[0140] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. The term also refers to antibodies consisting of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as various forms, including full-length antibodies and antigen-binding portions thereof; for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), diabodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, bispecific antibodies, functionally active epitope-binding portions thereof, and / or bifunctional hybrid antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of a CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), and may be interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region is composed of three CDRs and four FRs, arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0141] Antibodies and / or antibody reagents can include immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab'), Fv, disulfide-linked Fv, scFv, single domain antibodies, diabodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, and functionally active epitope-binding portions thereof.

[0142] As used herein, the term "nanobody" or single domain antibody (sdAb) refers to antibodies containing small single variable domains (VHH) of antibodies derived from camelids and dromedaries. Antibody proteins derived from members of the camel and dromedary families (Camelus baclrianus and Calelus dromaderius), including members of new kingdoms such as llama species (alpacas, llamas, and vicunas), have been characterized for size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies from mammals of this family, as found in nature, lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains of antibodies derived from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published March 3, 1994, which is incorporated herein by reference in its entirety).

[0143] Regions of camelid antibodies, the small single variable domains specified as VHHs, can be genetically engineered to give rise to small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "camelid nanobodies". See U.S. Patent No. 5,759,808, issued June 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512-3520, each of which is incorporated by reference in its entirety. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, the amino acid sequences of camelid antibodies can be recombinantly modified to obtain sequences that more closely resemble human sequences, i.e., the nanobodies can be "humanized." Thus, the naturally low antigenicity of camelid antibodies to humans can be further reduced.

[0144] Camelid nanobodies have a molecular weight approximately one-tenth that of human IgG molecules, and the proteins have a physical diameter of only a few nanometers. One consequence of their small size is that they can bind to antigenic sites functionally invisible to larger antibody proteins, making them useful as reagents for detecting antigens that are otherwise obscure using classical immunological techniques, and as potential therapeutic agents. Thus, yet another consequence of their small size is that camelid nanobodies can bind to specific sites in grooves or narrow clefts of target proteins and thereby inhibit them, thus acting in a capacity more closely resembling that of classical low-molecular-weight drugs than that of classical antibodies. The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and proteolytic digestion, and poorly antigenic. See U.S. Patent Application 20040161738, published August 19, 2004, which is incorporated herein by reference in its entirety. These features, combined with low antigenicity to humans, indicate great therapeutic potential.

[0145] In some embodiments of any of the aspects, the method described herein relates to treating a subject who has cancer or has been diagnosed with cancer with the composition described herein.Subjects who have cancer can be identified by doctors using current methods for diagnosing cancer.The symptoms and / or complications of cancer that characterize these conditions and aid in diagnosis are well known in the art, and for example, in the case of breast cancer, include, but are not limited to, lumps or masses in breast tissue, swelling of all or part of the breast, skin irritation, breast cupping, breast or nipple pain, nipple involution, redness, scaly or irritation of the breast or nipple, and nipple discharge.For example, tests that can aid in the diagnosis of breast cancer include, but are not limited to, mammograms, X-rays, MRIs, ultrasounds, ductograms, biopsies, and ductal lavage.Family history of cancer or exposure to risk factors for cancer (e.g., smoking, radiation, pollutants, BRCA1 mutations, etc.).

[0146] In some embodiments of any of the aspects, the chimeric molecules described herein provide a therapeutic effect via immunotherapy, as opposed to simply directly killing tumor cells, for example. As described herein, certain chimeric molecules that directly kill tumors also induce an immune response in the tumor, and the combination of cytotoxic AsiC and immunomodulatory AsiC improves tumor suppression.

[0147] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissues. Cancer cells may also spread to other parts of the body through the blood and lymphatic system. There are several major types of cancer. Carcinoma is cancer that begins in the skin or in tissues lining or covering internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that begins in blood-forming tissues, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in cells of the immune system. Central nervous system cancer is cancer that begins in the tissues of the brain and spinal cord.

[0148] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion of and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or "metastasis." A metastatic tumor contains cells similar to those in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to a tumor that may grow larger but does not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0149] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0150] As used herein, the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, which growth exceeds and is uncoordinated with the growth of normal tissue. Thus, a neoplasm can be a benign neoplasm, a premalignant neoplasm, or a malignant neoplasm.

[0151] A subject with cancer or tumor is one who has objectively measurable cancer cells present in the subject's body. This definition includes malignant, actively growing cancers, as well as potentially dormant tumors or micrometastases. Cancers that migrate from their original location and disseminate to other vital organs may ultimately lead to the death of the subject through the deterioration of the affected organ's function.

[0152] Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia. Blood cancer; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., of the lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach Cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; man These include, but are not limited to, Toll cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0153] "Cancer cells" are cancerous, precancerous, or transformed cells, either in vivo, ex vivo, or in tissue culture, that undergo spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acid, or from the incorporation of exogenous nucleic acid, but can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is associated with, for example, morphological changes, cellular immortalization, aberrant growth control, foci formation, anchorage independence, malignant potential, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts, such as nude mice.

[0154] In some embodiments of any of the aspects, the cancer is an epithelial cancer. In some embodiments of any of the aspects, the cancer is breast cancer, colon cancer, or triple-negative breast cancer. In some embodiments of any of the aspects, the cancer is a HER2+ cancer. In some embodiments of any of the aspects, the cancer is not BRCA1 deficient, e.g., the patient does not have a BRCA1 mutation or oncomutation.

[0155] The compositions and methods described herein can be administered to a subject with or diagnosed with cancer. In some embodiments of any of the aspects, the methods described herein include administering to a subject an effective amount of a composition described herein to alleviate the symptoms of cancer. As used herein, "alleviating symptoms" of cancer refers to the amelioration of any condition or symptom associated with cancer. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or greater, as measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those skilled in the art. Such methods can include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. In some embodiments of any of the aspects, administration is subcutaneous.

[0156] The term "effective amount" as used herein refers to the amount of at least one chimeric molecule required to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a pharmacological composition sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of at least one chimeric molecule that is sufficient to provide a specific anti-cancer effect when administered to a typical subject. As used herein, the effective amount also includes an amount sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (for example, but not limited to, slow the progression of disease symptoms), or reverse disease symptoms in various situations. Therefore, it is generally not practical to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one skilled in the art using only routine experimentation.

[0157] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods exhibiting large therapeutic indices are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient that achieves a half-maximal suppression of symptoms) determined in cell culture or in an appropriate animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays, such as, inter alia, assays for tumor growth. Dosages can be determined by a physician and, if necessary, adjusted to suit the observed effects of treatment.

[0158] Effective dose, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the minimum effective dose and / or maximum tolerated dose. Dosage can vary depending on the dosage form used and the route of administration used. The therapeutically effective dose can be initially estimated from cell culture assays. Also, the dose can be formulated in animal models to achieve a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dosage can be monitored by suitable bioassays, for example, tumor growth and / or size assays, among others. Dosage can be determined by a physician and adjusted as needed to suit the observed effects of treatment.

[0159] In some embodiments of any of the aspects, at least one chimeric molecule described herein is administered as a monotherapy, eg, no other treatment for cancer is administered to the subject.

[0160] In some embodiments of any of the aspects, the methods described herein can further include administering a second agent and / or treatment to the subject, for example, as part of a combination therapy. In some embodiments of any of the aspects, the second agent is paclitaxel. In some embodiments of any of the aspects described herein, the second agent is a taxane (e.g., docetaxel or paclitaxel).

[0161] Non-limiting examples of second agents and / or treatments may include: radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa, and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; benzodopa aziridines such as carboquone, meturedopa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryothecins statins; kallistatins; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatins; chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidase nitrogen mustards such as cidohydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosaturates such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate;esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin n), puromycin, quelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, thiamiprine, and thioguanidine purine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; florinic acid Folic acid replenishers such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine;Maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);Retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)), and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0162] Additionally, the method of treatment can further include the use of radiation or radiotherapy. Additionally, the method of treatment can further include the use of surgical procedures.

[0163] In certain embodiments, an effective dose of a composition comprising at least one chimeric molecule described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising at least one chimeric molecule can be administered to a patient repeatedly. For systemic administration, a subject can be administered a therapeutic amount of a composition comprising at least one chimeric molecule, such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0164] In some embodiments of any aspect, after the initial treatment regimen, treatment can be administered less frequently.For example, after 3 months of treatment every other week, treatment can be repeated once a month for 6 months or 1 year or longer.Treatment by the methods described herein can reduce the level of markers or symptoms of condition, for example, tumor size or growth rate, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0165] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to suit the observed effects of treatment. Regarding the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment provides therapeutic benefit and whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosing schedule can vary from once a week to daily, depending on numerous clinical factors, such as the subject's sensitivity to at least one chimeric molecule. The desired dose or amount of activation can be administered all at once or divided into subdoses, e.g., 2 to 4 subdoses, administered at appropriate intervals over a period of time, e.g., throughout the day or other suitable schedule. In some embodiments of any of the aspects, administration can be chronic, e.g., one or more doses and / or treatments daily for a period of several weeks or months. Exemplary dosing and / or treatment schedules are daily, twice daily, three times daily, or four or more times daily for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or longer. A composition comprising at least one chimeric molecule can be administered over a period of time, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0166] The dosage range for administering at least one chimeric molecule according to the methods described herein depends, for example, on the form of the at least one chimeric molecule, its efficacy, and the degree to which the symptoms, markers, or indicators of the conditions described herein are desired to be reduced, for example, the desired percentage reduction in tumor size or growth rate. The dosage should not be so high as to cause adverse side effects. Generally, the dosage varies with the age, condition, and sex of the patient and can be determined by those skilled in the art. The dosage can also be adjusted by an individual physician in the unlikely event of any complications.

[0167] For example, the efficacy of at least one chimeric molecule in treating a condition described herein or in inducing a response described herein (e.g., reduction in tumor size and / or growth rate) can be determined by a skilled clinician. However, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically recognized symptoms are improved or even ameliorated, or the desired response is induced, for example, by at least 10% after treatment according to the methods described herein, the treatment is considered an "effective treatment," as that term is used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a condition treated according to the methods described herein, or any other suitable measurable parameter, such as cancer cell survival. Efficacy can also be measured by the absence of deterioration of an individual, as assessed by hospitalization, or the need for medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals), including: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount that, when administered to a subject in need thereof, is sufficient to result in effective treatment for the disease, as that term is defined herein. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response. It is well within the capabilities of one skilled in the art to monitor the efficacy of administration and / or treatment by measuring any one or any combination of such parameters. Efficacy can be assessed in animal models of the conditions described herein, e.g., the treatment of cancer. When using an experimental animal model, efficacy of treatment is demonstrated when a statistically significant change in a marker, e.g., a target gene in cancer cells, is observed.

[0168] Provided herein are in vitro assays and animal model assays that allow the evaluation of a given dose of at least one chimeric molecule.By way of non-limiting example, the effect of the dose of at least one chimeric molecule can be evaluated by cancer cell expression analysis or survival rate.The efficacy of a given dosage combination can also be evaluated in animal models, such as mouse models of cancer.

[0169] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the appended claims, definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0170] For convenience, certain terms used herein in the specification, examples, and appended claims are collected here.

[0171] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments of any of the aspects, "reduce," "reduction," or "reduce," or "inhibit" typically refer to a reduction of at least 10% compared to a reference level (e.g., a given level or the absence of an agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to the reference level. The decrease can preferably be down to a level that is considered to be within the normal range for individuals without the given disorder.

[0172] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments of any of the aspects, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% compared to a reference level, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0173] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaque monkeys, such as Rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments of any of the aspects, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0174] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects representing animal models of cancer. The subject can be male or female.

[0175] The subject can be a subject who has previously been diagnosed or identified as suffering from or having a condition (for example, cancer) that needs treatment or one or more complications related to such a condition, and optionally undergoes treatment for cancer or one or more complications related to cancer.Alternatively, the subject can also be a subject who has not previously been diagnosed with cancer or one or more complications related to cancer.For example, the subject can be a subject who shows one or more risk factors for cancer or one or more complications related to cancer, or a subject who does not show risk factors.

[0176] A "subject in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0177] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, although the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein to refer to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0178] In some embodiments of any of the aspects, it is further intended to encompass variants (naturally occurring or not), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" if the alterations result in the substitution of amino acids with chemically similar amino acids and retain the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0179] A given amino acid can be replaced by a residue with similar physicochemical properties, for example, by substituting one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for each other), or by substituting one polar residue for another (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity of the native or reference polypeptide, such as binding activity and specificity, is retained.

[0180] Amino acids can be grouped according to similarities in the properties of their side chains (AL Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class. Specific conservative substitutions include, for example: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0181] In some embodiments of any of the aspects, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described herein below. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0182] In some embodiments of any of the aspects, the polypeptides described herein can be variants of the sequences described herein. In some embodiments of any of the aspects, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of native nucleotide sequences. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide, but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding a variant polypeptide encompasses sequences that encode a variant protein or fragment thereof that contains one or more additions, deletions, or substitutions of nucleotides when compared with a native or reference DNA sequence, but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0183] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings).

[0184] Alterations to the native amino acid sequence can be achieved by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the required substitution, deletion, or insertion. Techniques for making such modifications are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, the entire contents of which are incorporated herein by reference. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or promote oligomerization.

[0185] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0186] The term "expression" refers to the cellular processes involved in producing RNA and proteins, and, where appropriate, secreting proteins, including, but not limited to, e.g., transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from one or more nucleic acid fragments of the invention, and / or the translation of mRNA into polypeptides.

[0187] In some embodiments of any of the aspects, expression of a biomarker, target, or gene / polypeptide described herein is tissue-specific. In some embodiments of any of the aspects, expression of a biomarker, target, or gene / polypeptide described herein is systemic. In some embodiments of any of the aspects, expression of a biomarker, target, or gene / polypeptide described herein is systemic.

[0188] "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0189] A "marker," in the context of the present invention, refers to an expression product, e.g., a nucleic acid or polypeptide, that is differentially present in a sample taken from a subject with cancer compared to a comparable sample taken from a control subject (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker."

[0190] In some embodiments of any of the aspects, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detecting" or "measuring" refers to observing a signal, for example, from a probe, a label, or a target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a specific labeled moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[0191] In some embodiments of any aspect, the polypeptide, nucleic acid or cell described herein can be engineered.As used herein, " engineered" refers to the aspect that is manipulated by human hands.For example, when at least one aspect of the polypeptide, for example, its sequence, is manipulated by human hands so that it is different from the aspect that exists in nature, the polypeptide is considered to be " engineered ".As is common practice and understood by those skilled in the art, even if actual manipulation is performed on the previous entity, the descendant of the engineered cell is still typically called " engineered ".

[0192] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment, where the objective is to reverse, alleviate, ameliorate, inhibit, slow, or stop the progression or severity of a condition associated with a disease or disorder, such as cancer. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a cancer-related condition, disease, or disorder. Treatment is generally "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" when the progression of the disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to those expected in the absence of treatment. Beneficial or desirable clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of disease, stable disease (i.e., not worsening), delay or slowing of disease progression, remission or palliation of the disease state, relief (partial or total), and / or reduced mortality. The term "treating" a disease also includes providing relief (including palliative treatment) from the symptoms or side effects of the disease.

[0193] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, within the scope of sound medical judgment, and without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient is not found to occur in nature.

[0194] As used herein, the term "administering" refers to the placement of a compound disclosed herein into a subject by a method or route that results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising a compound disclosed herein can be administered by any suitable route that results in effective treatment in a subject. In some embodiments of any of the aspects, administration includes physical human activity, such as injection, ingestion, application, and / or operation of a delivery device or machine. Such activity can be performed, for example, by a medical professional and / or the subject being treated.

[0195] As used herein, " contacting " refers to any suitable means for delivering or exposing an agent to at least one cell.Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery methods known to those skilled in the art.In some embodiments of any aspect, contacting includes physical human activity, such as injection; dispensing, mixing, and / or decanting; and / or operating a delivery device or machine.

[0196] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2SD) or greater.

[0197] Except in the Examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0198] As used herein, the term "comprises" means that other elements may also be present in addition to the defined elements presented. The use of "comprises" indicates inclusion rather than limitation.

[0199] The term "consisting of" refers to the compositions, methods, and each component thereof described herein, excluding any element not recited in that description of the embodiment.

[0200] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0201] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, in which a first entity binds to a second target entity with higher specificity and affinity than it binds to a third, non-target entity. In some embodiments of any of the aspects, specific binding can refer to an affinity of a first entity for a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or higher than its affinity for a third, non-target entity. A reagent specific for a given target is one that exhibits specific binding to that target under the conditions of the assay being used.

[0202] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0203] Groupings of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member can be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is considered herein to contain the modified group and thus fulfill the written description of all Markush groups used in the appended claims.

[0204] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the appended claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Werner Luttmann's Immunology, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WWNorton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; as well as Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the entire contents of which are incorporated herein by reference in their entirety.

[0205] Those skilled in the art can readily identify useful chemotherapeutic agents (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0206] In some embodiments of any of the aspects, the disclosure described herein does not concern processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or animals that may cause suffering without any substantial medical benefit to humans or animals, nor processes for modifying the genetic identity of animals that result from such processes.

[0207] Other terms are defined herein within the description of various aspects of the invention.

[0208] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0209] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of, and examples of, the present disclosure are described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide additional embodiments. Aspects of the present disclosure can be modified, as appropriate, to use the compositions, functions, and concepts of the above-mentioned references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0210] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0211] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0212] Some aspects of the technology described herein can be defined according to any of the following numbered clauses: 1. An EpCAM-binding aptamer domain; A chimeric molecule comprising at least one inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; PTPN2; SMG1; TREX1; CMAS; and CD47. 2. The molecule of item 1, which is an aptamer-siRNA chimera (AsiC). 3. The molecule of any one of items 1 to 2, wherein the inhibitory nucleic acid specifically binds to a gene product of a selected gene. 4. The molecule of any of items 1 to 3, wherein the EpCam-binding aptamer domain comprises a sequence of any of SEQ ID NOs: 63 to 68. 5. The molecule of any of paragraphs 1-4, wherein the inhibitory nucleic acid domain comprises a sequence selected from SEQ ID NOs: 1-62 and 69-126, or a reverse complement thereof. 6. The molecule of any of paragraphs 1 to 5, wherein the chimeric molecule comprises a first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain. 7. The molecule of paragraph 6, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise different sequences but each inhibit expression of the same gene. 8. The molecule of paragraph 6, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain each inhibit expression of a different gene. 9. The molecule of paragraph 8, wherein at least the second inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of PLK1 and MCL1. 10. The molecule of any of paragraphs 1 to 9, comprising one of the sequences of SEQ ID NOs: 127 to 137. 11. The molecule of any one of items 1 to 10, wherein the 3' end of the chimeric molecule contains dTdT. 12. The molecule of any of paragraphs 1 to 11, wherein the chimeric molecule comprises at least one 2'-F pyrimidine. 13. The molecule of any one of paragraphs 1 to 12, wherein the chimeric molecule further comprises a chemotherapeutic agent. 14. A pharmaceutical composition, kit, or combination comprising the chimeric molecule of any one of items 1 to 13, and optionally a pharmaceutically acceptable carrier. 15. The composition, kit, or combination of paragraph 14, comprising at least two chimeric molecules, the chimeric molecules having different aptamer domains or inhibitory nucleic acid domains. 16. The composition, kit, or combination of paragraph 15, wherein the different inhibitory nucleic acid domains recognize different targets. 17. The composition, kit, or combination of paragraph 15, wherein the different inhibitory nucleic acid domains have different sequences and recognize the same target. 18. a. any of the chimeric molecules of paragraphs 1 to 13 of paragraph 1; b. a second chimeric molecule, i. any of the chimeric molecules of paragraphs 1-13, wherein the inhibitory nucleic acid domain of the second chimeric molecule inhibits expression of a different gene than the first chimeric molecule; or ii. an EpCAM-binding aptamer domain; and an inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of PLK1 and MCL1; and a chimeric molecule comprising a second chimeric molecule; and c. optionally, a pharmaceutically acceptable carrier A pharmaceutical composition, kit, or combination comprising: 19. A method for treating cancer in a subject in need thereof, comprising the step of administering to the subject a chimeric molecule, composition, kit, or combination of any of items 1 to 18. 20. The method of paragraph 19, wherein the cancer is epithelial cancer, breast cancer, colon cancer, or triple-negative breast cancer. 21. The method of any one of items 19 to 20, wherein the administration is subcutaneous. 22. The method of any of paragraphs 19 to 21, wherein the subject is further administered an additional cancer treatment. 23. The method of paragraph 22, wherein the cancer treatment is paclitaxel. 24. A method of treating cancer in a subject in need thereof, comprising administering to the subject the chimeric molecule. 19. The chimeric molecule, composition, or kit of any one of items 1 to 18 for use in 25. The chimeric molecule, composition, or kit of item 24, wherein the cancer is epithelial cancer, breast cancer, colon cancer, or triple-negative breast cancer. 26. The chimeric molecule, composition, or kit of any of items 24 to 25, wherein the administration is subcutaneous. 27. The chimeric molecule, composition, or kit of any of paragraphs 24 to 26, wherein the subject is further administered an additional cancer treatment. 28. The kit of any of paragraphs 24 to 26, further comprising an additional cancer treatment in the same formulation as the chimeric molecule or in a separate formulation. 29. The chimeric molecule, composition, or kit of any of paragraphs 27 to 28, wherein the cancer treatment is paclitaxel.

[0213] Some aspects of the technology described herein can be defined according to any of the following numbered clauses: 1. An EpCAM-binding aptamer domain; at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47; A chimeric molecule comprising: 2. The molecule of item 1, wherein the gene is selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. 3. The molecule of item 1, wherein the gene is selected from the group consisting of UPF2; PD-L1; MCL1; and CD47. 4. The molecule of any of the preceding clauses, which is an aptamer-siRNA chimera (AsiC). 5. The molecule of any of the preceding clauses, wherein the inhibitory nucleic acid specifically binds to the gene product of a selected gene. 6. The molecule of any of the preceding paragraphs, wherein the EpCam-binding aptamer domain comprises the sequence of any of SEQ ID NOs: 63-68. 7. The molecule of any of the preceding clauses, wherein the inhibitory nucleic acid domain comprises a sequence selected from SEQ ID NOs: 1-62, 69-126, and 149-162, or the reverse complement thereof. 8. The molecule of any of the preceding clauses, wherein the chimeric molecule comprises a first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain. 9. The molecule of paragraph 8, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise different sequences but each inhibit expression of the same gene. 10. The molecule of paragraph 8, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain each inhibit expression of a different gene. 11. The molecule of paragraph 10, wherein at least the second inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of PLK1 and MCL1. 12. The molecule of any of the preceding clauses, comprising one of the sequences of SEQ ID NOs: 127-137 or 163-168. 13. A molecule of any of the preceding clauses that is a single-stranded nucleic acid. 14. The molecule of any of paragraphs 1 to 12, comprising a double-stranded portion. 15. The molecule of paragraph 14, wherein the double-stranded portion comprises two separate nucleic acids hybridized to each other, or comprises a single nucleic acid (e.g., a hairpin structure), and two portions of the single nucleic acid hybridize to each other. 16. The molecule of any of the preceding clauses, wherein the 3' end of the chimeric molecule comprises dTdT. 17. The molecule of any of the preceding clauses, wherein the chimeric molecule contains at least one 2'-F pyrimidine. 18. The molecule of any preceding clause, wherein the chimeric molecule comprises one or more of a 2' sugar modification, a phosphothiorate backbone modification, and a 5' unlocked nucleic acid modification. 19. The molecule of any of the preceding clauses, wherein the chimeric molecule is conjugated or attached to cholesterol, PEG, or a liposome. 20. The molecule of any of the preceding clauses, wherein the chimeric molecule further comprises a chemotherapeutic agent. 21. A pharmaceutical composition, kit, or combination comprising the chimeric molecule of any of items 1 to 20, and optionally a pharmaceutically acceptable carrier. 22. The composition, kit, or combination of item 21, comprising at least two different chimeric molecules of any one of items 1 to 20, the chimeric molecules having different aptamer domains or inhibitory nucleic acid domains. 23. The composition, kit, or combination of paragraph 21, wherein the different inhibitory nucleic acid domains recognize different targets. 24. The composition, kit, or combination of paragraph 21, wherein the different inhibitory nucleic acid domains have different sequences and recognize the same target. 25. The chimeric molecule of paragraphs 1-20 of the first paragraph comprises an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47; and The chimeric molecule of paragraphs 1 to 20 of the second paragraph comprises an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47. 25. The composition, kit, or combination according to any one of items 21 to 24. 26. The chimeric molecule of paragraphs 1-20 of the first paragraph comprises an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47; and The chimeric molecule of paragraphs 1 to 20 of the second paragraph comprises an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. 26. The composition, kit, or combination according to any one of items 21 to 25. 27. The composition, kit, or combination of any of paragraphs 21 to 26, comprising at least six different chimeric molecules of paragraphs 1 to 20, collectively comprising inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47. 28. The chimeric molecule of paragraphs 1 to 20 of the first paragraph comprises an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PD-L1; MCL1; and CD47; and The chimeric molecule of paragraph 2, 1 to 20, further comprises an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PD-L1; MCL1; and CD47. 28. The composition, kit, or combination according to any one of items 21 to 27. 29. The composition, kit, or combination of any of paragraphs 21 to 28, comprising at least four different chimeric molecules of paragraphs 1 to 20, collectively comprising inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PD-L1; MCL1; and CD47. 30. a. Any chimeric molecule of paragraphs 1-20 of paragraph 1; b. a second chimeric molecule, i. any of the chimeric molecules of paragraphs 1-20, wherein the inhibitory nucleic acid domain of the second chimeric molecule inhibits expression of a different gene than the first chimeric molecule; or ii. an EpCAM-binding aptamer domain; and an inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of PLK1 and MCL1; and a chimeric molecule comprising a second chimeric molecule; and c. optionally, a pharmaceutically acceptable carrier A pharmaceutical composition, kit, or combination comprising: 31. The composition, kit, or combination of any of items 21 to 30, further comprising an immune checkpoint inhibitor. 32. The composition, kit, or combination of paragraph 31, wherein the immune checkpoint protein is PD-1 or PD-L1. 33. The composition, kit, or combination of paragraph 32, wherein the immune checkpoint protein is PD-1. 34. The composition, kit, or combination of item 33, wherein the immune checkpoint inhibitor is pembrolizumab; nivolumab; pidilizumab; or AUNP12. 35. A method of treating cancer in a subject in need thereof, comprising administering to the subject a chimeric molecule, composition, kit, or combination of any of items 1 to 34. 36. The method of paragraph 35, wherein the cancer is epithelial cancer, breast cancer, or colon cancer. 37. The method of paragraph 36, wherein the breast cancer is HER2+ breast cancer or triple-negative breast cancer (TNBC). 38. The method of paragraph 36, wherein the breast cancer is not BRCA1 deficient. 39. The method of any of items 35 to 38, wherein the administration is subcutaneous. 40. The method of any of paragraphs 35 to 39, wherein the subject is further administered an additional cancer treatment. 41. The method of paragraph 40, wherein the cancer treatment is paclitaxel. 42. A method of treating cancer in a subject in need thereof, comprising administering to the subject the chimeric molecule. 35. The chimeric molecule, composition, or kit of any one of items 1 to 34 for use in 43. The chimeric molecule, composition, or kit of paragraph 42, wherein the cancer is epithelial cancer, breast cancer, or colon cancer. 44. The chimeric molecule, composition, or kit of paragraph 43, wherein the breast cancer is HER2+ breast cancer or triple-negative breast cancer (TNBC). 45. The chimeric molecule, composition, or kit of paragraph 44, wherein the breast cancer is not BRCA1 deficient. 46. ​​The chimeric molecule, composition, or kit of any of paragraphs 42 to 46, wherein the administration is subcutaneous. 47. The chimeric molecule, composition, or kit of any of paragraphs 42 to 46, wherein the subject is further administered an additional cancer treatment. 48. The kit of any of paragraphs 42 to 47, further comprising an additional cancer treatment in the same formulation as the chimeric molecule or in a separate formulation. 49. The chimeric molecule, composition, or kit of any of paragraphs 42 to 48, wherein the cancer treatment is paclitaxel. [Example]

[0214] Example 1: Enhanced immunotherapy of triple-negative and HER+2 breast cancer using EpCAM aptamer-siRNA-mediated gene knockdown The present invention describes a conjugate of EpCAM aptamer and siRNA, which is two RNA molecules. The two ends are complementary and bind to each other. When the conjugate enters cells, the enzyme Dicer destroys the double-stranded RNA, freeing the siRNA to knock out its complementary mRNA, effectively shutting down the target gene. Triple-negative breast cancer and HER2 breast cancer are not adequately treated by current technology, and this need is addressed by the composition described herein.

[0215] Triple-negative breast cancer (TNBC) and HER2+ breast cancer (BC) are particularly aggressive tumors with the worst prognosis. They tend to recur and metastasize after chemotherapy or targeted therapy. Immunotherapy, which has achieved significant therapeutic benefit in some cancers, offers a promising but unproven alternative approach for treating poor-prognosis BC. BC have a relatively low nonsynonymous mutation rate, which makes many of them poorly immunogenic. Novel strategies for increasing the immunogenicity of BC cells and improving tumor antigen-specific T cell responses are crucial for enhancing the efficacy of BC immunotherapy.

[0216] Described herein is a method for increasing the immunogenicity of breast tumors by utilizing the unique strengths of small interfering RNA (AsiC) carried by the EpCAM aptamer. EpCAM-AsiC can specifically knock down any gene product, including intracellular and undruggable targets, selectively in EpCAM+ BC tumor cells. The immunomodulatory EpCAM-AsiC targets genes involved in various functional processes of the cancer-immune cycle, thereby making invasive BC visible to T cells and improving T cell tumor recruitment and function, thus enhancing anti-tumor immune responses.

[0217] These subcutaneously (sc) administered AsiCs are selectively taken up by cells at distant sites in the body that possess the receptor recognized by the aptamer. Intracellularly, AsiCs are cleaved by the RNA interference nuclease Dicer, liberating active siRNAs, which result in efficient gene knockdown. EpCAM is a tumor-specific antigen expressed at levels several logs higher on all epithelial cancers, including 97% of BC and its "cancer stem cells," than on normal epithelia. High-affinity EpCAM-AsiCs are described herein using an EpCAM aptamer that binds to both mouse and human EpCAM with low nanomolar affinity. These EpCAM-AsiCs selectively accumulate in EpCAM+ BC but not in normal tissues. To be clinically useful, EpCAM-AsiCs must be taken up by distant tumors. Sc-injected EpCAM-AsiCs were concentrated in distant EpCAM+ TNBC xenografts, but not in EpCAM- TNBC xenografts, in mice and persisted there for at least 4 days.

[0218] The use of EpCAM-AsiC for BC immunomodulation was explored by knocking down genes controlling various functional processes. 1) Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs), which can result from various genetic mutations and frameshifts. When translated, these mRNAs produce truncated proteins with abnormal functions. UPF2 is a key enzyme in NMD. Knockdown of UPF2 in EpCAM+ BC cells results in the expression and presentation of neoantigens recognized by T cells. As described herein, UPF2 EpCAM-AsiC can enhance antitumor T cell responses.

[0219] 2) PARP1 is involved in DNA damage detection, DNA repair, and maintaining genomic stability. PARP1 inhibition can lead to chromosomal aberrations and contribute to overall genomic instability. The primary function of APE1 is to repair abasic sites during base excision repair (BER). In addition, APE1 functions as a redox regulator, playing a critical role in tumor cell survival. Knockdown of the DNA repair enzymes PARP1 and APE1 in tumor cells according to the methods described herein results in more DNA strand break-associated mutations, thus introducing tumor-specific neoantigens to the immune system. In addition, inhibiting the redox activity of APE1 can directly suppress tumor growth. It is demonstrated herein that both Parp1-AsiC and APE1-AsiC significantly suppress tumor progression and enhance the function of CD8+ tumor-infiltrating lymphocytes (TILs). PARP1-AsiC also outperforms the FDA-approved drug olaparib, further inhibiting the growth of 4T1E (a 4T1 cell line with high EpCAM expression) breast tumors.

[0220] 3) Tumor cells evade immune surveillance by upregulating CD47, which binds to signal-regulatory receptor protein (SIRP)α on macrophages and dendritic cells (DCs) and inhibits phagocytosis and antigen cross-presentation. Animal studies have shown that anti-CD47 therapy enhances antitumor immunity, suppresses tumor growth, and synergizes with chemotherapy and radiation therapy by promoting cross-presentation of TAs to T cells. It is demonstrated herein that CD47-AsiC enhances the ratio of CD8+ TILs to regulatory T cells (Tregs) and reduces the expression of co-inhibitors, improving the function of CM+ TILs and suppressing 4T1E tumor growth. Tumor-associated macrophages (TAMs) derived from tumors treated with CD47-AsiC exhibit improved phagocytic ability. It is further demonstrated that CD47-AsiC outperforms anti-CD47 antibodies in suppressing 4T1E tumor growth.

[0221] 4) Our data show that EpCAM-AsiC directly kills tumor cells by targeting PLK1 and MCL1, essential genes that BC cells depend on for survival. PLK1 is a serine-threonine kinase essential for mitosis and maintaining DNA integrity. MCL1 acts by sequestering the apoptotic effector Bak and other proapoptotic proteins and is a critical survival factor in TNBC. Both PLK1 and MCL1 are overexpressed in BC cells. Increased tumor cell death induced by knockdown of PLK1 or MCL1 promotes both tumor cell death and tumor antigen cross-presentation to CD8+ T cells, thereby improving antitumor T cell responses. Our data show that PLK1 and MCL1 EpCAM-AsiC slow tumor growth and enhance the number and function of CD8+ TILs.

[0222] Finally, we demonstrate that UPF2, CD47, PARP1, PLK1, and MCL1 AsiCs work synergistically to further slow tumor growth and even result in tumor regression compared with single AsiC treatment. The combination therapy significantly increased the amount of CD8+ TILs, reduced Tregs and myeloid-derived suppressor cells (MDSCs) in the tumor, and improved the function of both CD4+ and CD8+ TILs.

[0223] To the best of our knowledge, AsiC does not induce an antibody-like immune response. By modulating tumors rather than systemically activating T cells, we can avoid the troublesome autoimmune side effects that occur when multiple checkpoint inhibitors are combined. Cytotoxic siRNAs targeting tumor-dependent genes can be easily combined with immune-modulating siRNAs. EpCAM is highly expressed by all epithelial cancers and their stem cells. Therefore, the approach described herein for treating breast cancer can be applied to other undertreated solid tumors and any epithelial cancer, including lung, colon, pancreas, prostate, bladder, stomach, head and neck, esophageal, and bile duct cancers.

[0224] Immunomodulatory EpCAM-AsiC has great potential to revitalize immune responses and treat BC with poor immunogenicity.Importantly, EpCAM-AsiC conjugates possess high affinity and tumor selectivity, which reduces toxicity compared with checkpoint blocking antibodies.In addition, these drugs are small molecules that diffuse into poorly vascularized tumors.This data shows that these novel EpCAM-AsiCs, with their small size and high selectivity, have great potential to improve therapeutic efficacy and reduce toxicity for BC patients compared with current checkpoint inhibitors.

[0225] Example 2 AsiC solves the problem of delivery beyond the liver and provides exquisitely specific drug uptake and knockdown only in target cells. Subcutaneous administration results in penetration into all tissues, like small molecules, and AsiC is stable in serum for several days. AsiC has the same favorable pharmacodynamics as other siRNA-based drugs, providing sustained knockdown without any apparent toxicity or immunogenicity. Chemical synthesis is performed using existing available manufacturing methods, and the same chemistry can be used to link more than one siRNA, miRNA, mRNA, toxin, or chemotherapeutic drug to create multifunctional drugs.

[0226] The AsiC described herein contains an aptamer specific for EpCAM, which is highly expressed in epithelial cancers (Figure 1). The knockdown effect achieved by EpCAM-AsiC provides an antitumor effect that correlates with EpCAM expression (Figure 2), and TNBC cells internalize EpCAM-AsiC at a higher rate than normal breast tissue (Figure 3). Alexa750-EpCAM-AsiC is selectively internalized into EpCAM+ tumors (Figure 6). EpCAM-AsiC inhibits in vitro cancer stem cell assays of EpCAM+ breast cancer cell lines (Figure 4). Ex vivo treatment of EpCAM+ TNBC cells blocks tumor initiation (Figure 5). EpCAM-AsiC targeting PLK1 inhibits EpCAM+ TNBC tumor growth (Figure 7).

[0227] EpCAM-AsiC knocks down genes in epithelial breast cancer cells and tumor-initiating cells within them, sparing normal epithelial cells. Subcutaneously injected EpCAM-AsiC localizes to distant tumors. PLK1 EpCAM-AsiC suppresses tumor growth in vitro and in vivo and eliminates tumor-initiating cells. AsiC does not induce innate immunity. The most common epithelial tumors are EpCAM+ (colon, lung, prostate, pancreas). Similar results are obtained in HCT116 colon cancer xenografts.

[0228] Certain AsiCs described herein are intended to manipulate antitumor immunity (Figure 8). Knockdown of UPF2 to inhibit the RNA quality control pathway enhances antitumor immunity (Figures 9A-9E). A similar effect was observed in 4T1 tumors, which had increased levels of CD8+ TILs after UPF2 EpCAM-AsiC treatment (Figure 10).

[0229] Disruption of DNA repair by knockdown of PARP1 and APE1 improves tumor immunity (Figure 11). PARP1-AsiC works better than the PARP1 inhibitor olaparib (an approved drug for a small subset of breast cancers) and similarly to olaparib plus a checkpoint inhibitor (anti-PDL1). Treatment with PARP1-AsiC results in an increase in CD8+ TILs and greater cytokine production by these cells.

[0230] Targeting the phosphatase PTPNT2 enhances tumor-mediated interferon signaling (Figure 12). PNPT2 suppresses interferon signaling, and loss of PNPT2 improves tumor antigen presentation and T cell responsiveness to tumors. Treatment with PTPNT2-AsiC suppressed tumor growth, induced CD8+ TILs, increased tumor antigen presentation, and increased the function of CD8+ and CD4+ TILs (Figures 12A-12E).

[0231] AsiC-mediated CD47 inhibition also reduces tumor growth (Figure 13). CD47-AsiC increases TAM in vivo phagocytosis of 4TE-eGFP tumors (Figure 14) and induces antitumor responses (Figures 15A-15B). Additionally, TILs express fewer inhibitory receptors after CD47-AsiC treatment (Figure 16). CD47-AsiC provides a more effective treatment than anti-CD47 antibodies in phase II clinical trials (Figure 17).

[0232] When combined, AsiC exhibits unexpected synergy (Figures 18A-18E, 21). This synergy is also evident in the treatment of 4T1E tumor-bearing mice (Figure 22) and ErbB2ΔEx16+ mice (Figures 23 and 24).

[0233] Because only the tumor is targeted, AsiC therapy is effective and well tolerated. Additionally, the choice of target can be tailored to the patient's tumor. AsiC therapy does not show any obvious signs of drug resistance (e.g., no EpCAM inhibition (Figure 19)), and can be used to treat common solid tumors for which existing therapies are inappropriate.

[0234] EpCAM-AsiC is a flexible platform for selectively knocking down gene expression only in tumors, including in the most aggressive subset of cancer stem cells. Gene knockdown can be used to directly kill tumors (PLK1, MCL1). These AsiCs also enhance immune responses (data not shown). Beyond checkpoint blockade, genes in tumors can be knocked down to regulate multiple pathways to induce immune recognition, activate dysfunctional immune cells, and reduce immunosuppressive cells that interfere with defense. Cocktails of AsiCs are easy to assemble and can synergize to improve tumor control. As shown herein, AsiCs performed better than blocking antibodies or inhibitors.

[0235] Example 3: Immunotherapy for breast cancer by EpCAM aptamer-targeted gene knockdown in tumors Introduction Triple-negative breast cancer (TNBC) and HER2+ breast cancer (BC) are the most aggressive types of BC with the worst prognosis 1,2 There are no targeted therapies for TNBC, and the majority of patients relapse and develop metastases after chemotherapy. 3 Although HER2-targeted therapy has radically improved the treatment of HER2+ BC, more than 20% of patients develop recurrent disease within 5 years. 4,5 Therefore, novel strategies that can improve therapeutic efficacy for aggressive BC are urgently needed. Cancer immunotherapy has shown significant and durable responses in patients with multiple types of cancer. 6 Responsive cancers have high somatic mutation rates (i.e., approximately 100 / Mb for melanoma and non-small cell lung cancer), which are thought to contribute to their immunogenicity. 7 BC have previously been considered immunologically quiescent, which is related to their low nonsynonymous mutation burden (approximately 1 / Mb), and their sensitivity to immunotherapy has not been thoroughly studied in the clinic. 7-9 However, abundant evidence suggests that the BC tumor microenvironment (TME) is under immune surveillance and that immunotherapy has shown efficacy in some BC cases. Gene expression profiling of BC has shown that expression of lymphocyte-related genes or genes linked to type I interferon (IFN-I) activation in tumors is associated with a better prognosis. 10,11 Importantly, both TNBC and HER2+ BC harbor a higher mutational load and a greater number of patients harbor a robust tumor immune infiltrate compared to other BC subtypes. 12,13 Increased levels of tumor-infiltrating lymphocytes (TILs) are associated with better overall survival (OS) and disease-free survival (DFS) in TNBC and HER2+ BC, with each 10% increase in TIL numbers being associated with a 15-25% reduction in the risk of recurrence and death. 14-16Furthermore, the long-term efficacy of several conventional chemotherapy drugs, targeted therapies, and radiation therapy depends on their ability to induce antitumor T cells. 11 These findings highlight the opportunity to develop potent immunotherapeutic approaches to improve treatment outcomes for patients with invasive BC.

[0236] The use of immune checkpoint inhibitors, such as anti-PD-1 / PD-L1 antibodies, is one of the most promising immunotherapeutic approaches for treating aggressive BC. The anti-PD-L1 antibody atezolizumab in combination with the chemotherapy drug nab-paclitaxel was approved for patients with metastatic TNBC in 2019. However, the therapeutic benefit is limited to a minority of patients. 13 Responsiveness to checkpoint inhibitors correlates with tumor genome stability, tumor neoantigen expression, and immune recognition. 17-22 Many BCs do not respond to checkpoint blockade, primarily due to a low mutation rate that renders breast tumor cells poorly recognized by the immune system. In addition, checkpoint inhibitors can nonspecifically activate T cells systemically, potentially leading to autoimmune side effects. 23 To optimize the efficacy of immunotherapy for the treatment of BC, there are multiple challenges in the cancer-immunity cycle that must be overcome to elicit effective antitumor immunity. 24,25 For example, tumor cells must express neoantigens that can be released and taken up / presented by antigen-presenting cells (APCs) to prime and activate tumor antigen (TA)-specific T cells. Activated T cells must also infiltrate the TME and efficiently kill target tumor cells that present TAs.

[0237] To achieve this goal, we exploited the unique strength of small interfering RNA (siRNA) delivered by the EpCAM aptamer. EpCAM aptamer-siRNA chimera (AsiC) can specifically knock down any gene product, including intracellular and undruggable targets, selectively in EpCAM+ breast tumor cells, making invasive BC visible to T cells and thus enhancing antitumor immune responses. As a tumor-associated antigen, EpCAM is expressed at several-log higher levels than normal epithelium on all epithelial cancers, including 97% of BC and its "cancer stem cells." 31-34 EpCAM exhibits oncogenic properties, as its expression is associated with accelerated tumor progression, bone metastasis, and poor prognosis. 32 This may make it difficult for tumor cells to develop drug resistance by downregulating EpCAM. The high-affinity EpCAM aptamer described herein can bind to both mouse and human EpCAM with low nanomolar affinity. To be clinically useful, EpCAM-AsiC must be internalized by distant tumors. We found that subcutaneously (sc) injected EpCAM-AsiC, which targets PLK1, a serine-threonine kinase essential for BC cell survival, could be selectively enriched in distant EpCAM+ TNBC xenografts, but not in EpCAM- TNBC xenografts or normal tissues, in mice and persisted there for at least 4 days. 26 In these mice, all EpCAM+ tumors completely regressed, whereas EpCAM+ tumors and all EpCAM- tumors in mice treated with control AsiC continued to grow. Furthermore, EpCAM-AsiC did not induce measurable toxicity or stimulate innate immune responses in treated mice. 26 These experiments demonstrated the promise of developing EpCAM-AsiC as an immunomodulatory therapy for BC.

[0238] Here, to enhance the immunogenicity of BC cells, we used the EpCAM-AsiC platform to knock down genes involved in various functional processes of the cancer-immune cycle in EpCAM+ BC cells, aiming to make invasive BC visible to the immune system and improve antitumor immunity. These targets include: (1) regulator of nonsense transcripts 2 (UPF2) and DNA repair enzymes poly(ADP-ribose) polymerase 1 (PARP1) and apurinic / apyrimidinic endodeoxyribonuclease 1 (APEX1), which function in the nonsense-mediated mRNA decay (NMD) pathway to trigger the expression of tumor neoantigens; (2) CD47, a "don't eat me" signal, promotes phagocytosis of cancer cells and their antigen presentation by dendritic cells (DCs) and macrophages; and (3) CD8, which promotes TA cross-presentation. + Myeloid cell leukemia 1 (MCL1) is a critical survival factor in TNBC for activating T cells and thereby directly killing tumor cells to improve antitumor T cell responses 35-37 and (4) tumor-infiltrating PD-1 + Programmed death-ligand 1 (PD-L1) to improve T cell function. As demonstrated herein, these EpCAM-AsiCs can knock down target gene expression in EpCAM+ breast tumor cells with high efficiency and selectivity both in vitro and in vivo. Using a mouse orthotopic TNBC model, it has been demonstrated that each of the four EpCAM-AsiCs targeting UPF2, PARP1, CD47, and MCL1 significantly suppresses breast tumor growth. PARP1 AsiC and CD47 AsiC outperform the FDA-approved PARP1 inhibitor olaparib and an anti-CD47 antibody currently in multiple clinical trials, respectively, in inhibiting tumor growth. These immunomodulatory EpCAM-AsiCs also inhibit CD8 + Tumor-infiltrating lymphocytes (TILs) vs. CD4 + Increasing the ratio of regulatory T cells (Treg) and CD8 + TILs and CD4+ By increasing the function of TILs, anti-tumor immunity was strongly enhanced.

[0239] Mechanistically, UPF2 knockdown reduced NMD pathway activity in EpCAM+ tumor cells and promoted the production of splice variant mRNAs that could encode neoantigens. CD47 knockdown promoted tumor cell phagocytosis by tumor-associated macrophages (TAMs), increased the ratio of tumor-suppressive M1 TAMs to tumor-promoting M2 TAMs, and also enhanced the number and maturation of tumor-infiltrating DCs, all of which could promote antigen presentation to activate T cells. MCL1 knockdown directly reduced tumor cell viability, which may increase the release of TAs to stimulate TA-specific T cells. Furthermore, the four EpCAM-AsiCs acted synergistically, resulting in a more significant reduction in tumor growth compared to single AsiC treatment, and also strongly inhibited the growth of lung metastatic BC. Single-cell RNA sequencing (scRNA-seq) studies showed that combined AsiCs simultaneously improved the antitumor capacity of both monocytes / macrophages and TILs. The combined AsiC further synergized with anti-PD-1, resulting in more pronounced tumor suppression. Finally, we demonstrated that a cocktail of EpCAM-AsiC targeting six genes, UPF2, PARP1, APEX1, CD47, MCL1, and PD-L1, simultaneously suppressed tumor growth in a transgenic mouse model of aggressive HER2+ BC, demonstrating the potential of utilizing immunomodulatory EpCAM-AsiC as a potent immunotherapeutic approach to combat aggressive BC.

[0240] result EpCAM aptamer-siRNA selectively induces gene knockdown in EpCAM+ murine BC cell lines To investigate the use of EpCAM aptamers for cell-specific gene knockdown for BC immunotherapy, we first verified that fluorescently labeled EpCAM aptamers were internalized by murine EpCAM+ BC cell lines (4T1, 4T1E, and N202.1A) but not by EpCAM- mouse cell lines (L929, P815, and B16-F10) (data not shown). It was hypothesized that knockdown of genes in murine BC cell lines that could increase tumor neoantigen expression (Upf2, Parp1, and Apex), trigger tumor cell death (Mcl1), enhance tumor cell phagocytosis (Cd47), or suppress checkpoint inhibition (Cd274, the gene encoding PD-L1) could enhance antitumor immunity. To test this hypothesis, we designed EpCAM aptamer-siRNA chimeras (AsiC) to knockdown each of these genes using siRNAs, each of which caused approximately 90% knockdown in 4T1E TNBCs transfected with 100 nM siRNA (Fig. 32A). With the exception of Mcl1 siRNA, transfection of all of these siRNAs had no effect on cell viability or proliferation (Fig. 32B).

[0241] To construct EpCAM-AsiC, we linked the sense (passenger or inactive) strand of each selected siRNA to the 3' end of a 19-nt EpCAM aptamer via a UUU linker (Figure 25A, Table 5). This RNA strand was chemically synthesized with 2'-fluoropyrimidine substitutions and 3'-dTdT overhangs to enhance RNase resistance, and then annealed to the antisense (guide or active) strand of each siRNA similarly modified with fluoropyrimidines and 3'-dTdT overhangs. This configuration was stable in vitro in serum for more than 36 hours, did not induce innate immune IFN or inflammatory cytokine responses, and was cleaved by Dicer in cells to release the active siRNA from the aptamer.

[0242] EpCAM-AsiC, engineered to knock down Upf2, Parp1, Apex, Cd47, Mcl1, or Cd274, knocked down target gene expression in EpCAM+ 4T1E tumor cells in vitro by 50–90% when measured 72 hours later. As expected, EpCAM-AsiC did not affect target gene expression in EpCAM-L929 tumors (not shown). Subcutaneous injection of 125 μg (5 mg / kg) of AsiC into the nape of the neck in mice resulted in 50–70% knockdown of gene expression in 4T1E tumors orthotopically implanted in the fourth mammary gland, measured 72 hours after injection. The knockdown was specific, as injection of the EpCAM aptamer itself or EpCAM-AsiC against eGFP did not knock down the endogenous gene. Furthermore, knockdown did not occur in EpCAM-CD45- cells within the tumor.

[0243] EpCAM-AsiC targeting UPF2 or PARP1 inhibits tumor growth and enhances antitumor T cell immunity Knockdown of Upf2, which encodes a protein that binds to prematurely terminated mRNAs resulting from diverse genetic mutations and induces nonsense-mediated decay, has been hypothesized to induce tumor cell expression of neoantigens to facilitate tumor recognition by T cells. To verify that in vivo treatment of 4T1E orthotopic tumor-bearing mice with EpCAM-AsiC, which reduces tumor UPF2 mRNA and protein, reduced NMD activity in tumor cells, we compared the ratio of fully spliced ​​mRNA to its precursor pre-mRNA for four known NMD-targeted translocation products (Gadd45α, Gadd45β, Cdkn1a, and Nat9). An increase in this ratio indicates decreased NMD activity. 39,40 The mRNA / pre-mRNA ratios for all four genes were significantly higher in tumors from mice treated with UPF2 EpCAM-AsiC than in control mice treated with aptamer alone, indicating attenuated NMD activity.

[0244] To determine whether Upf2-targeted EpCAM-AsiC has antitumor activity, mice bearing palpable orthotopic 4T1E tumors were treated sc with 5 mg / kg of EpCAM aptamer or UPF2 EpCAM-AsiC every 3 days. 4T1E tumor growth was significantly inhibited in mice treated with UPF2 EpCAM-AsiC (Figure 25B). The effect of tumor-targeted Upf2 on tumor-infiltrating lymphocytes (TILs) was assessed by immunohistochemistry (IHC) and flow cytometry analysis of single-cell suspensions of tumors harvested 16 days after three AsiC or aptamer injections. UPF2 EpCAM-AsiC significantly inhibited CD8 T cell proliferation as measured by IHC. + It strongly increased the density of TILs by 3-fold (Fig. 25C), a parameter strongly associated with anti-tumor immunity and response to immunotherapy for invasive BC. 43,44 , CD8 + vs. CD4 + Foxp3 + T reg The ratio of CD8 to CD8+ was also increased three-fold in tumors treated with UPF2 AsiC by flow cytometry (Figure 25D). + TILs also produced more IFN-γ and TNF-α after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Figure 25E). After 6 hours of ex vivo co-incubation with UPF2 siRNA-treated 4T1E, these CD8 + TILs were also more degranulated, as measured by CD107a / b surface expression (Fig. 25F), and stained more for the cytotoxic effector molecules granzyme B and perforin (Fig. 25G). Indeed, CD8 from UPF2 AsiC-treated tumors was significantly higher than that from aptamer-treated tumors. + TILs were twice as effective at killing Upf2-knockdown 4T1E cells. Thus, UPF2 EpCAM-AsiC mediates antitumor CD8 + It significantly enhanced T cell immunity and slowed 4T1E tumor growth.

[0245] UPF2 knockdown induces novel mRNA transcripts To investigate whether UPF2 knockdown in BC generates novel mRNA isoforms, we performed a cytotoxic assay using EpCAM mice transfected with either a non-coding control or UPF2 siRNA for 72 h. hi We performed bulk RNA sequencing (RNA-seq) using the MDA-MB-231 human BC cell line. We identified 222 instances of differential exon usage (DEU) events within 281 genes (data not shown). For example, UPF2 knockdown significantly reduced exon 8 usage in RINL mRNA (transcript ID ENSG00000187994) (log2 fold change -15.2, adjusted p-value = 0.03) and significantly enhanced exon 6 usage in ATP11B mRNA transcript (ENSG00000058063), which was barely detectable in control cells (log2 fold change 14.5, adjusted p-value = 0.02). These DEU events could lead to the expression of novel polypeptides and novel T cell epitopes. The number and diversity of DEUs suggest that UPF2 knockdown could induce novel alternative splicing.

[0246] To test this, we deconvolved and identified UPF2 knockdown-mediated transcriptional diversity and estimated the abundance of transcript isoforms. Forty-two genes with potential differential isoform usage (DIU) were identified (data not shown). These included seven genes identified as having DEU (CENPH, PFKFB4, UCN2, SNHG8, CDKAL1, TRIM4, and TMEM242). These DIU events included examples of novel mRNA isoforms that may encode new polypeptides, such as DNAJC2 and TMPRSS5 LAT2 (Figure 33A). In addition, several genes with DIU, such as CENPH, SNRPA1, and EBPL, increased the abundance of mRNA isoforms known to be susceptible to NMD. For example, UPF2 knockdown increased the CENPH isoform, indicating that exon skipping events are predicted to have a premature stop codon that renders it susceptible to NMD (Figure 33B). Taken together, this data indicates that reducing NMD activity by knocking down UPF2 induces the expression of tumor neoantigens.

[0247] Knockdown of Parp1 reduces tumor growth and enhances antitumor immunity We hypothesized that inhibition of DNA repair in tumors might be another way to induce tumor neoantigen expression. PARP1 is a critical DNA damage repair protein that senses single- and double-strand DNA breaks and recruits and activates DNA repair machinery at the site of the break. 45Knockdown of PARP1 in tumor cells potentially resulted in more DNA break-associated mutations, thereby introducing tumor-specific neoantigens that could be recognized by T cells. To test whether PARP1 knockdown activates antitumor immunity, mice bearing palpable orthotopic 4T1E tumors were treated with the EpCAM aptamer, PARP1 EpCAM-AsiC, or the FDA-approved PARP1 inhibitor olaparib. PARP1 AsiC inhibited 4T1E tumor growth more effectively than olaparib, showing a trend toward inhibition that did not reach significance (Figure 26A). PARP1 AsiC also had a more pronounced effect on the antitumor properties of TILs than olaparib. It significantly reduced CD8+ TILs in tumors compared with tumors treated with the control aptamer. + / CD4 + T reg Parp1 induced a strong and significant increase in tumor cell proliferation (Figure 26B), activation-stimulated production of IFNγ and TNFα by CD8+ TILs (Figure 26C), and increased TNFα production by CD4+ TILs (Figure 26D). Olaraprib had more subtle effects on antitumor immunity, failing to reach significance except for increased TNFα production by CD4+ TILs. It is unclear why Parp1 knockdown has a stronger effect than PARP1 enzyme inhibition; however, ablation of PARP1 protein may interfere with the recognition and assembly of repair proteins at sites of DNA damage, whereas inhibition of PARP1's poly (ADP-ribosylation) activity may simply act downstream to inhibit repair. As a result, unrepaired DNA damage and genomic instability after Parp1 knockdown may be more widespread than after inhibiting PARP1 enzyme activity.

[0248] Knockdown of Apex1 leads to tumor growth APEX1 is a key endonuclease in base excision repair (BER), which repairs abasic sites formed by oxidative DNA damage, the most common type of DNA damage in cells. 47,48Apex1 gene deletion results in early embryonic lethality (E4-6.5), and Apex1-deficient cell lines do not proliferate. Notably, tumors do not mutate this essential gene. Therefore, we investigated Apex1 knockdown because it may also induce mutations that are directly cytotoxic and can activate T cell immunity. Perhaps because it appears to be an essential gene, in vivo Apex1 knockdown by EpCAM-AsiC was only 50%, less effective than other EpCAM-AsiCs. When administered at the same dose and schedule as other EpCAM-AsiCs, tumor-targeted Apex1 knockdown reduced 4T1E tumor growth, although the difference compared to mice treated with aptamer alone did not reach significance (Figure 26E).

[0249] CD47EpCAM-AsiC regulates EpCAM expression by macrophages + Promotes phagocytosis of BC cells and enhances anti-tumor T cell immunity Tumor cells alter the expression of many genes to evade immune elimination, a process called tumor editing. One strategy is tumor upregulation of the surface glycoprotein CD47, which binds to the signal-regulatory protein SIRPα on macrophages and DCs and acts as a potent "don't eat me" signal, inhibiting phagocytosis and antigen cross-presentation. 49 To evaluate the antitumor effect of CD47 knockdown, mice bearing orthotopic 4T1E tumors were treated with the EpCAM aptamer or CD47 EpCAM-AsiC. CD47 EpCAM-AsiC inhibited tumor growth compared with mice treated with the EpCAM aptamer (Figure 27A), and CD8 + / CD4 + T reg Promotes antitumor immunity as indicated by increased TIL ratio (Fig. 27B) and IFN-γ-producing CD8 + TILs and CD4 + Functional capacity of TILs (Figures 27C-27D), as well as CD8 of GzmB + increased the functional capacity of TIL expression (Figure 27E).

[0250] Next, we analyzed the effects of Cd47 knockdown in tumors on tumor-associated macrophages (TAMs) and dendritic cells (DCs). In response to tumor environmental cues, TAMs can polarize into either proinflammatory classically activated M1-like macrophages with antitumor properties or anti-inflammatory alternatively activated M2-like macrophages that are immunosuppressive and correlate with tumor progression, metastasis, and poor prognosis. 52-54 Although CD47 EpCAM-AsiC did not significantly alter the number of TAMs (not shown), the ratio of M1 / M2 TAMs was significantly increased in tumors treated with CD47 EpCAM-AsiC (Fig. 27F, Fig. 34B). In addition, CD11c TAMs among CD45+ hematopoietic cells in tumors were significantly increased. + DC205 + The percentage of DCs was significantly higher after CD47 EpCAM-AsiC treatment compared with aptamer treatment (Figure 27G). DCs in tumors treated with CD47 EpCAM-AsiC expressed more costimulatory CD80 and CD86 and surface MHC-II, suggesting that they were more effective APCs (Figure 27H). To determine whether TAM phagocytosis of tumor cells increased in vivo after aptamer or CD47 EpCAM-AsiC treatment, we replaced 4T1E tumors with 4T1E tumors stably expressing eGFP (4T1E-eGFP) and examined TAM GFP fluorescence. Significantly more TAMs expressed GFP in tumors treated with CD47 EpCAM-AsiC. + , indicating increased in vivo phagocytosis (Figure 27I). To confirm that the enhanced TAM phagocytosis was due to reduced CD47 expression on tumor cells, we co-cultured TAMs from tumors treated with CD47 EpCAM-AsiC with 4T1E-eGFP treated with non-targeting siRNA or CD47 siRNA. TAM phagocytosis of CD47-knockdown 4T1E-eGFP was four-fold greater than that in control tumors (Figure 27J).

[0251] To determine whether the tumor-suppressive effect of CD47 EpCAM-AsiC was mediated by TILs and / or TAMs, we performed CD8 immunohistochemistry using antibodies against CD4, CD8, or CSF1R, respectively, in mice bearing orthotopic 4T1E tumors before treatment with CD47 EpCAM-AsiC. + T cells or CD4 + T cells or macrophages were depleted (Figures 35A-35C). + T cell depletion completely abolished the antitumor effect of CD47EpCAM-AsiC, whereas CD4 + Depletion of T cells or macrophages had less of an effect (Figure 27K). However, macrophage depletion was less complete than T cell depletion, as approximately 30% of TAMs persisted after depletion. CD8 TAMs from tumors treated with CD47 EpCAM-AsiC, as assessed by IFN-γ and TNF-α production and degranulation in response to incubation with 4T1E, were significantly reduced. + The increased functionality of TILs was reduced to background levels in macrophage-depleted mice, which was consistent with the increased functionality of CD8 in tumors treated with CD47 AsiC. + The importance of TAMs in promoting TIL anti-tumor immunity is shown (Figure 27L).

[0252] To compare the efficacy of blocking antibodies and AsiC knockdown, we evaluated the antitumor effects of CD47 AsiC and anti-CD47 antibodies. Both treatments reduced tumor volume, especially at later time points, but only CD47 AsiC treatment significantly inhibited tumor growth (Figure 27M). CD8 cells from mice treated with CD47 EpCAM-AsiC and anti-CD47 were significantly reduced. + Both TILs produced more IFN-γ after PMA and ionomycin stimulation than those in control tumors, but only CD47 AsiCs produced more IFN-γ than those in control tumors. +CD47 EpCAM-AsiC significantly increased TNF-α production by TILs. Additionally, CD47 EpCAM-AsiC, but not anti-CD47, significantly reduced the number of immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and mononuclear (MO)-MDSCs infiltrating tumors compared with control tumors. Therefore, CD47 EpCAM-AsiC was more effective than anti-CD47 in controlling tumor growth and inducing antitumor immunity.

[0253] CD274(PD-L1)EpCAM-AsiC has a modest effect on tumor growth Checkpoint blockade induces protective immunity in several cancers, with dramatic and durable responses. While breast cancer is generally not sensitive to checkpoint inhibitors, last year, anti-PD-L1 therapy administered with protein-bound paclitaxel was shown to improve survival by several months in a subset of TNBC patients whose tumors express PD-L1, making it the first FDA-approved checkpoint blockade therapy for this subset of patients. 4T1E strongly and uniformly expresses PD-L1 (Figure 36A). Therefore, we evaluated the antitumor activity of CD274 EpCAM AsiC, which targets PD-L1. CD274 EpCAM-AsiC inhibited tumor growth, but the effect was not statistically significant (Figure 36B), indicating that combining CD274 EpCAM-AsiC with other therapies may be necessary to improve antitumor immunity.

[0254] MCL1 EpCAM-AsiC induces antitumor immunity Because TNBC is a heterogeneous cancer defined by exclusion, genome-wide siRNA screening to identify shared dependencies in human basal-A TNBC cell lines identified few shared dependency genes. 26,35 One of the strongest hits was MCL1, an anti-apoptotic BCL-2 family gene that is commonly amplified in TNBC and whose overexpression correlates with poor prognosis. 36We found that tumor cell death induced by Mcl1 knockdown (Fig. 32B) was mediated by CD8 + We hypothesized that MCL1 EpCAM-AsiC may promote cross-presentation of tumor antigens to T cells, thereby improving antitumor immunity. We first verified that MCL1 EpCAM-AsiC reduced 4T1E survival in vitro (Fig. 37A). After orthotopic 4T1E tumors became palpable, MCL1 EpCAM-AsiC injected sq every 3 days significantly slowed tumor growth (Fig. 37B). MCL1 EpCAM-AsiC also inhibited CD8 expression in tumors. + / CD4 + T reg The tumor cell ratio and antitumor CD4+ T cell and CD8+ T cell function were also significantly improved. We also observed a similar improvement in antitumor T cell immunity using another cytotoxic EpCAM-AsiC that targets Plk1, an essential gene encoding a kinase required for mitosis (data not shown). Thus, some cytotoxic EpCAM-AsiCs also promote effective antitumor immune responses.

[0255] Enhanced antitumor activity of the EpCAM-AsiC combination One advantage of AsiC for cancer is the relative ease of combining AsiCs targeting multiple genes to create drug cocktails that can have additive or synergistic effects in inhibiting tumor growth by knocking down genes that promote tumor immunity through different mechanisms. To examine the efficacy of EpCAM-AsiC combinations, mice bearing 4T1E orthotopic tumors were treated individually or in combination with four of the most effective EpCAM-AsiCs targeting Upf2, Parp1, Cd47, or Mcl1, with the EpCAM aptamer or eGFP EpCAM-AsiC as controls (Figures 28A-28B). While each EpCAM-AsiC significantly delayed tumor progression on its own, the cocktail was significantly better. The cocktail increased the number of CD8+ TILs by approximately fourfold (Figure 28C), and CD8 + / CD4 + T regThe TIL ratio improved approximately 5-fold (Figure 28C), and CD8 + TILs and CD4 + The combination of EpCAM-AsiC and EpCAM-AsiC increased the stimulated production of cytokines and cytotoxic molecules by TILs (Figures 28E-28G). The combined EpCAM-AsiC was also evaluated in mice bearing orthotopic 4T1E-eGFP tumors, where expression of the immunogenic foreign protein caused tumor regression beginning approximately two weeks after tumor implantation (Figure 28H). Tumors in mice treated with the AsiC cocktail grew much more slowly and began to regress more quickly. The combination therapy also potently enhanced T cell immunity in 4T1E-eGFP tumors (Figure 28I). Importantly, after five injections of the EpCAM-AsiC combination, EpCAM expression on 4T1E-eGFP tumors remained unchanged, indicating that tumors did not become resistant to EpCAM-AsiC due to downregulation of EpCAM.

[0256] We next investigated whether tumor inhibition by the EpCAM-AsiC cocktail could be improved by adding the checkpoint inhibitor anti-PD-1 to target exhausted T cells. Treatment of control mice receiving the EpCAM aptamer with anti-PD-1 only slightly, but not significantly, slowed 4T1E tumor growth. However, the combination of anti-PD-1 and the EpCAM-AsiC cocktail significantly reduced tumor growth compared with the AsiC cocktail alone. The EpCAM-AsiC cocktail inhibits CD44 + CD8 +Although □-PD-1 significantly reduced PD-1 levels on TILs when the same antibody clone (29F.1A12) was used for detection, the addition of □-PD-1 further reduced PD-1 staining, likely due to the bound therapeutic antibody blocking the staining (Figure 38A). As with CD47 EpCAM-AsiC itself, the combined AsiC also significantly reduced the expression of other inhibitory coreceptors (CTLA-4, TIM-3, and LAG-3) on CD44 CD8 TILs (Figure 38B). The addition of anti-PD1 did not have any significant additional effect on these inhibitory receptors. Furthermore, the addition of anti-PD-1 was able to reduce T cell responses, resulting in reduced expression of the costimulatory receptor 2B4 (CD244) on CD44 CD8 TILs compared to T cells from mice treated with the cocktail alone. However, the addition of anti-PD-1 to the AsiC cocktail did not significantly reduce the expression of CD8 TILs compared to mice treated with the AsiC cocktail alone. + The AsiC cocktail significantly increased the number of TILs (Figure 38C) and NK TILs and stimulated cytokine production by CD8+ TILs. Thus, the strategy of using the AsiC cocktail to target immune-evading tumor cells can synergize with checkpoint inhibitors against T cell inhibitory receptors.

[0257] The EpCAM-AsiC cocktail broadly enhances the antitumor functionality of tumor-infiltrating T cells and macrophages To unbiasedly evaluate the changes in tumor-infiltrating immune cells induced by treatment with the four EpCAM-AsiC cocktails, we analyzed sorted CD45 IgG from mice bearing 4T1E orthotopic tumors treated with the EpCAM aptamer or cocktail. +We performed scRNA-seq analysis on tumor-infiltrating cells (Figures 29A-29F). This analysis focused on tumor-infiltrating proliferating T cells and macrophages, which showed the greatest changes with EpCAM-AsiC therapy (Figures 29A-29F). Gene ontology analysis of differentially expressed genes (DEGs) in proliferating T cells revealed significantly increased expression of gene signatures related to migration / chemotaxis, immunological synapse formation, T cell activation, proliferation, and metabolism in EpCAM-AsiC-treated tumors compared to control tumors (Figures 29A-29F).

[0258] Expression of genes related to monocyte / macrophage migration, activation, and endocytosis was significantly upregulated in the "M1" macrophage subpopulation treated with EpCAM-AsiC, whereas genes involved in inflammation and production of type I IFN and chemokines, including genes regulating immune responses to tumors, were upregulated in the "M2" subpopulation (Figures 29A-29F). T cells in the AsiC-treated group expressed higher levels of genes involved in T cell activation and effector function compared with those in the control group (Figures 29A-29F). T cells in cluster 1 expressed higher levels of genes involved in early signaling events of T cell activation, such as Cd69, Zap70, Fos, and Junb, which were further upregulated by EpCAM-AsiC treatment (Figures 29A-29F). Proliferating T cells in AsiC-treated tumors express effector and memory and functional T cell genes, such as transcripts of effector molecules Ifng, Tnf, Il2, Gzmb, and Gzmk; costimulatory gene Icos; IL-2 receptor complex genes IL2ra, IL2rb, and IL2rg; and CD8 + Runx2, which promotes the long-term persistence of memory T cells, was upregulated. Several T cell function genes, such as Gzmb, Gzmk, Prf1, and Tnf, were also upregulated in cluster 1 T cells after EpCAM-AsiC treatment. In contrast, genes encoding co-inhibitory molecules, such as Pdcd1, Ctla4, Tigit, Lag3, and Havcr2, as well as Treg The signature gene Foxp3 was mostly downregulated in T cells from AsiC-treated tumors, particularly in the proliferating T cell cluster (Figures 29A-29F), indicating that EpCAM-AsiC rescues T cell exhaustion. Furthermore, macrophages in AsiC-treated tumors upregulated the expression of genes related to myeloid cell maturation (e.g., Cd74); genes related to M1 function (e.g., Nos2, Fcgr1, Cd68, Il12a, and Ccr7); genes related to phagocytosis and antigen processing (e.g., Lgals3, Il1b, Apoe, Cd14, and Ly75); and genes related to the production of inflammatory cytokines / chemokines (e.g., Tnf, Ccl2, Cxcl2, and Il12a) (Figures 29A-29F), indicating improved antitumor functionality.

[0259] EpCAM-AsiC reduces metastatic tumor growth All previously reported experiments treated orthotopic tumors immediately after they became palpable. However, BC patients often present with more advanced local or metastatic disease, which is more difficult to treat. Many BC patients also have evidence of microscopic metastases before metastatic disease becomes clinically apparent. Furthermore, metastatic disease is usually what kills the patient. Therefore, being able to target metastatic tumor cells is crucial for effective BC therapy. To determine whether EpCAM-AsiC has activity against metastatic TNBC, we generated a 4T1E cell line (4T1E-Luc) stably expressing firefly luciferase, which can be detected by bioluminescence imaging in live animals. Intravenous injection of 4T1E-Luc resulted in tumor cells lodge in the lungs, and tumors in the lungs could be detected 7–10 days later. Seven-day-old mice bearing metastatic 4T1E-Luc lung tumors were treated with the EpCAM aptamer or a cocktail of EpCAM-AsiC targeting Upf2, Cd47, Parp1, and Mcl1. EpCAM-AsiC significantly inhibited breast tumor growth in the lung (Figures 30A-30K). 20 days after tumor challenge, CD8 + T cells and CD4+ T cells were isolated from the lungs and analyzed for IFN-γ and TNF-α production stimulated with PMA and ionomycin. Significantly more CD4+ and CD8+ T cells from mice treated with EpCAM-AsiC produced these cytokines. Thus, EpCAM-AsiC inhibited metastatic tumor growth and enhanced antitumor immunity at the site of metastasis in the lung.

[0260] EpCAM-AsiC suppresses invasive breast cancer in Erb2ΔEx16 transgenic mice To evaluate the efficacy of EpCAM-AsiC in the challenging GEMM of aggressive Her2+ breast cancer, we used a doxycycline-inducible mouse model expressing eGFP and a truncated Her2 gene (Erb2ΔEx16 with a deletion of exon 16, resulting in a juxtamembrane 16-aa deletion and a constitutively active HER2 receptor) under the control of the MMTV promoter. 61At least 80% of these mice develop multifocal, rapidly growing, and metastatic HER2+ breast tumors that are uniformly EpCAM+ within approximately 10 to 28 days of doxycycline addition (Figures 31A-31D). Without either treatment, these tumors had few infiltrating CD4 or CD8 T cells (Figures 31A-31D). We treated these mice with either the EpCAM aptamer as a control or six EpCAM-AsiC combinations targeting Upf2, Parp1, Apex1, Cd47, Mcl1, and Cd274, starting 3 days after initiating doxycycline and every 3 days thereafter (Figures 31A-31D). Treatment with the EpCAM-AsiC cocktail did not alter the number of mice that developed detectable tumors (four of six mice in each group developed tumors within 10 days), but significantly inhibited tumor growth over four weeks of treatment (Figures 31A-31D). After eight treatments, EpCAM expression by GFP+ tumors remained unchanged (Figures 31A-31D). Furthermore, antitumor immune function was significantly enhanced in mice receiving the EpCAM-AsiC cocktail. More TAMs were GFP+, indicating increased tumor cell phagocytosis (Figures 31A-31D). The EpCAM-AsiC cocktail did not alter the number of TILs within spontaneously arising ErbB2ΔEx16 tumors (not shown), but stimulated the production of IFN-γ and TNF-α by CD4+ and CD8+ TILs (Figures 31A-31D), and GzmB and PFN expression was significantly increased by CD8+ TILs. + TIL, CD4 + The EpCAM-AsiC cocktail was increased in TILs, TILs, and NK TILs (Figures 31A-31D). Thus, the EpCAM-AsiC cocktail suppressed tumor growth and mobilized antitumor immunity in immunologically "cold," aggressive, spontaneous GEMM breast tumors.

[0261] Consideration Highly invasive TNBC, which has the worst survival rate of all BC subtypes and lacks a potent treatment strategy, represents a significant hurdle for BC therapy. 66Fortunately, TNBC, as well as HER2+ BC, another aggressive subtype of BC susceptible to drug resistance and recurrence, are more immunogenic with higher levels of tumor mutation burden and TILs compared with other BC subtypes, making them better targets for cancer immunotherapy. 67-69 In this study, we demonstrate that tumor cell-targeted gene knockdown with immunomodulatory EpCAM-AsiC can effectively improve tumor immunogenicity and potently inhibit tumor growth in both murine TNBC and HER2+ BC models, demonstrating the antitumor activity of EpCAM-AsiC as a powerful approach for BC immunotherapy. To our knowledge, this is the first study to demonstrate the immunostimulatory efficacy of EpCAM-AsiC against invasive BC. No evidence of toxicity or weight loss was detected in treated mice. Furthermore, combined AsiC synergized with PD-1 checkpoint inhibitors to further suppress tumor progression, highlighting its potential clinical benefit for the majority of BC patients who show limited response to immune checkpoint inhibitor monotherapy.

[0262] The EpCAM aptamer-mediated siRNA delivery system exploited tumor-specific surface overexpression of EpCAM to selectively target epithelial tumor cells and stem-like tumor-initiating cells. 34 Normal epithelial cells express much lower levels of EpCAM on the outer interface as part of the tight junction complex. 73 , thus escaping EpCAM-AsiC targeting. We demonstrated selective internalization of the EpCAM aptamer by human EpCAM+ tumors in vitro and at distant sites in vivo. 26 Herein, we demonstrate that EpCAM-AsiC can bind to and internalize into mouse EpCAM+ BC cells with high affinity and specificity for knockdown target genes. As an oncogenic signaling protein, the tumor-associated antigen EpCAM is essential for the proliferation and migration of BC cells. 74It is demonstrated herein that BC cells treated multiple times with EpCAM-AsiC do not downregulate EpCAM levels, likely due to its oncogenic properties.

[0263] EpCAM-AsiC also do not activate the receptor, presumably because they do not cross-link the receptor. 28 .

[0264] Despite its promise, AsiC can be further modified to enhance its therapeutic potential. Various biochemical modifications of AsiC have been developed to optimize its performance, for example, to reduce systemic clearance and extend its half-life, attenuate nuclease degradation, enhance its delivery and cellular uptake, and avoid immune sensor activation. 70,78,79 The EpCAM-AsiC described herein is chemically modified with 2'-fluoropymidine substitutions in the RNA aptamer and siRNA sense strand, as well as 3'-dTdT overhangs, which contribute to its RNase resistance, stability, and help reduce immune activation. In addition, AsiC was administered via sc injection, which exhibited a slower release rate into the circulation and could provide more time for recycling of cellular receptors that mediate uptake to improve the efficiency of siRNA delivery. 80,81 Additional modifications of EpCAM-AsiC, such as 2' sugar modifications of the siRNA guide strand, phosphorothioate (PS) backbone modifications, and 5' unlocked nucleic acid modifications of the canonical siRNA, have great potential to further improve gene knockdown efficiency, reduce the required AsiC dose, and reduce off-target RNAi activity. 70,82 Indeed, such changes result in a two-order of magnitude reduction in the administered dose of N-acetylgalactosamine (GalNac)-conjugated siRNA, while promoting RNAi activity and preserving the reagent's low toxicity profile. 83 Endosomal escape is a major obstacle to improving RNAi efficacy beyond the liver 84At a dose of 5 mg / kg, EpCAM-AsiC exhibited a favorable gene silencing profile in tumor cells in vivo, indicating that a certain number of AsiCs could leave the endosome for targeted knockdown. Furthermore, conjugating EpCAM-AsiC to bulky chemicals, i.e., cholesterol, liposomes, or PEG, could further extend its circulatory half-life and reduce systemic clearance, achieving superior therapeutic efficacy for cancer patients. 85-88 .

[0265] Tumor neoantigens, often generated by genetic instability in tumor cells, are not expressed by normal tissues and are therefore highly immunogenic. + and CD8 + and are ideal targets for cancer immunotherapy. 89,90 The lack of tumor neoantigen expression due to the low nonsynonymous mutation rate in BC cells represents a major challenge for BC immunotherapy. Using UPF2 AsiC, tumor neoantigen expression can be induced by reducing the NMD machinery in BC cells. NMD has traditionally been viewed as a key mechanism for mRNA quality control, and NMD-targeted transcripts can arise from various mRNA variations that cause PTC. The core NMD machinery contains three trans-acting factors, UPF1-3, in addition to SMG1-7. 91 .

[0266] UPF2 is a key NMD factor that mediates the interaction between the UPF3 / exon junction complex (EJC) and a UPF1-containing complex that subsequently phosphorylates UPF1 to induce mRNA decay activity. 92,93 Cells deficient in NMD activity have been shown to upregulate aberrant mRNA splicing variants. 94-96 In one study, NMD inhibition by UPF1 knockdown in N2A neuroblastoma cells resulted in altered expression of more than 200 exons. 97Similarly, we demonstrate herein that knockdown of UPF2 reduced NMD activity in BC cells grown in vitro and in vivo, induced DEU events in 281 genes, and generated numerous novel mRNA isoforms and NMD-sensitive transcripts that may encode tumor neoantigens. + This was associated with increased numbers and improved function of TILs and robust inhibition of breast tumor growth. These findings were supported by studies in which knockdown of UPF2 or SMG1 with PSMA-targeting aptamers suppressed PSMA-CT26 / B16F10 tumor growth in a T cell-dependent manner. 98 Although many neoantigens induced by NMD inhibition are generated by random mutations and are therefore tumor cell specific, there is also a set of bona fide NMD targets that are stabilized to express novel antigens upon NMD inhibition. 39,42,99 In particular, NMD has been reported to regulate many non-mutated transcripts involved in cellular stress response and nutrient homeostasis pathways. 42,100,101 Amino acid starvation and ER stress in tumors inhibit NMD activity, which is likely a strategy used by tumor cells to upregulate stress-responsive transcripts to adapt to these environmental challenges. 42,95,101 Interestingly, this study identified both DEU and DIU events upon UPF2 knockdown in the PFKFB4, UCN2, CDKAL1, and TRIM4 genes, all of which are involved in oxidative and ER stress regulatory pathways. 102-105 These changes were also observed in all three samples studied, suggesting that NMD inhibition could induce the expression of antigens shared among all or at least a portion of the tumor cells in which UPF2 was downregulated.

[0267] TNBC exhibits approximately 80% mutations in TP53, which leads to its high genomic instability 106,107In addition, the majority of TNBCs are characterized by defects in homologous recombination (HR), a high-fidelity DNA repair mechanism that is critical for the efficient repair of double-strand DNA breaks (DSBs). 108 Therefore, TNBC represents a good therapeutic target for PARP1 inhibitors (PARPi). PARP1 is well known to be involved in distinct DNA repair processes, such as BER, single-strand break (SSB) repair, and DSB repair. Olaparib primarily works in BRCA-mutated BC, which are defective in HR, because endogenously generated SSBs are no longer repaired in the presence of PARPi and are converted to DSBs during cell replication, which cannot be repaired in BRCA-deficient cases, resulting in cell death. 109 .

[0268] A similar mechanism of action may exist for PARP1 AsiC, whereby knockdown of PAPR1 expression in BC cells promotes cancer cell death in vivo. Dying cells may release more TAs and attract T cell tumor infiltration. Notably, both PARP1 AsiC and olaparib can also exert antitumor effects in the majority of TNBCs that are BRCA+ but contain other HR-related defects. 110 By reducing the expression of key DNA repair enzymes, PARP1 AsiC may also introduce more DNA damage, increase DNA mutation burden, and promote tumor neoantigen generation. PARP1 AsiC mediates the expression of CD8 + TIL vs CD4 + Treg ratio and CD8 + TILs and CD4 + It is demonstrated herein that olaparib strongly enhances cytokine production by both TILs and tumor cells, which may be due to its ability to induce both tumor cell death and tumor neoantigen expression. Surprisingly, olaparib did not achieve significant tumor suppression in the 4T1E TNBC model and failed to increase anti-tumor T cell immunity, which is in contrast to its therapeutic efficacy in the BRCA1-deficient tumor model. 111-113The immunomodulatory effects of olaparib depend on STING-mediated IFN-I production. 112,113 This may be insufficient in 4T1E tumors. The better stability and tumor penetration ability of PARP1 AsiC may also contribute to its improved efficacy. In addition, PARP1 is a known coactivator of NF-κB, which can induce tumor inflammation. 114 PARP1 knockout was able to strongly reduce inflammation-driven tumor formation. 115 PARP1 AsiC-mediated gene knockdown may result in similar effects that may not be achieved by olaparib-mediated inhibition of poly (ADP-ribosylation).

[0269] APC (macrophages and dendritic cells)-mediated phagocytosis and TA cross-presentation of dying cancer cells are crucial for initiating effective antitumor T cell immunity. Tumor cells ubiquitously upregulate CD47 expression, presumably to avoid endogenous "eat me" signals induced during programmed cell death and cell elimination, and to avoid recognition by the immune system. 49,50 Neutralization of the anti-phagocytic signaling of CD47 via anti-CD47 antibodies could restore cancer cell phagocytosis by either macrophages or DCs. + Although previously considered key players in presenting exogenous antigens to T cells, in the context of blocking the CD47-SIRP axis, both macrophages and DCs are unable to effectively express CD8 + demonstrated its antigen cross-priming ability to stimulate T cell responses 50,116,117 Our data indicate that the antitumor efficacy of CD47 AsiC is TAM-dependent, as anti-CSF1R-mediated TAM depletion, while only reducing TAM numbers by 70%, strongly attenuated the antitumor function of CD47 AsiC. + TILs almost completely abolished their effector functions, which is consistent with the fact that TAMs are CD8 +These findings suggest that CSF-1R plays a key role in cross-priming TIL immunity. Although anti-CSF1R is primarily used for macrophage depletion in vivo, CSF1R is also expressed by plasmacytoid and conventional DC subsets, and CSF-1 signaling is required for optimal DC differentiation. 118 Therefore, it is possible that anti-CSF1R antibodies also depleted a large number of DCs, which may have contributed to the reduced tumor-suppressive and immunostimulatory abilities of CD47AsiC. Indeed, CD47AsiC is a CD11c antigen in tumors that specializes in the uptake of extracellular antigens. + DEC205 + The percentage of DCs was increased and DC maturation was promoted, and the antigen cross-presentation capacity of these DCs is also likely to be improved by CD47 AsiC treatment.

[0270] Furthermore, studies have reported that the therapeutic potential of CD47 blockade requires STING-mediated tumor DNA sensing by host DCs. 116 Both tumor-infiltrating DCs (TIDCs) and TAMs produced more IFN-I upon antibody-mediated CD47 blockade, which may promote their antigen cross-presentation function. Improved DC maturation upon CD47 AsiC treatment also likely depends on increased IFN-I signaling. Additionally, CD47 AsiC treatment may also modify the tumor cytokine environment through promoting DC maturation, which may help increase the ratio of M1 TAMs to M2 TAMs and reduce the presence of MDSCs, creating a TME that is tumor-suppressive and immunostimulatory. Interestingly, CD47 AsiCs outperformed anti-CD47 antibodies in suppressing 4T1E tumor growth. Both treatments inhibited CD8 + Although CD47 AsiC increased the function of TILs, only CD47 AsiC increased the function of CD4 + Anti-CD47 antibody therapy promoted the function of TILs and reduced the number of MDSCs, suggesting its therapeutic potential in the 4T1E tumor model. + Did not improve T-cell function 50 On the other hand, CD4 +T cell depletion significantly impaired the therapeutic efficacy of CD47 AsiC, clearly demonstrating its importance for CD47 AsiC treatment. CD47 signaling can be directly reduced by gene knockdown rather than antibody-mediated signal blockade, and smaller CD47 AsiC with better tumor penetration ability leads to more efficient tumor suppression.

[0271] Biomarkers consistently identified by targeting each of the factors in the cancer-immune cycle with EpCAM-AsiC include CD8 + TIL vs CD4 + An upregulated ratio of Tregs has been reported as a favorable prognostic marker associated with improved clinical outcomes in patients with various types of cancer, including invasive BC. 44,119-121 This indicates the potential clinical benefit that may be provided by the therapeutic approach described herein. However, EpCAM-AsiC targeting PD-L1 did not significantly inhibit overall tumor growth, despite the clinical efficacy shown by anti-PDL1 antibodies in patients with TNBC. PD-L1 is actually expressed at higher levels on TICs than on tumor cells, and high PD-L1 expression on TICs alone is a favorable prognostic factor for cancer patients. 122,123 Therefore, EpCAM-AsiC-mediated PD-L1 + Targeting tumor cells alone may not achieve an effective antitumor effect. When simultaneously inducing tumor neoantigen expression, inducing cancer cell death, increasing TA release, and promoting antigen uptake and cross-presentation, AsiC cocktail therapy demonstrated the most potent efficacy in enhancing antitumor immunity and suppressing tumor growth. Immunomodulatory AsiC cocktails targeting more than one gene may be ideal for cancer immunotherapy to reduce the chance of developing drug resistance.

[0272] scRNA-seq data revealed that CD8 +An improved activation state and functional profile of both TILs and monocytes / macrophages was further revealed, which was associated with increased expression of CD8 + This corroborated immunological studies that identified enhanced cytokine production and cytotoxic function of TILs, as well as increased tumor cell endocytosis by TAMs. The proliferating TIL clusters, which showed the most significant functional improvement with AsiC cocktail treatment, were likely composed primarily of TILs that recognized TAs. AsiC cocktail treatment also reduced the expression of mRNA transcripts encoding various co-inhibitory molecules in proliferating TILs, suggesting that they were protected from overactivation / exhaustion. Reduction of PD-1 protein expression also significantly reduced CD44 expression in antigen-experienced AsiC cocktail-treated tumors. + CD8 + When AsiC cocktail was given together with PD-1 checkpoint inhibitors, the CD8 + Enhanced numbers and function of TILs and NK TILs, along with reduced co-inhibitor expression, were observed, indicating that the combinatorial approach provides additional therapeutic benefit. Finally, inducible genetically engineered mouse (GEM) tumors are relatively resistant to immunotherapeutic intervention, in part because they generally do not harbor many genetic mutations and are therefore poorly recognized. 124,125 The AsiC cocktail approach showed promising antitumor efficacy in GEM models of both highly invasive HER2+ BC and lung metastatic TNBC, demonstrating the great immunotherapeutic potential offered by immunomodulatory EpCAM-AsiC for patients with invasive BC.

[0273] material and method cell line Human MDA-MB-468, MCF7, T47D, SKBR3, and mouse L929 and P815 cell lines were obtained from ATCC. 4T1E cells were generated by sorting 4T1 cells for high E-cadherin expression. 4T1E-eGFP cells were generated with the pCAG-eGFP lentiviral vector. 4T1E cells stably expressing firefly luciferase (4T1E-Luc) were selected using puromycin after infection with the EF1a-luciferase (firefly)-2A-RFP-Puro lentiviral vector (amsbio). Cell lines were maintained in 10% heat-inactivated FBS (Gemini Bioproducts), 6 mM HEPES, 1.6 mM L-glutamine, 50 μM 2-mercaptoethanol, and 100 U ml -1 Penicillin G, and 100 μg ml -1 DMEM (4T1, 4TO7, 4T1E, 4T1E-eGFP, 4T1E-Luc, L929, P815, MCF10CA1a, EpCAM) supplemented with streptomycin sulfate (Sigma-Aldrich) hi Cells were cultured in RPMI 1640 (MDA-MB-231 cells), RPMI 1640 (MDA-MB-468, T47D cells), MEM (MCF7 cells), and McCoy's 5A (SKBR3) medium (Gibco, Thermo Fisher Scientific). All cell lines were verified to be mycoplasma-free by PCR and authenticated by morphology.

[0274] Mouse studies All animal experiments were conducted in compliance with all relevant ethical regulations and approved by the Harvard Medical School Institutional Animal Care and Use Committee. All mice were housed in the Harvard Medical School Animal Facility. Female BALB / c mice (6–8 weeks old) were purchased from The Jackson Laboratories. Transgene expression was confirmed by tail clipping and ErbB2ΔEx16 transfection. The tumor size was determined by genotyping using the TIFF2025169317000016.tif31152. Eight-week-old female ErbB2ΔEx16+ / -MTB+ / - mice received 2 mg / ml doxycycline (Sigma-Aldrich) in drinking water for tumor induction throughout the study. After tumor induction for 3 days, mice were randomly assigned to either a control or treatment group and treated every 3 days with either the EpCAM-AsiC cocktail (5 mg / kg each in PBS, for a total of 30 mg / kg) or the EpCAM aptamer (30 mg / kg). Tumor size was monitored by palpation, and tumor growth was assessed by measuring the perpendicular diameter of the tumor every other day. Mice were euthanized on day 28.

[0275] For orthotopic tumor burden, 4T1E cells (approximately 10 per mouse) were 5 cells) or 4T1E-eGFP cells (approximately 3 × 10 per mouse 5 EpCAM-AsiC) were injected into the four mammary fat pads of BALB / c mice. When tumors became palpable (approximately 3-4 days after tumor challenge), mice were subcutaneously injected every 3 days with medium alone (mock), 5 mg / kg of EpCAM aptamer or eGFP EpCAM-AsiC as a control treatment, or immunomodulatory EpCAM-AsiC. Tumor growth was monitored by measuring the perpendicular diameter of the tumor daily. When the mean diameter of tumors in the control group reached approximately 4-5 mm (approximately 2 weeks), all mice in the experiment were euthanized, and tumors were collected for analysis. To determine the longer-term antitumor efficacy of UPF2 EpCAM-AsiC, mice were injected with 5 × 10 44T1E cells were loaded, and treatment was initiated on day 8 after tumor challenge. Tumor growth was monitored for 25 days. For cell depletion assays, CD8 antibody (clone 2.43), CD4 antibody (clone GK1.5), CSF1R antibody (clone AFS98), or isotype control antibody (all from BioXCell) were injected intraperitoneally (ip, 300 μg / mouse) into mice loaded with 4T1E tumor cells. CD8 or CD4 antibody was administered starting on day 2 after tumor challenge for three consecutive days, and then every five days thereafter. For TAM depletion, anti-CSF1R antibody was administered starting on day 0 after tumor challenge and every other day thereafter. Immune cell depletion was verified by staining for CD4, CD8, CD11b, F4 / 80, and MHCII using peripheral blood mononuclear cells obtained 7 days after tumor challenge and / or tumor-infiltrating immune cells obtained at necropsy, as well as by flow cytometry. For anti-CD47 antibody (clone MIAP410, BioXcell), the antibody was injected i.p. (400 μg / mouse) starting on day 3 after tumor challenge and every 3 days thereafter. For PARP1 inhibitor treatment, olaparib (LC Laboratories) was dissolved in DMSO to 50 mg / ml. It was further diluted in 10% 2-hydroxypropyl-cyclodextrin / PBS (Sigma-Aldrich) and given to mice by i.p. injection at 50 mg / kg daily, starting on day 3 after tumor challenge, for a total of 12 injections. For immunotherapy with PD-1 inhibitors, anti-PD-1 antibody (clone 29F.1A12, BioXCell) was given starting on day 10 after tumor challenge and every 3 days thereafter (200 μg / mouse).

[0276] To evaluate the antitumor efficacy of EpCAM-AsiC against lung metastatic breast tumors, 4T1E-Luc cells were first mixed with 150 μg / ml of D-luciferin (PerkinElmer), and their luciferase activity was checked by luminescence imaging using an IVIS Lumina II system (Caliper Life Sciences). BALB / c mice were inoculated with 4T1E-Luc cells (approximately 3 × 10 per mouse). 5Mice were intravenously injected with EpCAM aptamer or EpCAM-AsiC cocktail starting on day 7 after tumor challenge. After ip injection of 150 mg / kg D-luciferin, whole-body luminescence images were taken immediately after tumor challenge and every 5 days thereafter for 20 days. Lungs were isolated at necropsy for analysis.

[0277] RNA A 19-nt EpCAM aptamer with a 2'-fluoropyrimidine and an EpCAM aptamer with fluorescent Cy3 conjugated to its 5' end (EpCAM-Cy3) (Trilink Biotechnologies or Dharmacon) were used as control RNA oligos. Candidate mouse or human gene-specific or mouse and human gene cross-reactive siRNAs were predesigned with ON-TARGETplus siRNAs and / or designed using the siDESIGN tool (both from Dharmacon). The siRNAs used in the EpCAM-AsiC construct were selected by comparing their gene knockdown efficiency in vitro in mouse and / or human BC cell lines using qRT-PCR. ON-TARGETplus non-targeting pool siRNA was used as a negative control (Dharmacon). The siRNA sequence with the best gene knockdown potential and lowest Tm value was selected. For EpCAM aptamer-siRNA conjugation, a long strand of AsiC containing the EpCAM aptamer, a UUU linker, and the sense strand of siRNA was synthesized with a 2'-fluoropyrimidine and a dTdT overhang at its 3' end. It was annealed to the antisense strand of siRNA using a two-fold molar excess of a short strand (both from Trilink Biotechnologies). The long RNA oligo was first heated to 95°C for 10 minutes. The short RNA was then added and annealed to the long strand at 65°C for 7 minutes. The mixture was allowed to cool at room temperature for 20 minutes. The annealed EpCAM-AsiC duplex was further purified using an Illustra MicroSpin G-25 column (GE Healthcare Life Sciences). The sequences of siRNA and EpCAM-AsiC are provided in Tables 5 and 6.

[0278] RNA uptake by mouse and human BC cell lines Mouse and human BC cell lines were plated at 30,000 cells per well in 96-well plates. Cells were incubated with a range of concentrations of EpCAM-Cy3 (0–1000 nmol / L) for 6 hours in Opti-MEM medium supplemented with 5 mM MgCl2, 0.1 mg / ml tRNA, and 0.1 mg / ml salmon sperm DNA (all from ThermoFisher). Complete culture medium supplemented with 20% FBS was then added, and the cells were cultured for 72 hours. Surface-bound EpCAM-Cy3 was washed away by incubation and washing with a wash buffer (DPBS supplemented with 5 mM MgCl2, 0.5 M NaCl, and 0.2 N acetic acid) at 4°C. The resulting cell suspension was stained for live cells with live / dead fixable aqua dead cell stain (ThermoFisher), and the amount of EpCAM-Cy3 internalization was analyzed by flow cytometry. The kinetic parameter Kd for the EpCAM-Cy3 uptake capacity of each BC cell line was calculated from nonlinear regression analysis of a one-binding site hyperbola using GraphPad Prism 8.

[0279] Gene knockdown and qRT-PCR For in vitro siRNA-mediated gene silencing, cells were used immediately after seeding at 10,000 cells per well in 96-well plates. Cells were transfected with 6.25 nmol / L to 100 nmol / L of siRNA using Dharmafect I according to the manufacturer's protocol (Dharmacon). Cells were transfected for 6–8 hours in serum- and antibiotic-free medium, after which culture medium supplemented with 20% FBS was added. RNA was extracted after 24–48 hours, and gene knockdown was assessed by qRT-PCR. For in vitro EpCAM-AsiC-mediated gene silencing, cells were incubated with 4 □mol / L of EpCAM aptamer or EpCAM-AsiC in WIT-T medium. Gene knockdown was assessed by measuring mRNA and protein levels by qRT-PCR and flow cytometry, respectively, after 72–96 hours of treatment. Cell viability was measured by CellTiter-Glo (Promega) 24–96 h after treatment as indicated. Cell proliferation was measured by CellTiter 96 A. queous The tumors were collected from mice treated with EpCAM aptamer or EpCAM-AsiC for in vivo gene silencing experiments. Single-cell suspensions were prepared by tumor digestion and homogenization. Dead cells were removed, and CD45 - EpCAM + Tumor cells were enriched by negative selection with CD45 microbeads and positive selection with CD326 (EpCAM) microbeads according to the manufacturer's protocol (Miltenyi Biotec). - EpCAM -Cells were collected as controls. Gene knockdown in both cell subsets was measured at the mRNA and protein levels by qRT-PCR and flow cytometry, respectively. For qRT-PCR, total RNA was extracted with TRIzol (ThermoFisher) and the Direct-zol RNA miniprep kit (ZYMO Research), and RNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription kit (ThermoFisher). qRT-PCR of the cDNA was performed using primers corresponding to the target gene or the housekeeping gene GAPDH (IDT), SsoFast EvaGreen Supermix, and a Bio-Rad C1000 Thermal Cycler (Bio-Rad).

[0280] Histology, IHC, and fluorescence microscopy Tumors were fixed in 10% formalin, stored in 70% ethanol, and embedded in paraffin. Sections (5 μm) were cut, air-dried, and fixed for hematoxylin-eosin (H&E) and IHC staining by the Dana-Farbar Cancer Institute Rodent Histopathology Core and the Dana-Farber / Harvard Cancer Center Specialized Histopathology Core as previously described. 113,126 Anti-CD8 antibody (clone 4SM15, ThermoFisher) was used at 5 μg / ml. Slides were scanned in the Aperio image analysis platform. CD8 + The number of T cells was visualized and digitally annotated in regions of interest (ROIs, 6 fields / slide) using ImageScope software (Aperio Technology), and the ROIs were analyzed using image analysis algorithms (Aperio Technology).

[0281] For confocal microscopy, 10,000 cells were seeded into each well of a 16-well chamber slide (ThermoFisher) and co-cultured with 1000 nmol / L EpCAM-Cy3 diluted in Opti-MEM medium supplemented with 5 mM MgCl2, 0.1 mg / ml tRNA, and 0.1 mg / ml salmon sperm DNA. Complete culture medium supplemented with 20% FBS was added after 6 hours. Cells were cultured for 72 hours and washed with ice-cold high-salt wash buffer (DPBS supplemented with 5 mM MgCl2, 0.5 M NaCl, and 0.2 N acetic acid) to remove surface-bound EpCAM-Cy3. Cells were then counterstained with CellMask Deep Red Plasma Membrane Stain (ThermoFisher), fixed with 3% paraformaldehyde and 0.5% glutaraldehyde, counterstained with Hoechst 33342, and mounted. Fluorescence was detected using a Zeiss LSM 800 confocal laser scanning microscope, and images were acquired using ZEN 2.3 imaging software (Carl Zeiss).

[0282] Isolation of immune cells from mice Peripheral blood mononuclear cells and TICs were collected as described 127 Briefly, blood was collected by submandibular puncture, and PBMCs were isolated by Histopaque gradient centrifugation (Sigma-Aldrich). Red blood cells were lysed with 1× RBC lysis buffer. To isolate TICs, tumors were cut into small pieces and treated with a digestion buffer consisting of RPMI supplemented with 2 mg / ml collagenase D, 100 μg / ml DNase I (both from Sigma-Aldrich), and 2% FBS for 30 minutes at 37°C with agitation. Samples were then homogenized and filtered through a 40 μm strainer, and immune cells were purified by Percoll gradient centrifugation (GE Healthcare) and washed with Leibovitz's L-15 medium (Gibco, ThermoFisher).

[0283] Antibody staining and flow cytometry Immune cells isolated from mice were immunoblotted with anti-CD45-PerCPCy5.5 or -PacBlue, CD3-PE-Cy7, -FITC, or -APC, CD8-PacBlue, -PerCPCy5.5, -Alexa700, -FITC, or -APC, CD4-PE-Cy7, -APC, or -PerCPCy5.5, CD19-FITC, CD25-PE, CD44-PerCPCy5.5 or PacBlue, Gr-1-FITC or -PE, CD11b-Alexa700, CD11c-APC or -PE-Cy7, DEC205-PE, CD49b-PerCPCy5.5, -PacBlue, or FITC, NKp46-APC, F4 / 80-PE-Cy7, MHCII-PacBlue, CD206-APC, TCR-□-FITC, TER-119- Cells were stained with FITC, EpCAM-PE-Cy7, CD47-FITC, CD40-APC, CD86-FITC, CD107a-APC, CD107b-APC, PD-1-PE-Cy7, 2B4-FITC, CTLA-4-PE, LAG-3-APC, and TIM-3-PerCPCy5.5 (all from Biolegend). Dead cells were excluded using live / dead fixable aqua dead cell stain (ThermoFisher) supplemented with cell surface antibodies.

[0284] Mouse TAMs were cultured in vitro. + CD45 + CD3 - CD19 - Ter119 - TCRβ - CD11b + F4 / 80 + defined as 128 M1 TAM, + CD45 + CD3 - CD19 - Ter119 - TCRβ - CD11b + F4 / 80 + CD206 - MHC + M2 MAM is defined as +CD45 + CD3 - CD19 - Ter119 - TCRβ - CD11b + F4 / 80 + CD206 + MHC + NOTE: TAMs that did not conform to any of these expression panels were not classified as M1 or M2 TAMs, as TAMs in mouse mammary tumors are primarily CD45 + CD11b + F4 / 80 + MHCII + This is consistent with previous studies that have shown that cells can be characterized as 129 For intracellular staining of UPF2, granzyme B, or perforin, cells were first stained with antibodies against cell surface markers at 4°C for 30 min, then fixed and permeabilized with fixation / permeabilization buffer (BD Pharmingen) and stained with a primary antibody against UPF2 (clone D3B10, Cell Signaling Technology) or rabbit monoclonal IgG isotype antibody (Abcam), anti-granzyme B-PacBlue or -APC (ThermoFisher), and perforin-PE (Biolegend). UPF2 was further detected with a goat anti-rabbit IgG H&L-APC secondary antibody (Abcam). For staining of Foxp3, cells were first stained for surface markers, then fixed and permeabilized with Foxp3 / transcription factor staining buffer and stained with Foxp3-PercpCy5.5 or -PE (ThermoFisher). For intracellular cytokine staining of ex vivo stimulated lymphocytes, approximately 10 cells per sample were used. 6Cells were cultured in RPMI medium containing 2% FBS and stimulated with PMA (50 ng / ml, Sigma), ionomycin (2 μg / ml, Sigma), and Golgiplug (1.5 μg / ml, ThermoFisher) for 4 hours. Cells cultured with medium and Golgiplug alone served as negative controls. After fixation and permeabilization, cells were stained with antibodies against IFN-γ-PacBlue or -APC and TNF-PE-Cy7. Cells were analyzed using a BD FACSCanto II (BD Biosciences), and data were analyzed using FlowJo V.10 (TreeScan).

[0285] CD8 + Degranulation and cytotoxicity assays of TILs Single-cell suspensions of tumor-infiltrating immune cells were purified using CD45 microbeads or CD8 microbeads (Miltenyi Biotec). + cells or CD8 + For the degranulation assay, freshly isolated CD45 cells were enriched for + CD8 in cells + The number of TILs was first determined by flow cytometry. +TILs were cocultured with autologous target tumor cells plated one day earlier in 48-well plates at a 1:3 ratio in RPMI medium containing 10% FBS. Antibodies against CD107a-APC and CD107b-APC (1 mg / ml each, Biolegend) and IL-2 (100 IU / ml) were added at the beginning of the coculture. Positive control cells were treated with PMA (50 ng / ml) and ionomycin (2 μg / ml), while negative control samples were treated with medium and IL-2. The cocultures were incubated at 37°C in a 5% CO2 incubator for 1 hour, followed by the addition of the secretion inhibitors monensin (1:1000, Biolegend) and Golgi Plug (1.5 μg / ml) for an additional 5 hours. TILs were washed from the co-cultures after stimulation, replated in 96-well plates, stained for viable cells, and then stained with antibodies against CD8, IFN-γ, and TNF after fixation / permeabilization. + For the TIL cytotoxicity assay, autologous target tumor cells were incubated with chromium-51 ( 51 Freshly isolated CD8 cells were labeled with Cr and plated in a 96-well plate one day prior. + TILs were co-cultured with target tumor cells at a 5:1 ratio for 30 hours in RMPI medium containing 10% FBS and supplemented with IL-2 (100 IU / ml). CD8 TILs were required to achieve efficient target tumor cell killing. + The time of TIL co-culture has been determined by previous studies. 130 . maximum 51 Cr release was measured by CD8 cells incubated with 1% SDS. + Spontaneous set up using TILs 51 Cr release was measured in CD8 cells cultured in medium and IL-2 alone. + TILs were used to set up the assay. The percentage of target cell lysis was calculated using the following formula: % specific lysis = ((test 51 Cr release)-(spontaneous 51 Cr release)) / ((max. 51 Cr release)-(spontaneous 51 Cr release))×100.

[0286] Ex vivo phagocytosis assay Dead cells were removed by a dead cell removal kit (Miltenyi Biotec), and TAMs were enriched from tumor-infiltrating immune cells using F4 / 80 microbeads (Miltenyi Biotec). + Live in cells + CD11b + F4 / 80 + The number of TAMs was first determined by flow cytometry. 4T1E-eGFP tumors were treated with either a negative control or CD47 siRNA 72 hours prior to treatment to knockdown CD47 expression. 50,000 TAMs were co-cultured with 200,000 4T1E-eGFP cells in RPMI serum-free medium for 3 hours at 37°C. Cells were then washed three times with DPBS supplemented with 0.5% BSA and 2 mM EDTA, stained with anti-CD45, CD11b, and F4 / 80, and analyzed by flow cytometry. TAMs with high GFP were considered phagocytic.

[0287] Single-cell RNA sequencing Sample preparation BALB / c mice, approximately 10 per mouse 5 Cells were orthotopically loaded. Three days after tumor challenge, mice were treated every 3 days with either the EpCAM aptamer or the EpCAM-AsiC cocktail targeting UPF2, PARP1, CD47, and MCL1 by sc injection. On day 14, tumors were harvested and Ca 2+ and Mg 2+ The cells were incubated with 100 μg / ml Liberase TL (Roche) diluted in RPMI medium (ThermoFisher) without ATP for 15 min at 37°C, followed by shaking for 10 min at 37°C. The samples were then filtered twice through a 40 μM strainer to remove dead cells and detect CD45. + Cells were enriched with CD45 microbeads at 4°C. More than 95% of the cells were CD45 as verified by flow cytometry. + The enriched cells were incubated in CaCl2+ containing 1% FBS.2+ and Mg 2+ The cells were diluted to 200,000 cells / ml in RPMI medium without ATP, and kept on ice until flowing into the microfluidic device. 6,000 cells per sample were encapsulated using inDrop technology; half of the sample was used for library preparation, and the other half was kept for backup purposes. Two biological replicates per condition were processed independently, and sequencing data from both samples were combined for data analysis. Single-cell encapsulation and RNA capture on the InDrop platform, as well as library preparation, were performed at the Harvard Medical School Single Cell Core as previously published. 131 Single-cell transcriptomes were barcoded within microfluidic droplets. After in-droplet reverse transcription, emulsions of approximately 3,000 cells were broken and used for library preparation. Libraries were indexed with V3 sequencing adapters, pooled from various samples in equimolar ratios, and sequenced on an Illumina NextSeq 500 system using a NextSeq 75 High Output Kit with standard Illumina sequencing primers, 61 cycles for read 1, 14 cycles for read 2, and 8 cycles each for index read 1 and index read 2.

[0288] Data Processing Raw data were processed using a previously published pipeline (github.com / indrops / indrops) in Python using default parameters. 132Briefly, reads were filtered and sorted by their corresponding library index. Valid reads were then demultiplexed and sorted by cell barcode. Cell barcodes containing fewer than 250 total reads were discarded, and the remaining reads were aligned to the reference mouse transcriptome (Ensembl GRCm38 release 87). The aligned reads were then quantified as an imputed count matrix, which was used for all downstream analyses.

[0289] Pre-clustering filtering, normalization, and batch correction Analysis of the processed data was performed in R version 3.5.2 using the Seurat package version 2.3. 133 All samples were merged together. The percentage of mitochondrial transcripts (percent.mito) and the average UMI of each gene (nUMI.nGene.ratio) for each cell were calculated. Low-quality cells were filtered using the following cutoffs: nGene - minimum 50, maximum 2000; percent.mito - minimum - infinity (-Inf), maximum 0.25; nUMI.nGene.ratio - minimum 1, maximum 5. The NormalizeData function was run with default parameters to remove differences in sequencing depth across cells. The ScaleData function was used to eliminate cell-to-cell variations in gene expression driven by batch and mitochondrial gene expression.

[0290] Dimensionality Reduction and Unsupervised Clustering Dimensionality reduction was performed in three stages of analysis: variable gene selection, PCA, and uniform manifold approximation and projection (UMAP). The FindVariableGenes function was applied to select 2274 highly variable genes covering most of the biological information contained in the whole transcriptome. The variable genes were then used for PCA, which was performed with the RunPCA function. Next, the RunUMAP function was run by selecting PCs 1-20 as input to obtain two-dimensional coordinates for each cell. The FindClusters function (resolution 0.4) was run to cluster cells using the Louvain algorithm based on the same PCs as the RunUMAP function.

[0291] DEG identification and GO analysis We used the FindMarkers or FindAllMarkers function (test.use=''t'', logfc.threshold=log(1.6)) based on the normalized data to identify differentially expressed genes (DEGs). P-value adjustment was performed using the Bonferroni correction based on the total number of genes in the dataset. DEGs with adjusted p-values ​​greater than 0.05 were filtered out. Gene Ontology (GO) analysis was performed using the R package clusterProfiler. 134 .

[0292] Bulk RNA sequencing EpCAM hiMDA-MB-231 cells were transfected with 100 nM of either negative control siRNA or ON-TARGETplus human UPF2 siRNA-SMARTpool (both from Dharmacon) for 72 hours. Transfection achieved greater than 80% UPF2 mRNA knockdown. Total RNA was extracted from each sample using TRIzol and Direct-zol RNA miniprep kits. Three biological replicates per condition were used for RNA sequencing library preparation. The RNA integrity number (RIN) of all samples was determined using an Agilent 2100 Bioanalyzer at the Harvard Medical School Biopolymers Facility. All RNA samples had an RIN greater than 9. Standard mRNA libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs) after polyA-mRNA isolation. Libraries for the negative control and UPF2 siRNA-transfected samples were pooled separately, and each pool was run on a single lane Illumina Hiseq X10 PE100 system, generating approximately 240 million mapped 150-bp paired-end reads per sample. Sequences were aligned to the reference genome GRCh38 (Ensembl release 98) using HISAT2. 135 A pipeline incorporating DEXSeq and HTSeq counts was used to identify differential exon usage (DEU) events using the reference GRCh38_98. 136,137 DEU analysis was limited to exons with at least 10 reads in at least three samples. DEU events were significant if they reached a multiple hypothesis adjusted p-value of less than 0.05. StringTie was used to assemble reads into novel annotated transcripts using a guided assembly approach on the GRCh38_98 reference. 138The assemblies per sample were then merged into a unified transcript reference using the merge function in StringTie. Transcript abundance was quantified from the StringTie-generated reference using Kallisto. 139 Differential isoform usage events (DIU) were identified using IsoformSwitchAnalyzeR. 136,140 IsoformSwitchAnalyzer also provided predictions of premature termination codons (PTCs) to read out potential NMD susceptibility. Changes in isoform usage were significant if they reached a q value less than 0.05.

[0293] statistical analysis Differences between two groups were determined using Student's t-test (two-tailed) or Mann-Whitney test. Differences between multiple populations were calculated using one-way or two-way analysis of variance. Differences between tumor growth curves were compared by first calculating the area under the curve value for each sample and then comparing different groups using Student's t-test or one-way analysis of variance. Comparisons of tumor volumes at various time points along tumor growth were determined by multiple t-tests with type I error correction. Type I error was corrected by the Holm-Sidak method. Significance was set at a p-value of 0.05 or less. For all figures, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p≦0.0001. All statistical analyses were performed using GraphPad Prism 8.

[0294] References TIFF2025169317000017.tif146160TIFF2025169317000018.tif238160TIFF2025169317000019.tif233160TIFF2025169317000020.tif238160 TIFF2025169317000021.tif238160TIFF2025169317000022.tif233160TIFF2025169317000023.tif238160TIFF2025169317000024.tif107159

[0295] (Table 5) TIFF2025169317000025.tif141168

[0296] (Table 6) Bold sequences indicate EpCAM aptamers TIFF2025169317000026.tif131168

[0297] (Table 7) TIFF2025169317000027.tif54168TIFF2025169317000028.tif242168

[0298] Sequence information SEQUENCE LISTING <110> THE CHILDREN'S MEDICAL CENTER CORPORATION <120> METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER <150> US 62 / 864,726 <151> 2019-06-21 <160> 202 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 ggucuagaga guugcgaau 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 gcauguaccu uguguagaa 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 cguuauguuu gguggaaga 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 caucagaguc agugcuaua 19 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 ggcuuuuguc ccagccaucu u 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 guggaaauuu aaaggaagau u 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 aaguauacgu aaaguggaau u 21 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 auacaaccuc cuaggaauau u 21 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 9 ugacuuuagu agugcaaaau u 21 <210> 10 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 cuaugagacc cuuacgugau uguua 25 <210> 11 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 gcacaugcau cuucuguaug gacaa 25 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 12 gaaaacaggu auuggauau 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 13 guucuuagcg cacaucuug 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 ccaauaggcu uaauccugu 19 <210> 15 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 15 ccgaguacag ugcgaguca 19 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 acggugaucg guagcaacaa a 21 <210> 17 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 ccgagaaauc ucuuaccuca a 21 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 ggacagagcc agaggccaau u 21 <210> 19 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 19 ggaagaagcc ccagauauau u 21 <210> 20 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 ggauuaagaa gaaaggauuu u 21 <210> 21 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 gagccuggau uaagaagaau u 21 <210> 22 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 caaaguuucu uacggcauau u 21 <210> 23 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 23 gucugguacg acuggagua 19 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 24 ccugccacac tcaagaucu 19 <210> 25 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 gaugggcuuc gagccuggau uaaga 25 <210> 26 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 26 caucaagucc ugagugguau u 21 <210> 27 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Syntheti...

Claims

1. an EpCAM-binding aptamer domain; at least one inhibitory nucleic acid domain that inhibits expression of a gene selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47; A chimeric molecule comprising:

2. The molecule of claim 1, wherein the gene is selected from the group consisting of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47.

3. 2. The molecule of claim 1, wherein the gene is selected from the group consisting of UPF2; PD-L1; MCL1; and CD47.

4. The molecule of any one of the preceding claims, which is an aptamer-siRNA chimera (AsiC).

5. 10. A molecule according to any one of the preceding claims, wherein the inhibitory nucleic acid specifically binds to a gene product of a selected gene.

6. The molecule of any one of the preceding claims, wherein the EpCam-binding aptamer domain comprises any of the sequences of SEQ ID NOs: 63 to 68.

7. 10. The molecule of any one of the preceding claims, wherein the inhibitory nucleic acid domain comprises a sequence selected from SEQ ID NOs: 1-62, 69-126, and 149-162, or a reverse complement thereof.

8. The molecule of any one of the preceding claims, wherein the chimeric molecule comprises a first inhibitory nucleic acid domain and at least one further inhibitory nucleic acid domain.

9. The molecule of claim 8, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain comprise different sequences but each inhibit expression of the same gene.

10. The molecule of claim 8, wherein the first inhibitory nucleic acid domain and at least one additional inhibitory nucleic acid domain each inhibit the expression of a different gene.

11. 11. The molecule of claim 10, wherein at least the second inhibitory nucleic acid domain inhibits expression of a gene selected from the group consisting of PLK1 and MCL1.

12. 10. The molecule of any one of the preceding claims, comprising one of the sequences of SEQ ID NOs: 127-137 or 163-168.

13. 10. The molecule of any one of the preceding claims, which is a single-stranded nucleic acid.

14. 13. The molecule of any one of claims 1 to 12, comprising a double-stranded portion.

15. 15. The molecule of claim 14, wherein the double-stranded portion comprises two separate nucleic acids hybridized to each other, or comprises a single nucleic acid (e.g., a hairpin structure), wherein two portions of the single nucleic acid are hybridized to each other.

16. The molecule of any one of the preceding claims, wherein the 3' end of the chimeric molecule comprises dTdT.

17. The molecule of any one of the preceding claims, wherein the chimeric molecule comprises at least one 2'-F pyrimidine.

18. The molecule of any one of the preceding claims, wherein the chimeric molecule comprises one or more of a 2' sugar modification, a phosphothiorate backbone modification, and a 5' unlocked nucleic acid modification.

19. 10. The molecule of any one of the preceding claims, wherein the chimeric molecule is conjugated or attached to cholesterol, PEG, or a liposome.

20. The molecule of any one of the preceding claims, wherein the chimeric molecule further comprises a chemotherapeutic agent.

21. A pharmaceutical composition, kit, or combination comprising the chimeric molecule of any one of claims 1 to 20, and optionally a pharmaceutically acceptable carrier.

22. 22. The composition, kit, or combination of claim 21, comprising at least two different chimeric molecules of any one of claims 1 to 20, wherein the chimeric molecules have different aptamer domains or inhibitory nucleic acid domains.

23. 22. The composition, kit, or combination of claim 21, wherein the different inhibitory nucleic acid domains recognize different targets.

24. 22. The composition, kit, or combination of claim 21, wherein the different inhibitory nucleic acid domains have different sequences and recognize the same target.

25. The chimeric molecule of first claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47; and The chimeric molecule of second claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; PTPN2; SMG1; TREX1; CMAS; and CD47.

25. The composition, kit, or combination of any one of claims 21 to 24.

26. The chimeric molecule of first claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47; and The chimeric molecule of second claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PARP1; APE1; PD-L1; MCL1; and CD47.

26. The composition, kit, or combination of any one of claims 21 to 25.

27. 27. The composition, kit, or combination of any one of claims 21 to 26, comprising at least six different chimeric molecules of claims 1 to 20, collectively comprising inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PARP1; APE1; PD-L1; MCL1; and CD47.

28. The chimeric molecule of first claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a gene selected from UPF2; PD-L1; MCL1; and CD47; and The chimeric molecule of second claims 1 to 20, comprising an inhibitory nucleic acid domain that inhibits expression of a second and different gene selected from UPF2; PD-L1; MCL1; and CD47.

28. The composition, kit, or combination of any one of claims 21 to 27.

29. 29. The composition, kit, or combination of any one of claims 21 to 28, comprising at least four different chimeric molecules of claims 1 to 20, collectively comprising inhibitory nucleic acid domains that inhibit the expression of each of UPF2; PD-L1; MCL1; and CD47.

30. a. the chimeric molecule of any one of claims 1 to 20; b. a second chimeric molecule, i. comprising the chimeric molecule of any one of claims 1 to 20, wherein the inhibitory nucleic acid domain of the second chimeric molecule inhibits expression of a different gene than the first chimeric molecule; or ii. an EpCAM-binding aptamer domain; and an inhibitory nucleic acid domain that inhibits the expression of a gene selected from the group consisting of PLK1 and MCL1; and a chimeric molecule comprising a second chimeric molecule; and c. optionally, a pharmaceutically acceptable carrier A pharmaceutical composition, kit, or combination comprising:

31. 31. The composition, kit, or combination of any one of claims 21 to 30, further comprising an immune checkpoint inhibitor.

32. 32. The composition, kit, or combination of claim 31, wherein the immune checkpoint protein is PD-1 or PD-L1.

33. 33. The composition, kit, or combination of claim 32, wherein the immune checkpoint protein is PD-1.

34. 34. The composition, kit, or combination of claim 33, wherein the immune checkpoint inhibitor is pembrolizumab; nivolumab; pidilizumab; or AUNP12.

35. 35. A method of treating cancer in a subject in need thereof, comprising administering to the subject the chimeric molecule, composition, kit or combination of any one of claims 1-34.

36. 36. The method of claim 35, wherein the cancer is epithelial cancer, breast cancer, or colon cancer.

37. 37. The method of claim 36, wherein the breast cancer is HER2+ breast cancer or triple-negative breast cancer (TNBC).

38. 37. The method of claim 36, wherein the breast cancer is not BRCA1 deficient.

39. The method of any one of claims 35 to 38, wherein the administration is subcutaneous.

40. 40. The method of any one of claims 35 to 39, wherein the subject is further administered an additional cancer treatment.

41. 41. The method of claim 40, wherein the cancer treatment is paclitaxel.

42. A method of treating cancer in a subject in need thereof, comprising administering to said subject said chimeric molecule.

35. The chimeric molecule, composition or kit of any one of claims 1 to 34 for use in

43. 43. The chimeric molecule, composition, or kit of claim 42, wherein the cancer is epithelial cancer, breast cancer, or colon cancer.

44. 44. The chimeric molecule, composition, or kit of claim 43, wherein the breast cancer is HER2+ breast cancer or triple-negative breast cancer (TNBC).

45. 45. The chimeric molecule, composition, or kit of claim 44, wherein the breast cancer is not BRCA1 deficient.

46. 47. The chimeric molecule, composition, or kit of any one of claims 42 to 46, wherein said administration is subcutaneous.

47. 47. The chimeric molecule, composition, or kit of any one of claims 42 to 46, wherein the subject is further administered an additional cancer treatment.

48. 48. The kit of any one of claims 42-47, further comprising an additional cancer treatment in the same formulation as the chimeric molecule or in a separate formulation.

49. 49. The chimeric molecule, composition, or kit of any one of claims 42 to 48, wherein the cancer treatment is paclitaxel.

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