Therapeutic compounds for erythrocyte-mediated delivery of active pharmaceutical ingredients to target cells

Abstract

To provide a therapeutic compound for erythrocyte-mediated delivery of an active pharmaceutical ingredient to a target cell.SOLUTION: Therapeutic compounds for erythrocyte-mediated delivery of an active pharmaceutical ingredient to a target cell are described. The therapeutic compound is configured to bind to the CD47 on the surfaces of red blood cells and then to be transferred to the CD47 on the surfaces of target cells, such that the therapeutic compound is eventually internalized into the target cells by endocytosis. The target cell may be a cancer cell, a virus-infected cell, or a fibrotic cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 281,370, filed November 19, 2021, and U.S. Provisional Patent Application No. 63 / 392,323, filed July 26, 2022, each of which is incorporated by reference in its entirety.

[0002] Technical Field The present invention relates generally to therapeutic compounds configured to bind to CD47, and more particularly to such compounds configured to bind to CD47 on the surface of red blood cells and then be transferred to CD47 on the surface of target cells, and therefore ultimately be internalized into the target cells by endocytosis. [Background technology]

[0003] Background technology The integrin-associated protein, cluster of differentiation 47 ("CD47"), is a multispanning plasma membrane protein involved in the process of phagocytic clearance or inhibiting neutrophil migration. Signal-regulatory protein alpha ("SIRPα"), a transmembrane protein expressed by innate immune cells such as macrophages and dendritic cells, is the primary receptor for CD47. Binding of SIRPα to CD47 induces a SIRPα inhibitory signal, which acts as a "don't eat me" signal to recipient macrophages, thereby preventing their phagocytic activation. Thus, the SIRPα-CD47 interaction functions as a negative checkpoint for innate and subsequent adaptive immunity. Other proteins, such as signal-regulatory protein gamma ("SIRPγ") and thrombospondin-1 ("TSP-1"), can also bind to CD47, thereby inhibiting aspects of the immune response.

[0004] Mammalian cells typically express low levels of CD47 to protect them from phagocytosis. However, cancer cells overexpress CD47 as an escape mechanism to escape immune surveillance and attack by phagocytes. Some human solid tumors overexpress CD47. That is, the cells of these solid tumors generally express more CD47 than normal cells (Willingham et al. PNAS 109(17):6662-6667 (2012), this reference is hereby incorporated by reference in its entirety). Therefore, CD47 has emerged as a promising new therapeutic target for cancer immunotherapy (Willingham et al. PNAS 109(17):6662-6667 (2012); Weiskopf, Eur. J. Cancer 76:100-109 (2017); Weiskopf et al. J Clin Invest 126(7):2610-2620 (2016), each of these references , which is hereby incorporated by reference in its entirety.

[0005] Additionally, virus-infected cells also express high levels of CD47, including cells infected with SARS-CoV-2, the virus that causes COVID-19 (Cham et al. Cell Rep 14;31(2):107494 (2020) doi:10.1016 / j.celrep.2020.03.058 and McLaughlin et al. bioRxiv 2021.03.01.433404 (2021) doi:10.1101 / 2021.03.01.433404, each of which references is hereby incorporated by reference in its entirety). (Incorporated herein by reference to the text.) Blockade of CD47 inhibitory signaling has been demonstrated to enhance innate and adaptive immune responses to viral infections.

[0006] Furthermore, increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by increasing phagocytosis of profibrotic fibroblasts and by abolishing their suppressive effects on adaptive immunity (Cui et al. Nat Commun 11:2795 (2020); Wernig et al. PNAS 2017;114(18):4757-62; Boyd J Cyst Fibros Suppl 1:S54-S59 (2020); Lerbs et al. JCI Insight 2020;5(16):e140458 (2020), each of which references is hereby incorporated by reference in its entirety). Therefore, CD47 is a promising target for the treatment of cancer, viral infections, and fibrotic diseases such as cystic fibrosis. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Willingham et al. PNAS 109(17):6662-6667 (2012) [Non-patent document 2] Weiskopf, Eur. J. Cancer 76:100-109 (2017) [Non-patent document 3] Weiskopf et al. J Clin Invest 126(7):2610-2620 (2016) [Non-patent document 4] Cham et al. Cell Rep 14;31(2):107494 (2020) doi:10.1016 / j.celrep.2020.03.058 [Non-patent document 5] McLaughlin et al. bioRxiv 2021.03.01.433404 (2021) doi:10.1101 / 2021.03.01.433404 [Non-patent document 6] Cui et al. Nat Commun 11:2795 (2020) [Non-Patent Document 7] Wernig et al. PNAS 2017;114(18):4757-62 [Non-patent document 8] Boyd J Cyst Fibros Suppl 1:S54-S59 (2020) [Non-Patent Document 9] Lerbs et al. JCI Insight 2020;5(16):e140458 (2020) Summary of the Invention [Means for solving the problem]

[0008] Overview of the embodiment According to one embodiment of the present invention, there is provided a therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, the therapeutic compound comprising a CD47 binding protein conjugated to an active pharmaceutical ingredient ("API") to form a conjugate; the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), homologs of any of the foregoing, and combinations thereof, and the therapeutic compound is delivered by the circulatory system of the subject. The conjugate is configured to bind to CD47 on the subject's red blood cells to enable transport of the conjugate to a target cell where the conjugate is located, such that (i) the CD47 binding protein configured to bind the conjugate to CD47 on the red blood cells binds to CD47 on the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell, and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. The mammalian subject may be a human.

[0009] According to another embodiment of the present invention, there is provided a therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), homologs of any of the foregoing, and combinations thereof, enabling transport of the conjugate to the target cells by the circulatory system of the subject. wherein the conjugate is configured to bind to CD47 on the subject's red blood cells to form a conjugate-CD47 binding protein, such that (i) the CD47 binding protein configured to bind the conjugate to CD47 on the red blood cells binds to CD47 on the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell, and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. The mammalian subject may be a human.

[0010] According to an embodiment of the present invention, there is provided a therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is an anti-CD47 antibody, the anti-CD47 antibody comprising either (a) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:932, SEQ ID NO:933, and SEQ ID NO:934, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:935, SEQ ID NO:936, and SEQ ID NO:937, respectively; or (b) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:940, SEQ ID NO:941, and SEQ ID NO:942, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945, SEQ ID NO:946, SEQ ID NO:947, SEQ ID NO:948, SEQ ID NO:949, SEQ ID NO:950, SEQ ID NO:951, SEQ ID NO:952, SEQ ID NO:953, SEQ ID NO:954, SEQ ID NO:955, SEQ ID NO:956, SEQ ID NO:957, SEQ ID NO:958, SEQ ID NO:959, SEQ ID NO:960, SEQ ID NO:961, SEQ ID NO:962, SEQ ID NO:963, SEQ ID NO:964, SEQ ID NO:965, SEQ ID NO:966, SEQ ID NO:967, SEQ ID NO:968, SEQ ID NO:9 (c) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:943, SEQ ID NO:944 and SEQ ID NO:945, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:948, SEQ ID NO:949 and SEQ ID NO:950, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:951, SEQ ID NO:952 and SEQ ID NO:953, respectively; or (d) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:956, SEQ ID NO:957 and SEQ ID NO:958, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:959, SEQ ID NO:960 and SEQ ID NO:961, respectively;The conjugate is configured to bind to CD47 on the subject's red blood cells to enable transport of the conjugate to the target cell via the subject's circulatory system, whereby (i) the CD47 binding protein configured to bind the conjugate to CD47 on the red blood cells binds to CD47 on the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. The mammalian subject may be a human.

[0011] The CD47 binding protein can be conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and a combination thereof. The CD47 binding protein can be conjugated to the API by a linker, and the linker can be cleavable. The linker can be configured to be cleaved by a lysosomal degrading enzyme.

[0012] In some embodiments, the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins, and small molecules. The RNA may be selected from the group consisting of siRNA, shRNA, miRNA, antimiR, and mRNA.

[0013] The target cell may be a cell selected from the group consisting of a cancer cell, a virally infected cell, a fibrotic cell, and a combination thereof. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a virally infected cell. In some embodiments, the target cell is a fibrotic cell.

[0014] In some embodiments, the cancer cells are in a tumor caused by a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer; blood cancer, including acute / chronic leukemia, malignant lymphoma, and multiple myeloma; bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

[0015] In some embodiments, the cancer cells are selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine endometrioid carcinoma, prostate cancer, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal carcinoma, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, invasive breast cancer, gastric adenocarcinoma, bladder urothelial carcinoma, bile duct carcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, cutaneous melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, Gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, acute leukemia of ill-defined lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor (NOS), neuroblastoma, papillary cell carcinoma of the kidney, hepatocellular carcinoma, chromophobe renal cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell / sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma / rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms' tumor, myofibromytosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma (NOS), embryonal rhabdomyosarcoma The tumor is caused by a cancer selected from the group consisting of uveal melanoma, Ewing's sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-dependent soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

[0016] In some embodiments, the virally infected cells are infected with the SARS-CoV-2 virus. In some embodiments, the fibrotic cells are associated with cystic fibrosis.

[0017] In some embodiments, the API is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-747 and 771-824; (b) the sense RNA strand is 19-29 nucleotides in length and complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-747 and 771-824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 38-39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43. In another embodiment, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 44-51.

[0018] In some embodiments, the API is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765, (b) the sense RNA strand is 19-29 nucleotides in length and complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 482-486 and 748-765.

[0019] In some embodiments, the API is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770, (b) the sense RNA strand is 19-29 nucleotides in length and complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43 and 766-770.

[0020] The API is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. and a fourth region having a fourth sequence, wherein (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complement of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-747 and 771-824, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-747 and 771-824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 38-39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43. In another embodiment, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 44-51.

[0021] In some embodiments, the API is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length and which comprises, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. and a fourth region having a fourth sequence, wherein (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complement of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang. In another embodiment, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 482-486 and 748-765.

[0022] In some embodiments, the API is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length and comprising, in 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. and a fourth region having a fourth sequence, wherein (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complement of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang. In another embodiment, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43 and 766-770.

[0023] In some embodiments, the API is an miRNA selected from the group consisting of SEQ ID NOs: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.

[0024] In some embodiments, the API is an antimiR, wherein the antimiR is a single-stranded nucleic acid molecule 12-25 nucleotides in length, wherein the antimiR has a sequence of 12-25 contiguous nucleotides that are complementary to contiguous nucleotides in a target mature miRNA product sequence, wherein the mature miRNA product sequence is selected from the group consisting of SEQ ID NOs: 884-908, and wherein the contiguous nucleotides in the mature miRNA product sequence include, in the 5' to 3' direction, nucleotides 2-8 of the mature miRNA product sequence.

[0025] In some embodiments, the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents, such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA damaging agents, such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand breakers (e.g., calicheamicin), cross-linking agents (e.g., pyrrolobenzodiazepine dimers-PBD), and alkylating agents (e.g., duocarmycin and indolinobenzodiazepine dimers-IGN).

[0026] In some embodiments, the API is a protein, and the protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof. In other embodiments, the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof.

[0027] In some embodiments, the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, wherein the mRNA is configured to be translated in a target cell to produce a protein comprising the amino acid sequence. In other embodiments, the mRNA is configured to be translated in a target cell to produce a protein consisting of the amino acid sequence. In some embodiments, the mRNA is codon-optimized.

[0028] According to one embodiment of the present invention, there is provided a method of treating cancer in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

[0029] According to another embodiment of the present invention, there is provided a method of treating a viral infection in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

[0030] According to an embodiment of the present invention, there is provided a method of treating a fibrotic disease in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

[0031] According to another embodiment of the present invention, a pharmaceutical composition comprising a therapeutic compound described herein.

[0032] The above-described features of the embodiments will be more readily understood by reference to the following detailed description, taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1A shows a FAM-tagged vSIRPα-siRNA conjugate according to an embodiment of the present invention. Figure 1B is an illustration of incubation of red blood cells with the FAM-tagged vSIRPα-siRNA conjugate of Figure 1A. Figure 1C shows fluorescence microscopy images of red blood cells that were not incubated with the FAM-tagged vSIRPα-siRNA conjugate shown in Figure 1A (left) and that were incubated with the FAM-tagged vSIRPα-siRNA conjugate shown in Figure 1A (right).

[0034] [Figure 2] Figure 2A shows flow cytometry results of CaCO2 cells before and after incubation with red blood cells bound with FAM-tagged vSIRPα-siRNA, and Figure 2B shows flow cytometry results of CT26.CL25 cells before and after incubation with red blood cells bound with FAM-tagged vSIRPα-siRNA, according to an embodiment of the present invention.

[0035] [Figure 3]Figure 3A shows flow cytometry results of CaCO2 cells before and after incubation with red blood cells bound with Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody, and Figure 3B shows flow cytometry results of CT26.CL25 cells before and after incubation with red blood cells bound with Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody, according to an embodiment of the present invention.

[0036] [Figure 4] FIG. 4 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with red blood cells bound with FAM-tagged vSIRPα-siRNA, according to an embodiment of the present invention.

[0037] [Figure 5] FIG. 5 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with red blood cells bound with Cy5.5-labeled vSIRPα, according to an embodiment of the present invention.

[0038] [Figure 6] FIG. 6 shows flow cytometry results of CT26.CL25 cells before ("unstained") and after incubation with red blood cells conjugated with Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody, according to an embodiment of the present invention.

[0039] [Figure 7] FIG. 7 shows flow cytometry results of CT26.CL25 cells before ("unstained") and after incubation with red blood cells bound with a CD47mAb-miR21-Cy5 conjugate, according to an embodiment of the present invention.

[0040] [Figure 8]FIG. 8 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with red blood cells bound to Cy5.5-labeled mouse thrombospondin-1, according to an embodiment of the present invention.

[0041] [Figure 9] FIG. 9 shows red blood cells isolated from untreated mice, mice injected with fluorescently labeled siRNA conjugates, and mice injected with fluorescently labeled vSIRPα-siRNA conjugates, according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description of Specific Embodiments As used in this specification and the appended claims, the following terms shall have the meanings indicated unless the context requires otherwise.

[0043] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to cover both the singular and the plural unless otherwise indicated herein or where the context clearly contradicts. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth herein. No language herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0044] A "set" contains at least one member.

[0045] The term "mammal" and the like refers to any animal species of the class Mammalia. Examples of mammals include humans; laboratory animals such as rats, mice, monkeys, and guinea pigs; domestic animals such as rabbits, cows, sheep, goats, cats, dogs, horses, pigs, etc.

[0046] "Active pharmaceutical ingredient," "API," and the like refer to the non-CD47 binding protein and non-linker portions of a therapeutic compound, according to embodiments of the present invention, that are biologically active. Suitable active pharmaceutical ingredients ("APIs") include RNA (siRNA, miRNA, shRNA, and mRNA), DNA, antimiR oligonucleotides (RNA, DNA, and derivatives thereof), RNA and DNA derivatives (including, but not limited to, modified RNA and DNA containing modified backbones, sugars, and / or bases), proteins, and small molecules.

[0047] As used herein, a "homolog" of such a given protein shall mean a protein having at least 95% sequence identity with the given protein.

[0048] "Complementarity," as used herein in reference to a nucleic acid sequence, refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through Watson-Crick base pairing or wobble base pairing. Percent complementarity refers to the percentage of nucleotides in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with the nucleotides of a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary" and the like means that all of the contiguous nucleotides of a nucleic acid sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleic acid sequence (i.e., the nucleic acid sequence has 100% complementarity). "Complementary," as used herein without further proviso, means that contiguous nucleotides of a nucleic acid sequence have a percent complementarity selected from the group consisting of 95%, 96%, 97%, 98%, 99%, and 100% complementarity with contiguous nucleotides of a second nucleic acid sequence over a region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more nucleotides of the nucleic acid sequence. For example, a nucleic acid sequence that is 19 nucleotides in length and complementary to 14 nucleotides of a second nucleic acid sequence means that the contiguous 14 nucleotides of the nucleic acid sequence have a percent complementarity selected from the group consisting of 95%, 96%, 97%, 98%, 99%, and 100% complementarity with the contiguous nucleotides of the second nucleic acid sequence. A nucleic acid sequence that is 19 nucleotides in length and that is complementary to contiguous nucleotides in a second nucleic acid sequence means that the 19 contiguous nucleotides of the nucleic acid sequence have a percent complementarity with the contiguous nucleotides of the second nucleic acid sequence selected from the group consisting of 95%, 96%, 97%, 98%, 99% and 100% complementarity.

[0049] "Codon-optimized" means that the coding sequence of an mRNA transcript contains the most or second most preferred codons for a given target / host cell species for at least 60% of the codons in the coding sequence; therefore, a codon-optimized sequence is translated more efficiently in the target / host cell than a non-optimized sequence.

[0050] The term "antibody" refers to an immunoglobulin molecule typically composed of two identical pairs of polypeptide chains, each pair having one "heavy" (H) chain and one "light" (L) chain. Human light chains are classified as kappa (κ) and lambda (λ). Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region of IgD, IgG, and IgA is composed of three domains, CH1, CH2, and CH3, while the heavy chain constant region of IgM and IgE is composed of four domains, CH1, CH2, CH3, and CH4. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH / VL) typically form the antigen-binding site of the antibody. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes monoclonal antibodies, recombinant antibodies, and polyclonal antibodies.

[0051] The term "human antibody" refers to an antibody consisting of amino acid sequences exclusively of human immunoglobulin sequences. Human antibodies may contain mouse carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Human antibodies can be prepared by a variety of methods known in the art.

[0052] The term "humanized antibody" refers to an antibody whose framework and constant regions contain amino acid residues derived from human antibody sequences, while containing some or all of the CDRs from a non-human animal antibody. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody onto human framework sequences, followed by back-substitution of certain human framework residues for the corresponding mouse residues from the original antibody. The term "humanized antibody" also refers to antibodies of non-human origin from which one or more epitopes, typically in one or more variable regions, that are likely to constitute human T-cell and / or B-cell epitopes have been removed to reduce immunogenicity. The epitope amino acid sequence can be completely or partially removed. However, typically, the amino acid sequence is altered by substituting one or more of the amino acids that constitute the epitope for one or more other amino acids, thereby changing the amino acid sequence to one that does not constitute a human T-cell and / or B-cell epitope. The amino acids are optionally substituted with amino acids present at the corresponding positions in the corresponding human variable heavy or light chain.

[0053] The term "pharmaceutically acceptable carrier" refers to a solvent, carrier agent, diluent, etc. commonly used in administering pharmaceutical compounds.

[0054] CD47 is overexpressed on various cancer cells, virus-infected cells, and fibrotic cells, making it a promising target for the treatment of various cancers, viral infections, and fibrotic diseases. Blocking CD47 signaling and even CD47 expression on these cells can suppress immune system evasion of these cells, allowing for their elimination and subsequent recovery from the disease in patients.

[0055] For example, intravenous injection of a variant SIRPα ("vSIRPα") conjugated to a probe with high affinity for CD47 has been shown to be taken up by tumorigenic cells in mice. Additionally, vSIRPα conjugated to siRNA targeting CD47 has been shown to enhance phagocytosis of CT26.CL25 cancer cells in culture (Ko et al. Control Release 323:376-386 (2020) and U.S. Patent Application Publication No. 2021 / 0015931, each of which is hereby incorporated by reference in its entirety).

[0056] The present inventors have surprisingly discovered that CD47, which is present on the cell surface of red blood cells ("RBCs") and on the cell surface of target cells such as cancer cells, virus-infected cells, and fibrotic cells, can be utilized to deliver various APIs to said target cells by a novel mechanism.

[0057] Here, the inventors describe novel therapeutic compounds for RBC-mediated delivery of an API to CD47-expressing target cells in a mammalian subject. The therapeutic compound is a conjugate comprising a CD47-binding protein conjugated to an API. The CD47-binding protein of the conjugate binds the conjugate to CD47 present on the surface of the subject's red blood cells, thereby enabling transport of the conjugate to the target cell via the subject's circulatory system. The conjugate is transferred from the RBC to CD47 present on the surface of the target cell. Binding of the conjugate to CD47 on the target cell reduces the target cell's ability to evade attack by the subject's immune system. Furthermore, binding of the conjugate to CD47 on the target cell causes the conjugate-CD47 complex to be internalized by endocytosis, thereby further reducing the target cell's ability to evade attack by the subject's immune system and delivering the API to the target cell. Surprisingly, even though the conjugate binds to CD47 on the surface of the RBC, the conjugate-CD47 is not internalized by the RBC. In some embodiments, the mammalian subject is a human.

[0058] The CD47 binding protein can be conjugated to the API via a linker. The linker connects the CD47 binding protein to the API. The linker can be a cleavable linker that is cleaved upon internalization of the conjugate by the target cell, thereby releasing the API from the CD47 binding protein.

[0059] In some embodiments, the target cells are cancer cells and the therapeutic compounds can be used to treat cancer in a mammalian subject.

[0060] The cancer cells can be in a tumor caused by a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer; blood cancer, including acute / chronic leukemia, malignant lymphoma, and multiple myeloma; bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

[0061] Cancer cells include ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine endometrioid carcinoma, prostate cancer, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, invasive breast cancer, gastric adenocarcinoma, and urinary tract cancer. Epithelial carcinoma, cholangiocarcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, cutaneous melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, idiopathic leukemia The tumor may be caused by a cancer selected from the group consisting of acute leukemia, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neuroblastoma, papillary cell carcinoma of the kidney, hepatocellular carcinoma, chromophobe renal cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell / sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma / rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms' tumor, myofibromatosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing's sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-dependent soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. See Gupta et al. Cancer Drug Resist 3:550-62 (2020), which is hereby incorporated by reference in its entirety.

[0062] In other embodiments, the target cells are virally infected cells and the therapeutic compounds can be used to treat viral infections in mammalian subjects. The virally infected cells can be infected with the SARS-CoV-2 virus.

[0063] In some embodiments, the target cells are fibrotic cells and the therapeutic compounds can be used to treat a fibrotic disease in a mammalian subject. The fibrotic cells can be fibrosing fibroblasts. In some embodiments, the fibrotic disease is cystic fibrosis.

[0064] CD47-binding protein

[0065] Suitable CD47 binding proteins for the conjugates described herein include wild-type ("wt") SIRPα (SEQ ID NO: 1), variant SIRPα ("vSIRPα") (SEQ ID NO: 3), wt TSP-1 (SEQ ID NO: 7), wt SIRPγ (SEQ ID NO: 4), variant SIRPγ-1 ("vSIRPγ-1") (SEQ ID NO: 5), variant SIRPγ-2 ("vSIRPγ-2") (SEQ ID NO: 6), and homologs of any of the foregoing. ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and their homologs are also suitable CD47 binding proteins for the conjugates described herein. ALX148 is a SIRPα D1 variant fused to an Fc domain monomer. See, e.g., U.S. Patent No. 10,696,730, which is hereby incorporated by reference in its entirety. TTI-661 is the IgV domain of human SIRPα variant 2 fused to the constant region of a human IgG1 antibody, and TTI-662 is the IgV domain of human SIRPα variant 2 fused to the constant region of a human IgG4 antibody. See, e.g., U.S. Patent No. 9,969,789, which is hereby incorporated by reference in its entirety.

[0066] Other suitable CD47 binding proteins include anti-CD47 antibodies.

[0067] In some embodiments, the anti-CD47 binding protein is an anti-CD47 antibody, such as B6H12, 5F9, 8B6, C3, and Hu5F9-G4, as described in U.S. Pat. Nos. 9,017,675 and 9,623,079, each of which is hereby incorporated by reference in its entirety.

[0068] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 930 and a light chain variable region comprising SEQ ID NO: 931. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 938 and a light chain variable region comprising SEQ ID NO: 939. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 946 and a light chain variable region comprising SEQ ID NO: 947. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 954 and a light chain variable region comprising SEQ ID NO: 955.

[0069] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively.

[0070] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944 and SEQ ID NO: 945, respectively.

[0071] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:948, SEQ ID NO:949 and SEQ ID NO:950, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:951, SEQ ID NO:952 and SEQ ID NO:953, respectively.

[0072] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957 and SEQ ID NO: 958, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960 and SEQ ID NO: 961, respectively.

[0073] In some embodiments, the anti-CD47 antibody is a human antibody. In some embodiments, the anti-CD47 antibody is a humanized antibody.

[0074] Each of these anti-CD47 binding proteins binds to CD47 present on the surface of target cells.

[0075] In some embodiments, the CD47 binding protein is conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and a combination thereof. Examples of linkers that covalently bond the CD47 binding protein to the API are described below.

[0076] Linker

[0077] Suitable linkers include, but are not limited to, cleavable linkers, such as hydrazone linkers, imine linkers, oxime linkers, carbonate linkers, acetal linkers, orthoester linkers, silyl ether linkers, disulfide linkers, trioxolane linkers, beta-glucuronide linkers, beta-galactoside linkers, pyrophosphate linkers, phosphoramidate linkers, aryl sulfate linkers, heptamethine cyanine linkers, nitrobenzyl linkers, arylboronic acid linkers, boronate linkers, thioether linkers, maleimidocaproyl-containing linkers, enzymatically cleavable peptide linkers, and para-aminobenzyl carbamate-containing linkers, as well as non-cleavable linkers, such as polyethylene glycol.

[0078] The CD47 binding protein-API conjugates described herein can be prepared by linking the CD47 binding protein to the API via a linker using coupling reactions such as bis(vinylsulfonyl)piperazine-disulfide coupling, N-methyl-N-phenylvinylsulfonamide-cysteine ​​coupling, platinum(II) compound-histidine coupling, and tetrazine-trans-cyclooctene coupling. Suitable linkers and coupling reactions are known to those skilled in the art. See, for example, Su et al. Acta Pharmaceutica Sinica B (2021), ISSN 2211-3835; Pan et al. Med Res Rev. 40:2682-2713 (2020); Khongorzulet al. Mol Cancer Res 18:3-19 (2020); Bargh et al. Chem Soc Rev 48:4361-4374 (2019); and Smith et al. Pharm Res 32:3526-3540 (2015). Each of these references is hereby incorporated by reference herein in its entirety.

[0079] According to embodiments of the present invention, various enzymatically cleavable peptide linkers, such as those described below, can be used to couple a CD47 binding protein to an API to form a CD47 binding protein-API conjugate. These linkers contain amino acid residues and are cleaved by specific enzymes within cells, such as lysosomal degradative enzymes. See, e.g., Kong et al. J Biol Chem 290:7160-7168 (2015); Poreba FEBS J 287:1936-1969 (2020); and Singh et al., Current Medicinal Chemistry 15(18) (2008), each of which is hereby incorporated by reference in its entirety. Exemplary peptide linkers suitable for use with embodiments of the present invention are described below, which are incorporated herein by reference.

[0080] For example, a dipeptide linker is composed of two amino acid residues that serve as a recognition motif for cleavage by the enzyme cathepsin B, which cleaves the amide bond between a carbonyl and an amine after the second amino acid residue. Dipeptide linkers cleaved by cathepsin B include Phe-Arg, Phe-Cit, Phe-Lys, Ala-Arg, Ala-Cit, Val-Ala, Val-Arg, Val-Lys, Val-Cit, and Arg-Arg. Cathepsin B similarly recognizes and cleaves the tetrapeptide linkers Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu after the fourth amino acid residue.

[0081] Additionally, the tripeptide linker Ala-Ala-Asn is cleaved after the last amino acid residue by the enzyme legumain, and the tetrapeptide linkers Lys-Ala-Gly-Gly, Leu-Arg-Gly-Gly, and Arg-Lys-Arg-Arg are cleaved by papain-like protease enzymes.

[0082] The peptide linkers Arg-Arg-X, Ala-Leu-X, Gly-Leu-Phe-Gly-X, Gly-Phe-Leu-Gly-X, and Ala-Leu-Ala-Leu-X, where X is any amino acid, are cleaved by the enzymes cathepsins B, H, and L. Cathepsins B, H, and L are involved in the lysosomal degradation of proteins.

[0083] The peptide linkers Phe-Ala-Ala-Phe(NO2)-Phe-Val-Leu-OM4P-X and Bz-Arg-Gly-Phe-Phe-Pro-4mβNA, where X is any amino acid, are cleaved by the enzyme cathepsin D.

[0084] Serum plasminogen activator is produced in many tumor cells. Plasminogen is converted to plasmin, resulting in high levels of plasmin in tumor cells. This plasmin is rapidly degraded in plasma, so tissues far from the tumor are not exposed to plasmin. Plasmin is involved in fibrinolysis and plasma protein degradation, cleaving the peptide linkers D-Val-Leu-Lys-X, D-Ala-Phe-Lys-X, and D-Ala-Trp-Lys-X (where X is any amino acid).

[0085] Tissue plasminogen activator (tPA) and urokinase (uPA) are involved in activating plasmin formation and each can cleave the peptide linker Gly-Gly-Gly-Arg-Arg-Arg-Val-X, where X is any amino acid.

[0086] Prostate specific antigen is involved in the liquefaction of semen and cleaves the peptide linker morpholinocarbonyl-His-Ser-Ser-Lys-Leu-Gln-Leu-X, where X is any amino acid.

[0087] Matrix metalloproteinases (MMP-2 and MM-9) are involved in the degradation of extracellular matrix and collagen and cleave the peptide linkers Ac-Pro-Leu-Gln-Leu-X and Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X, where X is any amino acid.

[0088] API

[0089] According to some embodiments, API can be small molecule.For example, the small molecule API useful in cancer treatment includes but is not limited to methotrexate; doxorubicin; vinca alkaloid; camptothecin analogue; microtubule disrupting agent, for example, auristatin (for example, MMAE and MMAF) and maytansinoid (for example, DM1 and DM4); and DNA damaging agent, for example, DNA topoisomerase I inhibitor (for example, SN-38 and exatecan), double-strand breaker (for example, calicheamicin), crosslinking agent (for example, pyrrolobenzodiazepine dimer-PBD), and alkylating agent (for example, duocarmycin and indolinobenzodiazepine dimer-IGN). See, e.g., Khongorzulet al. Mol Cancer Res 18:3-19 (2020); Salomon et al. Mol Pharm 16(12):4817-4825 (2019); and Drago et al. Nat Rev Clin Oncol 18, 327-344 (2021), each of which references is hereby incorporated by reference. The entirety of which is incorporated herein by reference.

[0090] In another embodiment, API can be small interfering RNA (" siRNA "). siRNA is a double-stranded RNA molecule that can reduce the expression of a specific gene by causing the degradation of the mRNA transcript of that gene that shares partial complementarity with the strand of the double-stranded siRNA molecule. The process of using siRNA to reduce gene expression is called RNA interference (" RNAi "). See, for example, U.S. Patent No. 7,056,704, U.S. Patent No. 7,078,196, U.S. Patent No. 8,372,968, each of which is hereby incorporated by reference in its entirety into this specification.

[0091] Several genes are involved in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes and their mRNA transcripts are listed in Table 1. Reducing the expression of one or more genes from Table 1 can inhibit cancer progression and cancer cell proliferation. Therefore, the transcripts of the genes listed in Table 1 represent essential targets for cancer treatment using siRNA-mediated RNAi.

[0092] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 8-747 and 771-824, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0093] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-747 and 771-824; (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0094] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 22-37, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0095] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 38-39; (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0096] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0097] In some embodiments, the API useful for treating cancer in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 44-51; (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0098] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] [Table 1-20]

[0099] Using siRNA-mediated RNAi to reduce gene expression is also useful in the treatment of viral infection.For example, reducing the expression of the gene set that allows infected cells to evade host immune system or the gene set that is necessary for viral replication prevents the virus from growing in host.Such genes and their mRNA transcripts are listed in Table 2.Therefore, the transcripts of the genes listed in Table 2 are the essential targets for the treatment of viral infection using siRNA-mediated RNAi.

[0100] Thus, in some embodiments, an API useful for treating a viral infection in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0101] In some embodiments, the API useful for treating a viral infection in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the cDNA sequence of which is selected from the group consisting of SEQ ID NOs: 482-486 and 748-765, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0102] [Table 2]

[0103] Increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by increasing the phagocytosis of profibrotic fibroblasts and eliminating its suppressive effect on adaptive immunity. In addition to CD47, the expression of other genes listed in Table 3 has been associated with the promotion of fibrosis. Reducing the expression of these genes using siRNA-mediated RNAi is also useful in the treatment of fibrotic diseases. Such genes and their mRNA transcripts are listed in Table 3. Therefore, the transcripts of the genes listed in Table 3 represent essential targets for the treatment of fibrotic diseases using siRNA-mediated RNAi.

[0104] In some embodiments, the API useful for treating a fibrotic disease in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770; (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand; and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0105] In some embodiments, the API useful for treating a fibrotic disease in a mammal is an siRNA, wherein the siRNA is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40-43 and 766-770, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region 14-29 nucleotides in length and a 3' overhang region 0-5 nucleotides in length.

[0106] [Table 3]

[0107] In some embodiments, the API can be a short hairpin RNA ("shRNA"). A short hairpin RNA is a single-stranded RNA molecule that forms a stem-loop structure and can reduce the expression of a specific gene by causing the degradation of the mRNA transcript of that gene that shares partial complementarity with the region of the shRNA molecule. Similar to siRNA, the process of using shRNA to reduce gene expression is called RNAi. Generally, see Rao et al. Adv Drug Deliv Rev 61(9):746-59 (2009), which is hereby incorporated by reference in its entirety.

[0108] Several genes are involved in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes and their mRNA transcripts are listed in Table 1. Reducing the expression of one or more genes from Table 1 can inhibit cancer progression and cancer cell proliferation. Therefore, the transcripts of the genes listed in Table 1 represent essential targets for cancer treatment using shRNA-mediated RNAi.

[0109] Thus, in some embodiments, an API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-747 and 771-824; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0110] In some embodiments, the API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule of 44-71 nucleotides in length and comprises, in 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a 2-nucleotide sequence at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22-747 and 771-824; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

[0111] In some embodiments, the API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22-37; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0112] In some embodiments, the API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 38-39; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0113] In some embodiments, the API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40-43; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0114] In some embodiments, the API useful for treating cancer in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 44-51; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0115] Using shRNA-mediated RNAi to reduce gene expression is also useful in the treatment of viral infection.For example, reducing the expression of the gene set that allows infected cells to evade the host's immune system or the gene set that is necessary for viral replication prevents the virus from multiplying in the host.Such genes and their mRNA transcripts are listed in Table 2.Therefore, the transcripts of the genes listed in Table 2 are also the essential targets for the treatment of viral infection using shRNA-mediated RNAi.

[0116] Thus, in some embodiments, an API useful for treating a viral infection in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

[0117] In some embodiments, an API useful for treating a viral infection in a mammal is an shRNA, wherein the shRNA is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 482-486 and 748-765; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0118] As mentioned above, increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by increasing the phagocytosis of profibrotic fibroblasts and eliminating its suppressive effect on adaptive immunity. In addition to CD47, the expression of other genes listed in Table 3 has been associated with the promotion of fibrosis. Reducing the expression of these genes using shRNA-mediated RNAi is also useful in the treatment of fibrotic diseases. Such genes and their mRNA transcripts are listed in Table 3. Therefore, the transcripts of the genes listed in Table 3 also represent essential targets for the treatment of fibrotic diseases using shRNA-mediated RNAi.

[0119] Thus, in some embodiments, an API useful for treating a fibrotic disease in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region of 19-29 nucleotides at the 3' end of the single-stranded RNA molecule, immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

[0120] In some embodiments, the API useful for treating a fibrotic disease in a mammal is an shRNA, which is a single-stranded RNA molecule 44-71 nucleotides in length, comprising, in a 5' to 3' direction: a first region of 19-29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a second region at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region. (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40-43 and 766-770; and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, with the first region base-pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3' overhang.

[0121] MicroRNA ("miRNA")-based therapeutics include miRNAs (and miRNA mimics), and inhibitors of miRNAs ("antimiRs").

[0122] miRNAs are transcribed as single-stranded RNA precursors with stem-loop structures, and then processed by the Dicer enzyme in the cytosol to produce mature double-stranded products ("mature miRNA products"). These mature miRNA products are thought to have regulatory roles, including RNA silencing and post-transcriptional regulation of gene expression, depending on their complementarity with mRNA.

[0123] The expression of the miRNAs shown in Table 4 is known to be downregulated in various cancers, making supplementation of downregulated miRNAs a promising therapeutic approach for the treatment of cancer.

[0124] Thus, in some embodiments, an API useful for treating cancer in a mammal is a miRNA selected from the group consisting of SEQ ID NOs: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.

[0125] [Table 4-1] [Table 4-2]

[0126] In contrast to miRNAs, antimiRs (also known as "antagomirs") are single-stranded antisense oligonucleotides ("ASOs") with sequences complementary to those of regions of target mature miRNA products. Mature miRNA products are short, single-stranded RNA molecules produced after miRNA molecules are processed in the cytosol. Typically, two mature miRNA products are produced from miRNA molecules: the 5p RNA molecule (so named because it is processed from the 5' arm of the duplex formed as the stem of the miRNA) and the 3p RNA molecule (so named because it is processed from the 3' arm of the duplex formed as the stem of the miRNA). The 5p and 3p molecules can base-pair with each other to form a duplex, and each molecule can be functional—indeed, can perform separate functions—in the cell due to their complementarity with mRNA. In some cases, miRNA molecules are processed such that only a single functional mature miRNA product is produced.

[0127] By binding to their target mature miRNA product through complementary base pairing, antimiR can block the binding of mature miRNA product to its target, thereby inhibiting the function of mature miRNA product.Using antimiR to inhibit mature miRNA product is called miRNA knockdown.In general, see Quemener et al.Wiley Interdiscip Rev RNA (5):e1594 (2020), and this reference is hereby incorporated by reference in its entirety.

[0128] AntimiRs are 12-25 nucleotides in length and are complementary to consecutive nucleotides of the target mature miRNA product. Different types of nucleic acids can be used to generate antimiRs. Preferably, antimiRs comprise RNA, as RNA / RNA hybrids are highly stable. In addition, antimiRs may comprise DNA or may contain both RNA and DNA nucleotides (referred to herein as "chimeras"). AntimiRs should bind with high affinity through complementary base pairing to the "seed region" of the mature miRNA product, spanning nucleotides 2-8 from the 5' end of the mature miRNA product (Lennox et al. Gene Therapy 18: 1111-20 (2011), which is hereby incorporated by reference in its entirety).

[0129] Over the years, significant improvements in the binding affinity, stability, and target modulation effect of antimiRs have been achieved by chemical modifications to the oligonucleotide backbone. Therefore, antimiRs can be RNA or DNA derivatives. In some embodiments, antimiRs contain modifications that provide them with additional properties, such as resistance to endonucleases and RNase H, stability (e.g., in body fluids), and reduced toxicity. In some embodiments, the modifications are 2'-O-methyl-phosphorothioate oligonucleotide modifications, 2'-O-methoxyethyl oligonucleotide modifications, and combinations thereof. In some embodiments, antimiRs contain peptide nucleic acids, locked nucleic acids, or morpholino phosphorodiamidates. Generally, see Wahlestedt et al. PNAS 97, 5633-5638 (2000); Elayadi et al. Curr Opin Investig Drugs 2, 558-61 (2001); Larsen et al. Biochim Biophys Acta 1489, 159-166 (1999); Braasch et al. Biochemistry 41, 4503-4510 (2002); Summerton et al. Antisense Nucleic Acid Drug Dev 7, 187-195 (1997), which Each of the above references is hereby incorporated by reference herein in its entirety.

[0130] Expression of the miRNAs shown in Table 5 is known to be upregulated in various cancers, and their mature miRNA products, as shown in Table 6, are preferred targets for miRNA knockdown for the treatment of cancer.

[0131] Thus, in some embodiments, an API useful for treating cancer in a mammal is an antimiR, wherein the antimiR is a single-stranded nucleic acid molecule 12-25 nucleotides in length, wherein the antimiR has a sequence of 12-25 contiguous nucleotides that are complementary to contiguous nucleotides in a target mature miRNA product sequence, wherein the mature miRNA product sequence is selected from the group consisting of SEQ ID NOs: 884-908, and wherein the contiguous nucleotides in the mature miRNA product sequence include, in the 5' to 3' direction, nucleotides 2-8 of the mature miRNA product sequence.

[0132] [Table 5]

[0133] [Table 6]

[0134] In other embodiments, APIs useful for treating cancer in mammals are proteins with anti-cancer properties. Anti-cancer properties include inhibiting cancer cell growth, inhibiting tumor growth, causing cancer cell death, reducing tumor size, or causing tumor disappearance. The proteins listed in Table 7 have anti-cancer properties. Thus, proteins having an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof are suitable APIs for use in embodiments of the present invention. In other embodiments, proteins consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof are suitable APIs for use in embodiments of the present invention.

[0135] In some embodiments, an API suitable for treating cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, wherein the mRNA is configured to be translated in a mammalian target cell to produce a protein comprising the amino acid sequence. In other embodiments, an API suitable for treating cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, wherein the mRNA is configured to be translated in a mammalian target cell to produce a protein consisting of the amino acid sequence. The mRNA may be codon-optimized for translation in a mammalian target cell.

[0136] [Table 7] [Example]

[0137] Example 1 vSIRPα-siRNA conjugate binds to erythrocytes

[0138] FAM-tagged siRNA (FIG. 1A) (SEQ ID NO: 965 (sense strand) and SEQ ID NO: 966 (antisense strand)) was mixed with vSIRPα at a 1:1 molar ratio (50 pmol vSIRPα and 50 pmol siRNA). The siRNA was modified at its 3' end with maleimide. vSIRPα was designed to have a cysteine ​​near its C-terminus. The thiol from the cysteine ​​reacted with the maleimide via click chemistry at neutral pH. The reaction was kept overnight in a shaker at 4°C, resulting in the vSIRPα-siRNA conjugate shown in FIG. 1A.

[0139] The vSIRPα-siRNA conjugate was isolated using a NAP-5 column using the manufacturer's protocol.

[0140] To assess the ability of vSIRPα-siRNA conjugates to bind to red blood cells ("RBCs"), the vSIRPα-siRNA conjugates were mixed with mouse RBCs as depicted in Figure 1B. For this experiment, 6 x 10 siRNA conjugates were mixed with mouse RBCs in a volume of 2 μl of phosphate-buffered saline ("PBS"). 6 of red blood cells were mixed with 2.5 μl of PBS containing vSIRPα-siRNA conjugate (50 pmol total vSIRPα-siRNA conjugate) and 5.5 μl of PBS. The negative control was 6×10 red blood cells in a volume of 2 μl of PBS. 6 The red blood cells were mixed with 8 μl of PBS, and each mixture was then incubated at room temperature (20–25°C) for 2 hours (Figure 1B).

[0141] After incubation, 5 μl of each mixture was diluted 20-fold with PBS to a total volume of 100 μl per mixture, and each dilution was placed in a glass-bottom dish and fluorescently imaged at 494–567 nm using a microscope (excitation wavelength of 488 nm).

[0142] As can be seen in Figure 1C, the negative control showed no fluorescence, whereas the mixture containing the vSIRPα-siRNA conjugate showed a strong fluorescent signal associated with red blood cells, indicating that the vSIRPα-siRNA conjugate binds to RBCs.

[0143] Example 2 vSIRPα-siRNA conjugates are transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0144] 500 pmol of vSIRPα-siRNA conjugate from Example 1 was diluted to 5×10 in Dulbecco's Phosphate Buffered Saline ("DPBS") 3 The mixture was incubated with mouse RBCs in a total volume of 20 μl for 30 minutes at room temperature (20–25°C).

[0145] After 30 minutes, the mixture was washed with DPBS by centrifugation at 500 × g for 10 minutes, and the supernatant was removed. The RBCs bound to the vSIRPα-siRNA conjugate were then resuspended in 20 μl of PBS.

[0146] Two cell lines were used: CT26.CL25 (ATCC CRL-2639) and CaCO2 (ATCC HTB-37). CT26.CL25 is a murine colon carcinoma cell line. CaCO2 is a human colon adenocarcinoma cell line that lacks CD47 expression and was therefore used as a negative control (Liu et al. J Biol Chem 276(43):40156-66 (2001), which is hereby incorporated by reference in its entirety). (Can be).

[0147] Each cell line, grown independently in a cell culture dish, was detached from the dish with trypsin-EDTA. Cells from each dish were then washed with DPBS, counted, and collected at 10 5 cells / 100 μl PBS. 4 μl FAM-vSIRPα-siRNA / RBC resuspension (total 10 3 100 μl of RBCs and 100 pmol of vSIRPα-siRNA were mixed with 100 μl of each dilution of CT26.CL25 cells and CaCO2 cells, independently. Each mixture was incubated for 30 minutes at room temperature (20-25°C).

[0148] After 30 minutes, each mixture was centrifuged, the supernatant was removed, and the cell pellet was resuspended in 200 μl of flow cytometry buffer for flow cytometry analysis.

[0149] Figures 2A and 2B show flow cytometry results for CaCO2 and CT26.CL25 cells, respectively. Flow cytometry was performed using a 488 nm laser for excitation of the FAM tag and detection was performed at wavelengths of 525–565 nm. After incubation with RBCs bound to FAM-tagged vSIRPα-siRNA, each cancer cell line exhibited different levels of fluorescence shift. These results show that CT26.CL25 exhibited a significant fluorescence shift after incubation with RBCs, while CaCO2 exhibited little shift after incubation with RBCs, indicating that the degree of fluorescence shift was dependent on the level of CD47 present on the cancer cells. These results strongly suggest that FAM-tagged vSIRPα-siRNA was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0150] Example 3 Anti-CD47 antibodies are transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0151] First, 0.1 μg of Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody (Biolegend, #127510) was added to 4 × 10 cells in a total volume of 100 μl of DPBS. 3 The antibody-RBC mixture was incubated with 100 μl of mouse RBCs for 30 minutes at room temperature. After incubation, the RBC-antibody mixture was centrifuged at 500 g for 5 minutes. The supernatant was then decanted and the cells were resuspended in 20 μl of DPBS.

[0152] Next, we used 2 × 10 5 of CT26.CL25 cells (in 100 μl of DPBS) were mixed with 10 μl of the resuspended RBC-antibody mixture to a total volume of 110 μl (Mixture #1). 5 of CaCO2 cells (in 100 μl of DPBS) were mixed with 10 μl of the resuspended RBC-antibody mixture to a total volume of 110 μl (mixture #2).

[0153] Mixtures #1 and #2 were incubated for 30 minutes at room temperature. After incubation, 1 ml of DPBS was added to each mixture, followed by centrifugation at 500 g for 5 minutes. After centrifugation, the supernatant was decanted, and the remaining cells were resuspended in 100 μl of DPBS and subjected to flow cytometry (Beckman, laser 488 nm, detection 650-670 nm).

[0154] Figures 3A and 3B show these flow cytometry results for CaCO2 cells and CT26.CL25 cells, respectively. Although not as pronounced as the fluorescence shift observed in Example 2, a significant fluorescence shift was observed for CT26.CL25 cells after incubation with RBCs compared to the fluorescence shift for CaCO2 cells after incubation with RBCs. This again indicates that the extent of the fluorescence shift is dependent on the level of CD47 on the cancer cells. These results suggest that the Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0155] Example 4 vSIRPα-siRNA conjugates are transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0156] The vSIRPα-siRNA-FAM conjugate of Example 1 was further tested for its ability to be transferred from RBCs to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 2, except that 100 pmol of the conjugate, rather than 500 pmol, was incubated with RBCs.

[0157] As shown by the flow cytometry results in Figure 4, a significantly greater percentage of CT26.CL25 cells showed FAM fluorescence compared to CaCO2 cells, again strongly suggesting that FAM-tagged vSIRPα-siRNA was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0158] Example 5 Cy5.5-labeled vSIRPα is transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0159] Cy5.5-labeled vSIRPα was tested for its ability to be transferred from RBCs to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 2, except that 200 pmol of labeled vSIRPα, rather than 500 pmol of conjugate, was incubated with RBCs.

[0160] As shown by the flow cytometry results in Figure 5, a significantly greater percentage of CT26.CL25 cells showed Cy5.5 fluorescence compared with CaCO2 cells, strongly suggesting that Cy5.5-labeled vSIRPα was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0161] Example 6 Anti-CD47 antibodies are transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0162] An anti-mouse CD47 monoclonal antibody (Biolegend, #127510) labeled with Alexa Flour® 647 was tested for its ability to transfer from RBCs to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 3, except that 1 μg of labeled antibody was incubated with RBCs instead of 0.1 μg.

[0163] As shown in the flow cytometry results in Figure 6, a significantly greater percentage of CT26.CL25 cells exhibited Alexa Fluor® 647 fluorescence compared to unstained cells, strongly suggesting that the anti-CD47 antibody conjugated to Alexa Fluor® 647 was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells. This experiment also demonstrates that a monoclonal antibody against CD47 can be transferred from RBCs to CD47 present on the surface of cancer cells.

[0164] Example 7 Antibody-miR21 conjugates are transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0165] An anti-CD47 monoclonal antibody (Bioxcell, #BE0270) conjugated to Cy5-labeled miR21 (SEQ ID NO: 878) was tested for its ability to transfer from RBCs to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 3, except that 15.8 μg of the antibody conjugate was incubated with the RBCs instead of 0.1 μg.

[0166] As shown by the flow cytometry results in Figure 7 , a greater percentage of CT26.CL25 cells exhibited Cy5 fluorescence compared with unstained cells, strongly suggesting that the CD47mAb-miR21-Cy5 conjugate was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0167] Example 8 Thrombospondin-1 is transferred from RBCs to cancer cells as demonstrated by flow cytometry

[0168] Thrombospondin-1 (TSP-1) is a matricellular protein that inhibits angiogenesis and endothelial cell proliferation. TSP-1 binds to CD47. The TSP-1 signaling pathway has been shown to be involved in various conditions, including renal disease, cardiovascular disease, inflammation, and cancer. The mechanisms and pathways underlying TSP-1 are still not fully understood. However, the function of TSP-1 on several essential receptors, CD36 / VEGF and CD47, has been demonstrated. Particularly in cancer, activated TSP-1 and CD47 pathways have been shown to reduce tumor growth and metastasis. Kale et al. Int See J Mol Sci, 22(8) (2021) and Kaur et al. J Biol Chem, 285(50), 38923-38932 (2010), where the inventors demonstrated that labeled mouse TSP-1 binds to RB. C to CT26.CL25 cancer cells.

[0169] Cy5.5-labeled murine TSP-1 (7859-TH, R&D Systems) was tested for its ability to transfer from RBCs to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 2, except that 5 μg of Cy5.5-labeled TSP-1 was incubated with RBCs instead of 500 pmol of the conjugate.

[0170] As shown by the flow cytometry results in Figure 8, a significantly greater percentage of CT26.CL25 cells exhibited Cy5.5 fluorescence compared with CaCO2 cells, strongly suggesting that Cy5.5-labeled TSP-1 was transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

[0171] Example 9 vSIRPα-siRNA conjugates bind to RBCs in vivo

[0172] Five nmol of the vSIRPα-siRNA conjugate from Example 1 and 5 nmol of unconjugated siRNA from Example 1 were each stained with the intercalating dye YOYO™-1 iodide at a 1:1 molar ratio in a total volume of 120 μl of RNAse-free water. After incubation for 30 minutes at room temperature, the stained conjugate and siRNA were injected into mice as detailed below.

[0173] Three Balb / c mice were used in the experiment: one untreated mouse served as a negative control; one mouse was intravenously injected with 5 nmol of unconjugated dyed siRNA via the tail vein; and one mouse was intravenously injected with 5 nmol of dyed vSIRPα-siRNA conjugate via the tail vein.

[0174] 45 minutes after injection, blood was collected from the mice. 50 μl of whole blood from each sample was washed twice with 1 ml of DPBS by centrifugation at 500 × g for 10 minutes and resuspended in DPBS. The resuspended blood was imaged by confocal microscopy using a confocal dish (SPL 100350), and the RBC-associated YOYO™-1 iodide fluorescence signal was compared between different groups (control, siRNA, and conjugate). A 488 nm laser was used (YOYO™-1 iodide has an excitation wavelength of 491 nm).

[0175] As shown in Figure 9, RBCs from mice injected with the vSIRPα-siRNA conjugate showed fluorescent puncta (arrows in Figure 9), indicating that binding of the vSIRPα-siRNA conjugate to RBCs occurs in vivo.

[0176] Possible claims

[0177] Various embodiments of the present invention may be characterized by the potential claims, listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form part of the written specification of this application. Accordingly, the subject matter of the following potential claims may be presented as actual claims in a later proceeding involving this application or any application claiming priority from this application. The inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Accordingly, a decision not to present these potential claims in a later proceeding should not be construed as an offering of that subject matter to the public.

[0178] Possible subject matter (prefaced with the letter "P" to avoid confusion with the actual claims set forth below) that may be claimed includes, but is not limited to: (P1) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cells by the circulatory system of the subject; and thus (i) the CD47 binding protein, configured to bind the conjugate to the CD47 of the red blood cell, binds to the CD47 of the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (P2) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cells by the circulatory system of the subject, and thus (i) administering the conjugate to a the CD47 binding protein, which is configured to bind to the CD47 of the red blood cell, binds to the CD47 of the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (P3) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is an anti-CD47 antibody, (a) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively; (b) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944 and SEQ ID NO: 945, respectively; (c) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 948, SEQ ID NO: 949 and SEQ ID NO: 950, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 951, SEQ ID NO: 952 and SEQ ID NO: 953, respectively; or (d) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957 and SEQ ID NO: 958, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960 and SEQ ID NO: 961, respectively; the conjugate is configured to bind to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cell by the subject's circulatory system, such that (i) the CD47 binding protein configured to bind the conjugate to the CD47 on the red blood cell binds to the CD47 on the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (P3.5) The therapeutic compound of claim P3, wherein the anti-CD47 antibody is a humanized antibody. (P4) 10. The therapeutic compound of any one of the preceding claims, wherein the CD47 binding protein is conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, van der Waals interactions, and combinations thereof. (P5) 10. The therapeutic compound of any one of the preceding possible claims, wherein the CD47 binding protein is conjugated to the API by a linker. (P6) The therapeutic compound of claim P5, wherein the linker is cleavable. (P7) The therapeutic compound of any one of claims P5 and P6, wherein the linker is configured to be cleaved by a lysosomal degrading enzyme. (P8) 10. The therapeutic compound of any one of the preceding possible claims, wherein the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins, and small molecules. (P9) The therapeutic compound of any one of claims P1 to P7, wherein the API is selected from the group consisting of siRNA, shRNA, miRNA, antimiR, and mRNA. (P10) 10. The therapeutic compound of any one of the preceding possible claims, wherein the target cell is a cell selected from the group consisting of a cancer cell, a virus-infected cell, a fibrotic cell, and combinations thereof. (P11) The therapeutic compound of any one of claims P1 to P9, wherein the target cell is a cancer cell. (P12) The therapeutic compound of any one of claims P1 to P9, wherein the target cell is a virally infected cell. (P13) The therapeutic compound of any one of claims P1 to P9, wherein the target cells are fibrotic cells. (P14) The therapeutic compound of claim P11, wherein the cancer cells are in a tumor caused by a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, gastric cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer; blood cancer including acute / chronic leukemia, malignant lymphoma, and multiple myeloma; bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer. (P15) The cancer cells are selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine endometrioid carcinoma, prostate cancer, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, invasive breast cancer, gastric adenocarcinoma, and bladder urothelial carcinoma. , bile duct cancer, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, cutaneous melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, acute leukemia of unclear lineage, brown The therapeutic compound of claim P11, caused by a cancer selected from the group consisting of pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neuroblastoma, renal papillary cell carcinoma, hepatocellular carcinoma, chromophobe renal cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell / sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma / rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms' tumor, myofibromatosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing's sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-dependent soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. (P16) The therapeutic compound of claim P12, wherein the virally infected cell is infected with the SARS-CoV-2 virus. (P17) The therapeutic compound of claim P13, wherein the fibrotic cells are associated with cystic fibrosis. (P18) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8 to 747 and 771 to 824; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P19) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 747 and 771 to 824; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P20) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 37; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P21) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 38 to 39; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P22) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40 to 43; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P23) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 44 to 51; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P24) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P12, and P16. (P25) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 482 to 486 and 748 to 765; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) The therapeutic compound of any one of claims P1 to P7, P10, P12, and P16, wherein the double-stranded RNA molecule has a double-stranded region 14 to 29 nucleotides in length and a 3' overhang region 0 to 5 nucleotides in length. (P26) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8 to 21, 40 to 43, and 766 to 770; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P13, and P17. (P27) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40 to 43 and 766 to 770; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; A therapeutic compound according to any one of claims P1 to P7, P10, P13, and P17. (P28) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-747 and 771-824; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P29) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22-747 and 771-824; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P30) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 37; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P31) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence. and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the mRNA sequence is selected from the group consisting of SEQ ID NOs: 38-39; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P32) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40 to 43; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P33) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 44 to 51; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14, and P15. (P34) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P12, and P16. (P35) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 482-486 and 748-765; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P12, and P16. (P36) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P13, and P17. (P37) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40-43 and 766-770; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. A therapeutic compound according to any one of claims P1 to P7, P10, P13, and P17. (P38) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is a miRNA selected from the group consisting of SEQ ID NOs: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883. (P39) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is an antimiR, wherein the antimiR is a single-stranded nucleic acid molecule 12 to 25 nucleotides in length, wherein the antimiR has a sequence of 12 to 25 contiguous nucleotides that are complementary to consecutive nucleotides in a target mature miRNA product sequence, wherein the mature miRNA product sequence is selected from the group consisting of SEQ ID NOs: 884-908, and wherein the consecutive nucleotides in the mature miRNA product sequence comprise, in the 5' to 3' direction, nucleotides 2 to 8 of the mature miRNA product sequence. (P40) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents, such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA damaging agents, such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand breakers (e.g., calicheamicin), cross-linking agents (e.g., pyrrolobenzodiazepine dimers-PBD), and alkylating agents (e.g., duocarmycin and indolinobenzodiazepine dimers-IGN). (P41) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is a protein, and the protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof. (P42) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is a protein, and the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof. (P43) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein comprising the amino acid sequence. (P44) The therapeutic compound of any one of claims P1 to P7, P10, P11, P14, and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein consisting of said amino acid sequence. (P45) The therapeutic compound of any one of claims P43 and P44, wherein the mRNA is codon-optimized. (P46) A method of treating cancer in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of claims P1 to P11, P14, P15, P18 to P23, P28 to P33, and P38 to P45. (P47) A method of treating a viral infection in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of claims P1 to P10, P12, P16, P24, P25, P34, and P35. (P48) A method of treating a fibrotic disease in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of claims P1 to P10, P13, P17, P26, P27, P36, and P37. (P49) The therapeutic compound of any one of claims P1 to P45, wherein the mammalian subject is a human. (P50) The method of any one of claims P46 to P48, wherein the mammalian subject is a human. (P51) A pharmaceutical composition comprising a therapeutic compound of any one of claims P1 to P45 and P49 and a pharmaceutically acceptable carrier.

[0179] The embodiments of the present invention described above are intended to be exemplary only, and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims. The present invention provides, for example, the following items. (Item 1) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: A CD47 binding protein conjugated to an API to form a conjugate Includes; the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cells by the circulatory system of the subject, and thus (i) administering the conjugate to a the CD47 binding protein, which is configured to bind to the CD47 of the red blood cell, binds to the CD47 of the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (Item 2) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: A CD47 binding protein conjugated to an API to form a conjugate Includes; the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cells by the circulatory system of the subject; and thus (i) the CD47 binding protein, configured to bind the conjugate to the CD47 of the red blood cell, binds to the CD47 of the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (Item 3) 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to target cells expressing CD47, comprising: a CD47 binding protein conjugated to an API to form a conjugate; the CD47 binding protein is an anti-CD47 antibody, (a) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively; (b) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944 and SEQ ID NO: 945, respectively; (c) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 948, SEQ ID NO: 949 and SEQ ID NO: 950, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 951, SEQ ID NO: 952 and SEQ ID NO: 953, respectively; or (d) a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957 and SEQ ID NO: 958, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960 and SEQ ID NO: 961, respectively; the conjugate is configured to bind to CD47 on red blood cells of the subject to enable transport of the conjugate to the target cell by the subject's circulatory system, such that (i) the CD47 binding protein configured to bind the conjugate to the CD47 on the red blood cell binds to the CD47 on the target cell, thereby transferring the conjugate from the red blood cell to the target cell and forming a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the target cell; and (ii) the conjugate is taken up by the target cell by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the target cell and delivering the API to the target cell. therapeutic compounds. (Item 4) The therapeutic compound of any one of the preceding items, wherein the CD47 binding protein is conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, van der Waals interactions, and combinations thereof. (Item 5) The therapeutic compound of any one of the preceding items, wherein the CD47 binding protein is conjugated to the API by a linker. (Item 6) 6. The therapeutic compound of item 5, wherein the linker is cleavable. (Item 7) 7. The therapeutic compound of any one of items 5 and 6, wherein the linker is configured to be cleaved by a lysosomal degrading enzyme. (Item 8) 2. The therapeutic compound of claim 1, wherein the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins, and small molecules. (Item 9) 8. The therapeutic compound of any one of items 1 to 7, wherein the API is selected from the group consisting of siRNA, shRNA, miRNA, antimiR, and mRNA. (Item 10) The therapeutic compound of any one of the preceding items, wherein the target cell is a cell selected from the group consisting of a cancer cell, a virus-infected cell, a fibrotic cell, and combinations thereof. (Item 11) 9. The therapeutic compound according to any one of items 1 to 8, wherein the target cells are cancer cells. (Item 12) 9. The therapeutic compound of any one of items 1 to 8, wherein the target cell is a virus-infected cell. (Item 13) 9. The therapeutic compound of any one of items 1 to 8, wherein the target cells are fibrotic cells. (Item 14) 12. The therapeutic compound according to item 11, wherein the cancer cells are in a tumor caused by a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, gastric cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer; blood cancer including acute / chronic leukemia, malignant lymphoma, and multiple myeloma; bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer. (Item 15) The cancer cells are selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine endometrioid cancer, prostate cancer, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, invasive breast cancer, gastric adenocarcinoma, and bladder urothelium. Cancer, bile duct carcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, cutaneous melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, acute leukemia of unclear lineage, 12. The therapeutic compound according to item 11, wherein the cancer is selected from the group consisting of pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neuroblastoma, papillary cell carcinoma of the kidney, hepatocellular carcinoma, chromophobe renal cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell / sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma / rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms' tumor, myofibromatosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing's sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-dependent soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. (Item 16) 13. The therapeutic compound of item 12, wherein the virus-infected cell is infected with the SARS-CoV-2 virus. (Item 17) 14. The therapeutic compound of item 13, wherein the fibrotic cells are associated with cystic fibrosis. (Item 18) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8 to 747 and 771 to 824; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 19) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 747 and 771 to 824; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 20) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 37; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 21) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 38 to 39; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 22) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40 to 43; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 23) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 44 to 51; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 24) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 17. The therapeutic compound of any one of items 12 and 16. (Item 25) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 482 to 486 and 748 to 765; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 17. The therapeutic compound of any one of items 12 and 16. (Item 26) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8 to 21, 40 to 43, and 766 to 770; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 18. The therapeutic compound of any one of items 13 and 17. (Item 27) the API is an siRNA, the siRNA being a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand; (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40 to 43 and 766 to 770; (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand; (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length; 18. The therapeutic compound of any one of items 13 and 17. (Item 28) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-747 and 771-824; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 29) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22-747 and 771-824; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 30) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 22 to 37; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 31) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the mRNA sequence is selected from the group consisting of SEQ ID NOs: 38-39; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 32) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the mRNA sequence is selected from the group consisting of SEQ ID NOs: 40 to 43; (d) the therapeutic compound of any one of items 11, 14, and 15, wherein the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. (Item 33) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 44 to 51; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 16. The therapeutic compound of any one of items 11, 14 and 15. (Item 34) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 482-486, and 748-765; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 17. The therapeutic compound of any one of items 12 and 16. (Item 35) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 482-486 and 748-765; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 17. The therapeutic compound of any one of items 12 and 16. (Item 36) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 8-21, 40-43, and 766-770; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 18. The therapeutic compound of any one of items 13 and 17. (Item 37) The API is an shRNA, the shRNA being a single-stranded RNA molecule of 44 to 71 nucleotides in length, and having in its 5' to 3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides immediately adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides immediately adjacent to the second region, the third region having a third sequence; and a fourth region of two nucleotides at the 3' end of the single-stranded RNA molecule immediately adjacent to the third region, the fourth region having a fourth sequence; and (a) the first region has the same number of nucleotides as the third region; (b) the third sequence is the reverse complement of the first sequence; (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NOs: 40-43 and 766-770; (d) the single-stranded RNA molecule is configured to form a stem-loop structure, in which the first region base-pairs with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 18. The therapeutic compound of any one of items 13 and 17. (Item 38) 16. The therapeutic compound of any one of items 11, 14 and 15, wherein the API is a miRNA selected from the group consisting of SEQ ID NOs: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883. (Item 39) 16. The therapeutic compound of any one of items 11, 14, and 15, wherein the API is an antimiR, the antimiR is a single-stranded nucleic acid molecule 12-25 nucleotides in length, the antimiR having a sequence of 12-25 contiguous nucleotides that are complementary to consecutive nucleotides in a target mature miRNA product sequence, the mature miRNA product sequence being selected from the group consisting of SEQ ID NOs: 884-908, and the consecutive nucleotides in the mature miRNA product sequence comprise, in the 5' to 3' direction, nucleotides 2-8 of the mature miRNA product sequence. (Item 40) 16. The therapeutic compound of any one of items 11, 14 and 15, wherein the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents; and DNA damaging agents. (Item 41) 16. The therapeutic compound of any one of items 11, 14 and 15, wherein the API is a protein and the protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof. (Item 42) 16. The therapeutic compound of any one of items 11, 14 and 15, wherein the API is a protein, and the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof. (Item 43) 16. The therapeutic compound of any one of items 11, 14, and 15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909-929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein comprising the amino acid sequence. (Item 44) 16. The therapeutic compound of any one of items 11, 14, and 15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 909 to 929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein consisting of the amino acid sequence. (Item 45) 45. The therapeutic compound of any one of items 43 and 44, wherein the mRNA is codon-optimized. (Item 46) 10. A method of treating cancer in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of items 1 to 11, 14, 15, 18 to 23, 28 to 33, and 38 to 45. (Item 47) 36. A method of treating a viral infection in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of items 1 to 10, 12, 16, 24, 25, 34, and 35. (Item 48) 38. A method of treating a fibrotic disease in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of a therapeutic compound of any one of items 1 to 10, 13, 17, 26, 27, 36, and 37. (Item 49) 46. ​​The therapeutic compound of any one of items 1 to 45, wherein the mammalian subject is a human. (Item 50) 49. The method of any one of items 46 to 48, wherein the mammalian subject is a human. (Item 51) 50. A pharmaceutical composition comprising a therapeutic compound according to any one of items 1 to 45 and 49 and a pharmaceutically acceptable carrier.

Claims

Claim 1: A composition comprising a therapeutic compound for RBC-mediated delivery to cancer cells expressing CD47 in a mammalian subject, comprising: the therapeutic compound comprising vSIRPα (SEQ ID NO: 3) or a homolog thereof conjugated to monomethyl auristatin E (MMAE) to form a conjugate; The vSIRPα is configured to bind the conjugate to CD47 on red blood cells of the subject to enable transport of the conjugate to the cancer cells by the subject's circulatory system, such that (i) the vSIRPα configured to bind the conjugate to CD47 on the red blood cells binds to the CD47 on the cancer cells, thereby transferring the conjugate from the red blood cells to the cancer cells and forming a conjugate-CD47 complex on the cancer cells, thereby blocking CD47 and inhibiting CD47 activity as an immune evasion mechanism of the cancer cells, and (ii) the conjugate is taken up by the cancer cells by endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune evasion mechanism of the cancer cells and delivering the MMAE to the cancer cells.

2. The composition described in claim 1, wherein the CD47 binding protein is conjugated to the MMAE by a linker.

3. The composition described in claim 2, wherein the linker is cleavable.

4. The composition described in claim 3, wherein the linker is configured to be cleaved by a lysosomal degrading enzyme.

5. The composition of claim 3, wherein the linker is selected from the group consisting of a hydrazone linker, an imine linker, an oxime linker, a carbonate linker, an acetal linker, an orthoester linker, a silyl ether linker, a disulfide linker, a trioxolane linker, a beta-glucuronide linker, a beta-galactoside linker, a pyrophosphate linker, a phosphoramidate linker, an aryl sulfate linker, a heptamethine cyanine linker, a nitrobenzyl linker, an arylboronic acid linker, a boronate linker, a thioether linker, a maleimidocaproyl-containing linker, a peptide linker, and a para-aminobenzyl carbamate-containing linker.

6. The composition described in claim 3, wherein the linker comprises para-aminobenzyl carbamate.

7. The composition of claim 1, wherein the cancer cells are in a tumor caused by a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cancer, paranasal cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, airway cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer; blood cancer including acute / chronic leukemia, malignant lymphoma, and multiple myeloma; bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

8. The cancer cells are selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine endometrioid carcinoma, prostate cancer, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, invasive breast cancer, gastric adenocarcinoma, and bladder urothelium. Cancer, bile duct carcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, cutaneous melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, acute leukemia of unclear lineage 10. The composition of claim 1, wherein the cancer is caused by a cancer selected from the group consisting of pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neuroblastoma, renal papillary cell carcinoma, hepatocellular carcinoma, chromophobe renal cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell / sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma / rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms' tumor, myofibromatosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing's sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-dependent soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

9. A composition described in any one of claims 1 to 8 for treating cancer in a mammalian subject in need of cancer treatment.

10. The composition of claim 9, wherein the mammalian subject is a human.

11. A pharmaceutical composition comprising a composition described in any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

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