Nucleic acids encoding engineered chimeric fusion proteins

Abstract

What is disclosed herein are nucleic acids encoding a chimeric fusion protein comprising an extracellular domain that comprises an antigen binding domain, particularly a TROP2 binding domain. The nucleic acids disclosed here comprise codon-optimized coding sequence for the chimeric fusion protein and comprises a 5' UTR and a 3' UTR, wherein the 3' UTR comprises at least one microRNA binding site.

Description

WSGR Docket No. 56371-768.601NUCLEIC ACIDS ENCODING ENGINEERED CHIMERIC FUSION PROTEINSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 693,494, filed on September 11, 2024, U.S. Provisional Application No. 63 / 739,246, filed on December 27, 2024, U.S. Provisional Application No. 63 / 742,083, filed on January 06, 2025, U.S. Provisional Application No 63 / 799,015, filed on May 02, 2025, and U.S. Provisional Application No. 63 / 820,633, filed on June 09, 2025, each of which is incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION

[0002] Cellular immunotherapy is a promising new technology to treat diseases, such as cancer, persistent infections and certain diseases that are refractory to other forms of treatment. Although CAR-T cells continue to remain prospective tools for cancer therapy, several limitations have slowed the progress on CAR-T cells and dampened its promise in clinical trials.

[0003] Understanding the limitations of CAR-T cells is the key to leveraging the technology and continue innovations towards better immunotherapy models. Specifically, in T cell malignancies, CAR-T cells appear to have faced a major problem. CAR-T cells and malignant T cells share surface antigen in most T cell lymphomas (TCL), therefore, CAR-T cells are subject to cytotoxicity in the same way as cancer cells. In some instances, the CAR-T products may be contaminated by malignant T cells. Additionally, T cell aplasia is a potential problem due to prolonged persistence of the CAR-T cells. Other limitations include the poor ability for CAR-T cells to penetrate into solid tumors and the potent tumor microenvironment which acts to downregulate their anti-tumor potential. CAR-T cell function is also negatively influenced by the immunosuppressive tumor microenvironment (TME) that leads to endogenous T cell inactivation and exhaustion.

[0004] Myeloid cells, including macrophages, are cells derived from the myeloid lineage and belong to the innate immune system. They are derived from bone marrow stem cells which egress into the blood and can migrate into tissues. Some of their main functions include phagocytosis, the activation of T cell responses, and clearance of cellular debris and extracellular matrices. They also play an important role in maintaining homeostasis, and initiating and resolving inflammation. Moreover, myeloid cells can differentiate into numerous downstream cells, including macrophages, which can display different responses ranging from pro- inflammatory to anti-inflammatory depending on the type of stimuli they receive from the surrounding microenvironment. Furthermore, tissue macrophages have been shown to play a broad regulatory and activating role on other immune cell types including CD8+ and CD4+ TWSGR Docket No. 56371-768.601 effector cells, NK cells and T regulatory cells. Macrophages have been shown to be a main immune infiltrate in malignant tumors and have been shown to have a broad immunosuppressive influence on effector immune infiltration and function.

[0005] Although myeloid cells have many functions, including phagocytosis and their ability to activate T cells, harnessing these functions for therapeutic uses has remained elusive. Newer avenues are therefore sought for using other cell types towards development of improved therapeutics, including but not limited to T cell malignancies. Engineered myeloid cells can also be short-lived in vivo, phenotypically diverse, sensitive, plastic, and are often found to be difficult to manipulate in vitro. For example, exogenous gene expression in monocytes has been difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with transfecting myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells, such as monocytes / macrophages, comprise many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer into these cells an inefficient process. This is especially true of activated macrophages which undergo a dramatic change in their physiology following exposure to immune or inflammatory stimuli. Viral transduction of these cells has been hampered because macrophages are end-stage cells that generally do not divide; therefore, some of the vectors that depend on integration into a replicative genome have met with limited success. Furthermore, macrophages are quite responsive to “danger signals,” and therefore several of the original viral vectors that were used for gene transfer induced potent anti-viral responses in these cells making these vectors inappropriate for gene delivery. Even after successfully introducing the exogenous genetic materials to myeloid cells, it is difficult to express the genetic materials to a desirable level, let alone to maintain the expression level for an extended time to achieve desirable result, such as therapeutic effect.SUMMARY OF THE INVENTION

[0006] Provided herein is a composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding TROP2, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36. In some embodiments, the CFP comprises an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), wherein the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises a sequence of SEQ ID NO: 180, the HCDR2 comprises a sequence of SEQ ID NO: 181, and the HCDR3 comprises a sequence of SEQ ID NO: 182; and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises a sequence of SEQ ID NO: 189,WSGR Docket No. 56371-768.601 the LCDR2 comprises a sequence of SEQ ID NO: 190, and the LCDR3 comprises a sequence of SEQ ID NO: 191. In some embodiments, the VH comprises a sequence of SEQ ID NO: 175, and the VL comprises a sequence of SEQ ID NO: 176. In some embodiments, the CFP comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the CFP comprises an amino acid sequence of SEQ ID NO: 179. In some embodiments, the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5, 33, or 34. In some embodiments, the nucleic acid further comprises a 5’ UTR, and wherein the 5’ UTR comprises a sequence of SEQ ID NO: 7 or 8. In some embodiments, the nucleic acid further comprises a 3’ UTR, (i) wherein the 3’ UTR is a bGH 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 9; or (ii) wherein the 3’ UTR is a a globin 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises one or more microRNA binding site. In some embodiments, the 3’ UTR comprises four microRNA binding sites. In some embodiments, the microRNA binding site comprise a sequence that specifically binds to miR- 27b-5p, and wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11. In some embodiments, the microRNA binding site comprises a sequence of SEQ ID NO: 12 or 24. In some embodiments, the 3’ UTR comprises a sequence of SEQ ID NO: 13. In some embodiments, the microRNA binding site comprise a sequence that specifically binds to miR122-5p, and wherein 3’ UTR comprises a sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, the nucleic acid comprises a sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid is an RNA; optionally, wherein the RNA is an mRNA. In some embodiments, the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 33; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 36; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 36. In some embodiments, the composition further comprises (i) a nucleic sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38; or (ii) a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7. In some embodiments, the CFP for specifically binding TROP2 comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16. In some embodiments, the nucleic acid comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17.

[0007] Also provided herein is a composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding GPC3, wherein the nucleicWSGR Docket No. 56371-768.601 acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 41. In some embodiments, the CFP comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the nucleic acid comprises a sequence having at least 95% sequence identity to SEQ ID NO: 41; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 41. In some embodiments, the nucleic acid further comprises a 5’ UTR, and wherein the 5’ UTR comprises a sequence of SEQ ID NO: 42. In some embodiments, the nucleic acid further comprises a 3’ UTR, wherein the 3’ UTR is a a globin 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 43. In some embodiments, the 3’ UTR comprises one or more microRNA binding site. In some embodiments, the 3’ UTR comprises four microRNA binding sites. In some embodiments, the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p, and wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11. In some embodiments, the microRNA binding site comprises a sequence of SEQ ID NO: 12. In some embodiments, the 3’ UTR comprises a sequence of SEQ ID NO: 13. In some embodiments, the microRNA binding site comprise a sequence that specifically binds to miR122-5p, and wherein 3’ UTR comprises a sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 44, optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 44. In some embodiments, the nucleic acid is an RNA, optionally, wherein the RNA is an mRNA. In some embodiments, the composition further comprises (i) a nucleic acid sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38, or (ii) a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7.

[0008] Also provided herein is a pharmaceutical composition, comprising the composition of any one of the foregoing embodiments, and a pharmaceutical acceptable excipient. In some embodiments, the pharmaceutical composition further comprises a lipid nanoparticle, wherein the nucleic acid is encapsulated in the lipid nanoparticle.

[0009] Also provided herein is a pharmaceutical composition, comprising: (I) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises a sequence of GGFGSSYWYFDV; and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR3 comprises a sequence of QQHYITPLT; (b) a CD89 transmembrane domain operatively linked to the extracellular domain; and (II) a lipid nanoparticle delivery vehicle encapsulating the RNAWSGR Docket No. 56371-768.601 in (I). In some embodiments, the HCDR1 comprises a sequence of NYGMN, the HCDR2 comprises a sequence of WINTYTGEPTYTDDFKG; and wherein the LCDR1 comprises a sequence of KASQDVSIAVA, the LCDR2 comprises a sequence of SASYRYT. In some embodiments, the VH comprises a sequence of SEQ ID NO: 175, and the VL comprises a sequence of SEQ ID NO: 176. In some embodiments, the lipid nanoparticle delivery vehicle comprises an ionizable lipid, wherein the ionizable lipid is ALC-0366, and wherein the ionizable lipid comprises the chemical formula (Formula I):

[0010] In some embodiments, the lipid nanoparticle delivery vehicle comprises a PEG-lipid, wherein the PEG-lipid (i) is ALC-0159, or (ii) comprises the chemical formula (Formula II):wherein n is an integer between 46 and 52. In some embodiments, the lipid nanoparticle delivery vehicle comprises distearoylphosphatidylcholine (DSPC), and wherein the DSPC comprises the chemical formula (Formula III):. In some embodiments, the lipid nanoparticle delivery vehicle comprises cholesterol, and wherein the cholesterol comprises the chemical formula (Formula IV):

[0011] In some embodiments, the ratio of mRNA to ionizable lipid (N / P) is 6.0. In some embodiments, the concentration of the RNA is between about 1.5 to about 0.5 mg / mL or between about 1.3 to about 0.7 mg / mL when stored in a container. In some embodiments, the LNP comprises (a) an ionizable lipid present at a mol% of from about 35% to about 55%; (b)WSGR Docket No. 56371-768.601 distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%; (c) cholesterol present at a mol% from about 30% to about 50%; and (d) a PEG lipid present at a mol% of from about 1% to about 10%. In some embodiments, (a) the CFP has an amino acid sequence of SEQ ID NO: 39 or 179; (b) the LNP comprises: (i) an ionizable lipid, wherein the ionizable lipid is ALC-0366 or comprises the chemical formula:wherein the ionizable lipid is present in the LNP at a mol% of about 47.5%, (ii) a PEG lipid, wherein the PEG-lipid is ALC-0159 or comprises the chemical formula:wherein n is an integer between 46 and 52; wherein the PEG-lipid is present in the LNP at a mol% of about 2.5%, (iii) distearoylphosphatidylcholine (DSPC), wherein the DSPC is present in the LNP at a mol% of about 10%; and (iv) cholesterol, wherein the cholesterol is present in the LNP at a mol% of about 40%.

[0012] Also provided herein is a pharmaceutical composition comprising: (a) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an amino acid sequence of SEQ ID NO: 39 or 179; and (b) a lipid nanoparticle delivery vehicle (LNP) encapsulating the RNA, wherein the LNP comprises (i) an ionizable lipid, wherein the ionizable lipid is ALC-0366 or comprises the chemical formula:wherein the ionizable lipid is present in the LNP at a mol% of about 47.5%, (ii) a PEG lipid, wherein the PEG-lipid is ALC-0159 or comprises the chemical formula:WSGR Docket No. 56371-768.601

[0013] wherein n is an integer between 46 and 52; wherein the PEG-lipid is present in the LNP at a mol% of about 2.5%, (iii) distearoylphosphatidylcholine (DSPC), wherein the DSPC is present in the LNP at a mol% of about 10%; and (iv) cholesterol, wherein the cholesterol is present in the LNP at a mol% of about 40%. In some embodiments, wherein the sequence of the RNA encoding the CFP comprises a sequence according to any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36.

[0014] Also provided herein is a pharmaceutical composition, comprising: a nucleic acid, encapsulated in an LNP, wherein the nucleic acid comprises a sequence encoding an anti-TROP2 chimeric fusion protein (CFP) having an amino acid sequence of SEQ ID NO: 39 or 179; wherein the nucleic acid comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; and wherein the LNP comprises (a) an ionizable lipid present at a mol% of from about 35% to about 55%; (b) distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%; (c) cholesterol present at a mol% from about 30% to about 50%; and (d) a PEG lipid present at a mol% of from about 1% to about 10%.

[0015] Also provided herein a pharmaceutical composition, comprising: a nucleic acid, encapsulated in an LNP, wherein the nucleic acid comprises a sequence encoding an anti-TROP2 chimeric fusion protein (CFP), wherein the nucleic acid comprises a sequence of any one of SEQ ID NOs: 4, 5, or 34; wherein the LNP comprises (a) an ionizable lipid present at a mol% of from about 35% to about 55%; (b) distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%; (c) cholesterol present at a mol% from about 30% to about 50%; and (d) a PEG lipid present at a mol% of from about 1% to about 10%.

[0016] Also provided herein is a cell comprising the composition of any one of the foregoing embodiments. In some embodiments, the cell is an immune cell; optionally, wherein the cell is a myeloid cell, a lymphoid cell, a precursor cell, a stem cell or an induced pluripotent cell; optionally, wherein the cell is CD14+ CD16- cell.

[0017] Also provided herein is a method for treating a patient in need thereof, comprising administering the cell of any one of the foregoing embodiments to the patient.

[0018] Also provided herein is a method for treating a cancer in a subject in need thereof, comprising administering the pharmaceutical composition of any one of the foregoing embodiments to the patient.

[0019] Also provided herein is a method of treating a cancer in a subject in need thereof, comprising administering to the subject (i) a composition comprising a nucleic acid comprising aWSGR Docket No. 56371-768.601 sequence encoding a chimeric fusion protein (CFP), the CFP comprising an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain operatively linked to the extracellular domain, and a CD89 intracellular domain operatively linked to the transmembrane domain; and (ii) at least one prophylactic agent for preventing or treating cytokine release syndrome (CRS).

[0020] Also provided herein is a method of treating a cancer in a human in need thereof, the method comprising administering to the subject a combination of: (a) a first component comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2; (b) at least one prophylactic agent; and (c) at least one anti -cancer agent selected from: (i) a chemotherapeutic agent; (ii) a tumor target drug or (iii) an immune checkpoint inhibitor. In some embodiments, the prophylactic agent is administered before, after, or simultaneously with the composition comprising the nucleic acid. In some embodiments, the prophylactic agent is an anti-interleukin (IL) agent; optionally, wherein the anti-IL agent is an IL receptor inhibitor; optionally, wherein the IL receptor inhibitor comprises tocilizumab and / or anakinra. In some embodiments, the tocilizumab is administered (i) at least one hour before the administration of the composition comprising the nucleic acid, (ii) at the time of the administration of the composition comprising the nucleic acid, (iii) at a dose of 8 mg / kg (max 800 mg), and / or (iv) via IV over 60 minutes. In some embodiments, the anakinra is administered (i) before the administration of the composition comprising the nucleic acid, (ii) at the time of the administration of the composition comprising the nucleic acid, (iii) subcutaneously, (iv) once daily, and / or (v) every 12 hours. In some embodiments, the amount of the anakinra administered is 100 mg. In some embodiments, the anakinra is administered (i) for from about 3 to about 5 days; (ii) intravenously;(iii) in an amount of 2 mg / kg; and / or (iv) every 6 hours. In some embodiments, the CRS is reduced as compared to a control group lacking the administration of the prophylactic agent, and wherein the CRS comprises elevated liver enzymes, D-dimer, ferritin, or C-reactive protein (CRP), wherein the CRP is measured according to ASTCT CRS grading.

[0021] Also provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering the subject: (a) a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain, and a CD89 intracellular domain; and (b) an anti-cancer agent comprising: a chemotherapeutic agent; and (i) a tumor target drug, or (ii) an immune checkpoint inhibitor. In some embodiments, (i) the nucleic acid and the anti-cancer agent are administered at separate times; (ii) the nucleic acid is administered after administering the anti-cancer agent; (iii)WSGR Docket No. 56371-768.601 the nucleic acid is administered before administering the anti-cancer agent; or (iv) the nucleic acid and the anti-cancer agent overlaps for at least one administration. In some embodiments, the chemotherapeutic agent is selected from the group consisting of capecitabine, oxaliplatin, fluoropyrimidine, cisplatin, and carboplatin. In some embodiments, the chemotherapeutic agent comprises capecitabine and oxaliplatin (Capox). In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1, optionally, wherein the antibody is pembrolizumab or nivolumab. In some embodiments, the anti-cancer agent comprises a tumor target drug; optionally, wherein the tumor target drug is an antibody specifically binds to a tumor antigen. In some embodiments, the tumor antigen is HER-2. In some embodiments, the antibody is trastuzumab. In some embodiments, the chemotherapeutic agent is capecitabine and oxaliplatin (Capox), and wherein the oxaliplatin is administered (i) at a dose of 130 mg / mA2, (ii) intravenously (IV), and / or (iii) on Day 1 of each cycle. In some embodiments, the capecitabine is administered (i) at a dose of 1000 mg / mA2, (ii) orally, and / or (iii) twice daily on Days 1-14 of each cycle, every three weeks. In some embodiments, the cancer is PD-L1 -positive, and the anticancer agent comprises the immune checkpoint inhibitor. In some embodiments, the tumor target drug is (i) administered IV; and / or (ii) administered every 3 weeks. In some embodiments, the nucleic acid is administered (i) on the same day of the anti-cancer agent and is administered every 3 weeks; and / or (ii) before the anti-cancer agent on the same day. In some embodiments, the method comprises at least 3 cycles; optionally, wherein each of the at least 3 cycles comprises 3 weeks. In some embodiments, the nucleic acid is administered on Day 1 and Day 8 of Cycle 1, Day 1 of Cycle 2 and afterwards. In some embodiments, the oxaliplatin is administered on Day 8 of Cycle 1, and on Day 1 of Cycle 2 and afterwards. In some embodiments, the capecitabine is administered on Day 8 and Day 15 of Cycle 1, and Day 1 and Day 8 of Cycle 2 and afterwards. In some embodiments, the nivolumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards. In some embodiments, the trastuzumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards. In some embodiments, the method further comprises administering an additional drug, wherein the additional drug comprises an anti-emetic, analgesic, anti-pyretic, anti -diarrheal, anti-inflammatory drug. In some embodiments, the additional drug comprises an anti-emetic drug, and wherein the anti-emetic drug comprises olanzapine, 5-HT3 receptor antagonist, or NK- 1 receptor antagonist. In some embodiments, the additional drug further comprises a steroid, optionally wherein the steroid comprises methylprednisolone. In some embodiments, the 5-HT3 receptor antagonist is ondansetron or palonosetron. In some embodiments, the NK-1 receptor antagonist is aprepitant or fosaprepitant. In some embodiments, the CFP comprises a sequenceWSGR Docket No. 56371-768.601 having at least 80% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16. In some embodiments, the nucleic acid comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17. In some embodiments, the RNA is expressed predominantly in myeloid cells in vivo. In some embodiments, the administration of the pharmaceutical composition does not generate a cytokine response. In some embodiments, the method comprises administering the pharmaceutical composition to the subject (i) for at least one treatment cycle; (ii) for at least 2, 3, 4, or 5 treatment cycles; (iii) about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about or once every 9 weeks in each of the treatment cycle; (iv) about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle; (v) between 1 to 12 times for each treatment cycle; and / or (vi) between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle. In some embodiments, a second cycle follows a first cycle. In some embodiments, the method further comprises administering the pharmaceutical composition to the subject during the second cycle. In some embodiments, the pharmaceutical composition is administered to the subject (i) about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, or about once every 9 weeks during the second cycle, (ii) from about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times in the second cycle, and / or (iii) 3 times in the second cycle. In some embodiments, an effective amount of the pharmaceutical composition ranges from 0.01 mg / kg / dose to 3.0 mg / kg / dose, from 0.05 mg / kg / dose to 2.5 mg / kg / dose, or from 0.1 mg / kg / dose to 1.0 mg / kg / dose. In some embodiments, an effective amount of the pharmaceutical composition ranges from about 0.001 to about 0.0015, about 0.0015 to about 0.002, about 0.002 to about 0.0025, about 0.0025 to about 0.003, about 0.003 to about 0.0035, about 0.0035 to about 0.004, about 0.004 to about 0.0045, about 0.0045 to about 0.005, about 0.005 to about 0.0055, about 0.0055 to about 0.006, about 0.006 to about 0.0065, about 0.0065 to about 0.007, about 0.007 to about 0.0075, about 0.0075 to about 0.008, about 0.008 to about 0.0085, about 0.0085 to about 0.009, about 0.009 to about 0.0095, or about 0.0095 to about 0.01 mg / kg of the RNA. In some embodiments, the effective amount of the pharmaceutical composition is administered in a 60- minute intravenous (IV) infusion per dose. In some embodiments, the effective amount of the pharmaceutical composition is administered (i) for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IV infusion doses, (ii) at an interval of once in a week, (iii) at an interval of once every 10 days, (iv) at an interval of once every 2 weeks, and / or (v) for at least 5 doses. In some embodiments, the cancerWSGR Docket No. 56371-768.601 is selected from the group consisting of cervical cancer, colorectal cancer, esophageal, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, and urothelial cancer. In some embodiments, the nucleic acid comprising a sequence encoding a CFP comprises a sequence with at least 80% sequence identity of SEQ ID NO: 4, 5, or 34, and wherein the CFP comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the CFP comprises an amino acid sequence of SEQ ID NO: 179. In some embodiments, the nucleic acid comprises a sequence encoding a CFP comprises a sequence of SEQ ID NO: 4, 5, or 34. In some embodiments, the nucleic acid comprises a sequence encoding a CFP comprises a sequence with at least 80% sequence identity of SEQ ID NO: 14, 16, 33, or 36. In some embodiments, the nucleic acid comprises a sequence encoding a CFP comprises a sequence of SEQ ID NO: 14, 16, 33, or 36.

[0022] Also provided herein is a combination therapeutic kit for treating a cancer in a subject in need thereof, comprising a first therapeutic agent and at least one additional therapeutic agent, wherein the first therapeutic agent comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain; and wherein the at least one additional therapeutic agent comprises (i) a prophylactic agent or (ii) a chemotherapeutic agent and at least one of (a) a tumor target drug or (b) an immune checkpoint inhibitor.

[0023] Also provided herein is a combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) nivolumab.

[0024] Also provided herein is a combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) pembrolizumab.

[0025] Also provided herein is a combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) fluoropyrimidine and cisplatin, and (iii) pembrolizumab.

[0026] Also provided herein is a combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) fluoropyrimidine and cisplatin, and (iii) nivolumab.

[0027] Also provided herein is a combination therapeutic kit for treating a HER2-positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) trastuzumab.WSGR Docket No. 56371-768.601

[0028] Also provided herein is a combination therapeutic kit for treating a HER-2 positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) trastuzumab.

[0029] Also provided herein is a combination therapeutic kit for treating a cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) tocilizumab and anakinra.INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0032] FIG. 1 depicts an illustration of the structure and myeloid cell targeted expression of a chimeric fusion (CFP) receptor that comprises (i) an extracellular domain comprising a binding domain (e.g., an scFv) against a cancer cell surface antigen, (ii) a transmembrane domain that multimerizes with an endogenously expressed myeloid cell specific transmembrane protein, e.g., Fc receptor common gamma chain and is therefore expressed solely in cells that endogenously express a Fc receptor common gamma chain, for example, in vivo, a myeloid cell; and (iii) one or more intracellular signaling domains. The chimeric fusion protein is also designated in the figure as Fea fusion construct, as the exemplary transmembrane domain of the chimeric fusion protein is an Fea transmembrane domain. An armed Fea construct as depicted in the figure is one that is expressed in a myeloid cell, and has engaged in multimerization with an endogenous Fc gamma transmembrane receptor, and therefore functionalized (e.g., armed). The figure also depicts that a recombinant polynucleotide (e.g., mRNA) construct encoding the CFP having the structural features described, is designed for in vivo delivery encapsulated in a lipid nanoparticle (LNP), which upon entry into a myeloid cell is expressed on the surface of the myeloid cell via multimerization with the endogenous Fc gamma transmembrane receptor, is functionalized and is armed to kill a tumor cell via CFP receptor activation and phagocytosis of the tumor cell by the myeloid cell expressing the CFP. However, when taken up by a cell in vivo that does not expressWSGR Docket No. 56371-768.601 an endogenous Fc gamma transmembrane receptor (for example a T cell), the construct will not be expressed on the membrane and not functional.

[0033] FIG. 2 depicts the different CFP constructs for in vivo delivery, each comprising a cancer cell-specific extracellular antigen binding domain (ECD, e.g., an scFV against a target cancer antigen), the CD89 TMD and one or more intracellular signaling domains which constitute next generation in vivo receptors that incorporate additional signaling domains e.g., for potentiating the activation of the myeloid cell that expresses the CFP for active phagocytosis and killing of the tumor cell targeted by the ECD of the CFP. The intracellular signaling domains are FcR gamma (FcRg) intracellular signaling domains, PI3 kinase recruitment domains (PI3K), TRIF intracellular signaling domain (TRIF) and CD40 intracellular domain (CD40), or parts thereof, and combinations of these, and shown following the arrow. Collectively, these constructs, comprising the types of transmembrane domain capable of multimerization with a cell-specific endogenous protein for expression and function in a specific cell type and further comprising intracellular domain(s) or combinations of intracellular domains that potentiate intracellular signaling of the cell that expresses the CFP and activate efficient phagocytosis and killing of a target cell, are often designated throughout the disclosure as second generation CFP constructs (or, 2ndgen or variations thereof).

[0034] FIG. 3, top panel depicts diagrammatic view of the different CFP designs as transmembrane proteins being developed and tested for expression and activity in myeloid cells, for example in a monocyte cells, shown here are positions of the CFPs expressing on the cell membrane (e.g., on lipid bilayer shown in a section of a cell). FIG. 3, bottom panel shows data of expression of each of the CFP constructs in a monocyte cell line. These data show that the CFPs with additional intracellular signaling domains (e.g., FcR, CD40, FcR-PI3K, TRIF and FcR-TRIF) are well tolerated and they express well.

[0035] FIGs. 4A and 4B depict pro-inflammatory cytokine and chemokine production by transfected monocyte cells expressing the indicated next generation receptors (2ndGen) for in vivo expression. Data shows side by side comparison of the first generation (1stGen) of cellspecific CFP constructs that lack the intracellular signaling domains, with second generation CFP constructs in cell activation and the generation of IL-12p70 and IFN cytokines.

[0036] FIG. 5A shows data indicating cr-globin 3’UTR with four miR122-5p binding sites suppresses mRNA expression in hepatocytes (Huh-7 cells) but not in primary monocytes.

[0037] FIG. 5B shows data indicating cr-globin 3’UTR with four miR192-5p binding sites suppresses mRNA expression in hepatocytes but not in primary monocytes.

[0038] FIG. 5C shows data indicating cr-globin 3’UTR with four miR192-3p binding sites, no significant change was observed with and without the miRNA.WSGR Docket No. 56371-768.601

[0039] FIG. 5D shows data indicating cr-globin 3’UTR with four miR27b-5p binding sites prolongs mRNA expression specifically in primary monocytes.

[0040] FIG. 5E shows data indicating cr-globin 3’UTR with four miR27b-3p binding sites suppresses mRNA expression in both hepatocytes and primary monocytes (no cells specificity).

[0041] FIG. 5F shows data indicating cr-globin 3’UTR with four miR10a-5p binding sites. No significant effect with and without the miRNA was observed.

[0042] FIG. 5G shows cr-globin 3’UTR with four miR10a-3p binding sites. No significant effect with and without the miRNA was observed.

[0043] FIG. 6 shows purity and polyA length for the mRNAs by electrophoresis. bGH UTR: an mRNA encoding the TROP2 CFP with bGH 3’ UTR. agiobin UTR: an mRNA encoding the TROP2 CFP with a globin 3’ UTR. miR27b UTR: an mRNA encoding the TROP2 CFP with a globin 3’ UTR having miR27b binding sites.

[0044] FIG. 7A shows the flow cytometry result demonstrating the expression levels of the CFP construct comprising a TROP2 binder that was introduced to primary monocytes by electroporation.

[0045] FIG. 7B shows the changes of the mean fluorescence intensity (MFI) of the TROP2 binder on the CAR construct in FIG. 7A.

[0046] FIGs. 8A-8E depict exemplary CFP RNA constructs that have undergone codon optimization. FIG. 8A, depicts an exemplary CFP construct with a bGH 3’UTR (Construct #10). FIG. 8B depicts an exemplary CFP construct which was designed using X- 10 (L10) codon optimization and comprises a cr-globin 3’UTR (Construct #11). FIG. 8C depicts an exemplary CFP construct which was designed using L10 codon optimization and comprises an cr-globin 3’UTR with four miR27b binding sites (Construct #12). FIG. 8D depicts an exemplary CFP construct which was designed using a PreferredCodon (PC) codon optimization and comprises a cr-globin 3’UTR (Construct #13). FIG. 8E depicts an exemplary CFP construct which was designed using a PC codon optimization and comprises a cr-globin 3’UTR with four miR27b binding sites (Construct #14).

[0047] FIG. 9 depicts an electrophoresis result characterizing the constructs described in FIGs. 8A-8E. From left to right, each lane represents ladder, Construct #11, Construct #12, Construct #13, Construct #14, and Construct #10.

[0048] FIGs. 10A-10C depicts flow cytometry results measuring CFP expression in primary monocytes electroporated with the constructs of FIGs. 8A-8E. FIG. 10A depicts results for mock electroporated cells (left) and the construct shown in FIG. 8A (right). FIG. 10B depicts results for constructs that were L10 codon optimized. FIG. 10C depicts results for constructs that were PC codon optimized.WSGR Docket No. 56371-768.601

[0049] FIG. 11 is a quantification of the mean fluorescence intensity (MFI) of the results obtained in FIGs. 10A-10C.

[0050] FIGs. 12A-12C depict schematic diagram of clinical trial protocol, dosing regimens for Cohorts 1-3, Cohort 4 and Cohorts 5-7 respectively.

[0051] FIG. 13 depicts BOIN design decision tree.

[0052] FIG. 14 depicts a schematic diagram for combination therapy clinical trial protocol.

[0053] FIGs. 15A-15C depict graphic representation of planned dosing schedules for anti- TROP2 CFP-encoding nucleic acid in combination with one or more additional therapy. FIG. 15A depicts the dosing schedule for Cohort 1, showing 3 cycles- Cycle 1, Cycle 2 and Cycle 3. During Cycle 1, subjects receive DP (anti-TROP2 CFP-encoding nucleic acid formulation) on days 1 and 8, day 1 being the first day of administration of the therapy; they receive oxaliplatin on day 8, and capecitabine on days 8 and 15. During Cycle 2, subjects receive DP on day 1, oxaliplatin on day 1, and capecitabine on days 1 and 8. During Cycle 3, subjects receive DP on day 1, oxaliplatin on day 1, and capecitabine on days 1 and 8 respectively. As indicated, Cycle 1 includes a monotherapy dose. FIG. 15B depicts Cohort 2 dosing schedule for a combination therapy comprising the DP (anti- TROP2 CFP-encoding nucleic acid formulation), Capox and Nivolumab. During Cycle 1, subjects receive DP (anti- TROP2 CFP-encoding nucleic acid formulation) on days 1 and 8, oxaliplatin and nivolumab on day 8, and capecitabine on days 8 and 15. During Cycle 2, subjects receive DP on day 1, oxaliplatin and nivolumab on day 1, and capecitabine on days 1 and 8. During Cycle 3, subjects receive DP on day 1, oxaliplatin and nivolumab on day 1, and capecitabine on days 1 and 8 respectively. FIG. 15C depicts Cohort 3 dosing schedule for a combination therapy comprising the DP (anti- TROP2 CFP-encoding nucleic acid formulation), CapOx and Trastuzumab. During Cycle 1, subjects receive DP (anti- TROP2 CFP-encoding nucleic acid formulation) on days 1 and 8, oxaliplatin and trastuzumab on day 8, and capecitabine on days 8 and 15. During Cycle 2, subjects receive DP on day 1, oxaliplatin and trastuzumab on day 1, and capecitabine on days 1 and 8. During Cycle 3, subjects receive DP on day 1, oxaliplatin and trastuzumab on day 1, and capecitabine on days 1 and 8 respectively.

[0054] FIGs. 16A-16B show data characterizing the metabolism of lipid components in subjects receiving LNPs containing the anti-TROP2 CFP-encoding nucleic acid formulation. FIG. 16A shows metabolism of cationic lipids. FIG. 16B shows metabolism of PEG lipid components.

[0055] FIG. 17 shows data indicating IFNy and CXCL10 concentration-time profiles by dose level. (N=19). Meso Scale Discovery (MSD) analysis of serum cytokines and chemokines in samples from 19 participants demonstrated robust pharmacodynamic activity. As shown in the figure, each infusion triggered transient, dose-dependent increase in the Thl effector cytokineWSGR Docket No. 56371-768.601IFN-y and the chemokine C-X-C motif chemokine ligand 10 (CXCL10, also known as interferon gamma-induced protein 10 [IP- 10]) — both key mediators of adaptive anti -tumor immunity. The repeat increases observed after every dose, together with peak-activity-to-dose relationship, provides causal relationship and confidence of the outcome.

[0056] FIG. 18 shows data indicating intratumoral CD3, HLA-DR, and HLA-ABC levels at screening vs. Day 31. (N=8). CD3, cluster of differentiation; HLA-ABC, human leucocytic antigen (HLA-A, HLA-B and HLA-C isotypes). SCR, screening. Hyperion proteomic imaging data shows that the drug induces T cell accumulation into tumor (left), while increasing antigen presenting cells evidenced by increase in HLA-DR (middle graph) and HLA-ABCs (right graph) in paired tumor biopsies.

[0057] FIG. 19 depicts an exemplary construct for in vivo delivery comprising a cancer cellspecific extracellular antigen binding domain comprising an anti-TROP2 domain operatively linked to a CD89 transmembrane and cytoplasmic domain.

[0058] FIG. 20 depicts inhibition of tumor growth in mice following intravenous administration of mRNA-LNP composition, wherein the mRNA encodes an TROP2 CAR comprising a CD89 transmembrane domain.

[0059] FIG. 21 depicts expression of the TROP2 CAR in tumors following the intravenous administration of the mRNA-LNP composition in FIG. 20.

[0060] FIGS. 22A-22C depict treatment with the mRNA-LNP composition encoding an TROP2 CAR comprising a CD89 transmembrane domain resulted in increased activation and cytolytic activity of CD8+ / T cells in tumors in mice. FIG. 22A shows dot plots and bar graph depicting activation and exhaustion (TIM3 / PD-1). FIG. 22B shows dot plots and bar graph depicting proliferation (Ki-67). FIG. 22C shows dot plots and bar graph depicting cytolytic activity (Granzyme B).

[0061] FIGS. 23A-23D depict expression of an TROP2 CAR in myeloid cells form whole blood following intravenous infusion of Test composition in a TROP-2+ HCC-1954 subcutaneous xenograft model in NCG Mice. FIG. 23A depicts the total Ly6C+ cells; FIG. 23B depicts Ly6C+ CD1 lb+ cells; FIG. 23C depicts Ly6C+ CD1 lc+ cells; and FIG. 23D depicts Ly6G+ cells.

[0062] FIG. 24 depicts mean tumor volume following administration of the mRNA-LNP compositions in a TROP-2+ HCC-1954 subcutaneous xenograft model in NSG mice.

[0063] FIG. 25 depicts the frequency of TROP2 CARs in monocytes after intravenous infusion of the compositions in Cynomolgus monkeys.

[0064] FIGs. 26A-26E depicts CAR expression in various cell types following electroporation of mRNA encoding the CAR. TROP2 CAR was delivered to primary human monocytes via mRNAWSGR Docket No. 56371-768.601 electroporation and CAR expression was measured using flow cytometry (FIG. 26A). Data shown are representative of two different donors. Association of TROP2 CAR with endogenous FcRy was detected by co-immunoprecipitation (FIG. 26B). Western Blot was performed against the FcRy chain with aGAPDH as loading control. Lower panel demonstrates expression level and duration of TROP2 CAR are influenced by the presence of FcRy. TROP2 CAR mRNA was also transfected into Jurkat (FIG. 26C), Huh7 (FIG. 26D), or HEK293T cells (FIG. 26E) with or without co-transfection of FcRy mRNA. Expression of TROP2 CAR was measured using flow cytometry over 3 days.

[0065] FIG. 26F shows flow cytometry dot plots demonstrating expression levels of TROP2 CAR over 7 days post electroporation.

[0066] FIG. 27A shows data demonstrating TROP2 CAR induced NF-KB (left) and IFN-I (right) pathways activation as measured in the THPl-Dual™ reporter cell. Stimulation with the TLR4 agonist LPS or cGAS agonist 2’3’-cGAMP are shown as positive control. Statistical analysis was performed using two-way ANOVA.

[0067] FIG. 27B shows experimental data that FcRy is required for TROP2 CAR activity. Top panel shows expression levels of TROP2 CAR in THPl-Dual and Jurkat-Dual reporter cells 24 h post electroporation. TROP2 CAR did not induce NF-K and IFN-I pathways activation in Jurkat-Dual™ reporter cell which lacks FcRy (bottom panel). Stimulation with cytokines including TNFcr or IFNP are shown as positive control. Statistical analysis was performed using two-way ANOVA.

[0068] FIG. 27C shows expression and activity of TROP2 CAR. Top panel shows expression levels of TROP2 CAR in different immune cell subsets in PBMC 24h post electroporation. Bottom, co-culture of PBMCs electroporated with TROP2 CAR with SKOV3 tumor cells resulted in elevated tumor killing compared to mock electroporated PBMCs. Statistical analysis was performed using t-test.

[0069] FIG. 28 depicts data demonstrating production of pro-inflammatory cytokines by PBMCs electroporated with CAR in presence of SKOV3 tumor cells. Co-culture of PBMCs electroporated with TROP2 CAR with SKOV3 tumor cells resulted in production of pro- inflammatory cytokines and chemokines. Data shown are mean ± SD. Statistical comparison with p value <0.05 are labeled.

[0070] FIG. 29A shows in vivo CAR expression data in immune competent mice resulting from TROP2 CAR mRNA product infusion. Non-tumor bearing C57BL / 6 mice were given a single i.v. injection of TROP2 CAR mRNA product at 0.5 mg / kg or 1 mg / kg. Blood, spleen, bone marrow and liver were collected from treated mice at 6 hours post-infusion. Single cell suspensions were prepared and stained for CAR expression among immune subsets (T cells, BWSGR Docket No. 56371-768.601 cells, NK cells, Neutrophils, Monocytes, DCs) by FACS. Data shown were average and STD of each group (n=3 mice per group).

[0071] FIG. 29B shows in vivo CAR expression data in non-human primate resulting from TROP2 CAR mRNA product infusion. Naive cynomolgus monkeys were infused with TROP2 CAR mRNA product (i.v.) (0.5, or 1 mg / kg) for Ih. Blood was collected 12 hours after infusion and TROP2 CAR surface expression of different immune populations of PBMCs was analyzed by FACS. Data shown were average and STD of each group (10 monkeys (5 females and 5 males).

[0072] FIG. 29C shows CAR expression data in human blood resulting from in vitro transfection. Whole blood from healthy human donors was incubated with 125 pg / mL of TROP2 CAR mRNA product for 3 hours in presence of ApoE3 (at 1.5 mg / mL). Following RBC lysis and additional 3 hours culture, surface TROP-2 CAR expression was assessed by FACS for T cells, B cell, NK cells, Granulocytes and Monocytes. Data shown were average and STD of each group (n=3 donors).

[0073] FIG. 30 depicts data on effect of TROP2 CAR on TROP2-positive (TROP2+) xenograft models at Q4D dosing scheme. Top, the scheme of the in vivo study testing TROP2 CAR with an injection every 4 days. NCG mice with established HCC-1954 tumor (TROP2+, s.c.) were treated with TROP2 CAR mRNA product (1 mg / kg / dose, total 5 doses) or PBS every 4 days (Q4D). Tumor growth was monitored by caliper measurement twice a week. Bottom, data showing change in tumor volume over days post infection. Data shown were average and STD of each group (n= 5 mice per group).

[0074] FIG. 31 depicts CAR expression detected in monocytes in vivo following TROP2 CAR infusion. NCG mice with established HCC-1954 tumor (tumor volume approximately 500 mm3) were dosed i.v. with TROP2 CAR (1 mg / kg) or PBS. Six hours post infusion, blood, spleen, bone marrow and tumor were harvested. Single cell suspensions were prepared from collected tissues and stained for CAR expression. Data shown were average and STD of each group (n= 3 mice per group) for monocytes (CD45+Ly6C+ cells).

[0075] FIG. 32A depicts data on effect of TROP2 CAR on TROP2-positive (TROP2+) xenograft models at Q2W dosing scheme. Top, scheme of the in vivo study testing TROP2 CAR mRNA product with an injection every 2 weeks (Q2W). Bottom, Anti -tumor efficacy of biweekly injection of TROP2 CAR mRNA product in vivo. NCG mice with established HCC-1954 tumor (TROP2+, s.c.) were treated with TROP2 CAR mRNA product (1 mg / kg / dose, total 3 doses) or PBS every 2 weeks (Q2W). Tumor growth was monitored by caliper measurement twice a week. Data shown were average and STD of each group (n= 5 mice per group).WSGR Docket No. 56371-768.601

[0076] FIG. 32B shows change in body weight of tumor bearing mice treated with TROP2 CAR mRNA-LNP. NCG mice bearing HCC-1954 tumors were treated with vehicle, empty LNP or TROP2 CAR as described above. The body weight changes (as percentage to the baseline bodyweight right before treatments) were shown for animals treated with TROP2 CAR every 4 days (left) or every 2 weeks (right). Data shown were average and STD of each group (n= 5 mice per group).

[0077] FIG. 33 shows data indicating gp75 CAR mRNA / LNP shows anti-tumor activity in the B16 syngeneic melanoma model and remodels the TMEs. The data demonstrates anti -tumor efficacy of surrogate gp75 CAR mRNA / LNP in syngeneic mouse melanoma model. C57BL / 6 mice were inoculated s.c. with B16-F10 / OVA tumor cells on day 0. Upon tumor establishment, PBS (vehicle), empty LNP or gp75 CAR mRNA / LNP were injected i.v. at 2 mg / kg / dose every 2 days. Data shown are mean ± SEM (n=6).

[0078] FIG. 34 shows flow cytometry analysis of CD8+ T cell phenotypes in tumors 24 h post 4th dose. Treatment with gp75 CAR mRNA / LNP significantly increased the frequency of proliferating (CD8+ Ki67+) and cytolytic (Granzyme B+) CD8+ T cells, and simultaneously reduced the percentage of PDlhlTOX+ exhausted T cells. Statistical analysis was performed using two-way ANOVA. Treatment with gp75 CAR mRNA / LNP reduced the percentage of memory CD44+ CD8+ T cells with exhaustion phenotype indicated by high PD1 expression. Statistical analysis was performed using two-way ANOVA. Treatment with gp75 LNP significantly increased activated dendritic cells (CD40+ CD86+). Statistical analysis was performed using an unpaired t-test.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0079] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0080] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.WSGR Docket No. 56371-768.601

[0081] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0082] “Percent (%) identity” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment tools online can be used for calculating the percentage (%) identity, for example, Blast from NCBI.

[0083] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong. Although any recombinant polypeptides, methods and other aspects similar or equivalent to those described herein can also be used in the practice or testing of the recombinant polypeptides, methods and other aspects, representative illustrative recombinant polypeptides, methods and other aspects are now described.

[0085] As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0086] As used herein, a “therapeutically effective amount” or “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of an agent means anWSGR Docket No. 56371-768.601 amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

[0087] As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0088] A “myeloid cell” can refer broadly to cells of the myeloid lineage of the hematopoietic cell system, and can exclude, for example, the lymphocytic lineage. Myeloid cells comprise, for example, cells of the granulocyte lineage and monocyte lineages. Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of cellular ontogeny, activation, differentiation, and tissue-specific functions of myeloid cells have been revisited during the last years with surprising results. However, their enormous plasticity and heterogeneity, during both homeostasis and disease, are far from understood. Although myeloid cells have many functions, including phagocytosis and their ability to activate T cells, harnessing these functions for therapeutic uses has remained elusive. Newer avenues are therefore sought for using other cell types towards development of improved therapeutics, including but not limited to T cell malignancies.

[0089] Myeloid cells are typically differentiated from common progenitors derived from the hematopoietic stem cells in the bone marrow. Commitment to myeloid cell lineages may be governed by activation of distinct transcription factors, and accordingly myeloid cells may be characterized as cells having a level of plasticity, which may be described as the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. MyeloidWSGR Docket No. 56371-768.601 cells can be rapidly recruited into local tissues via various chemokine receptors on their surface. Myeloid cells are responsive to various cytokines and chemokines.

[0090] A myeloid cell, for example, may be a cell that originates in the bone marrow from a hematopoietic stem cell under the influence of one or more cytokines and chemokines, such as G-CSF, GM-CSF, Flt3L, CCL2, VEGF and S100A8 / 9. In some embodiments, the myeloid cell is a precursor cell. In some embodiments, the myeloid cell may be a cell having characteristics of a common myeloid progenitor, or a granulocyte progenitor, a myeloblast cell, or a monocyte- dendritic cell progenitor or a combination thereof. A myeloid can include a granulocyte or a monocyte or a precursor cell thereof. A myeloid can include an immature granulocyte, an immature monocyte, an immature macrophage, an immature neutrophil, and an immature dendritic cell. A myeloid can include a monocyte or a pre-m onocytic cell or a monocyte precursor. In some cases, a myeloid cell as used herein may refer to a monocyte having an MO phenotype, an Ml phenotype or an M2 phenotype. A myeloid can include a dendritic cell (DC), a mature DC, a monocyte derived DC, a plasmacytoid DC, a pre-dendritic cell, or a precursor of a DC. A myeloid can include a neutrophil, which may be a mature neutrophil, a neutrophil precursor, or a polymorphonucleocyte (PMN). A myeloid can include a macrophage, a monocyte-derived macrophage, a tissue macrophage, a macrophage of an MO, an Ml or an M2 phenotype. A monocyte or a macrophage exhibit polarization. “Polarization” as used herein may refer to a process by which macrophages exhibit distinct functional phenotypes in response to specific microenvironmental stimuli and signals, often referred to as physiological states. In some cases, macrophages can pass from one polarization state to another. For example, macrophages can be polarized into classically activated (Ml) and alternatively activated (M2) macrophages. M2 macrophages are divided into M2a, M2b, M2c, and M2d subcategories. These macrophages differ in their cell surface markers, secreted cytokines and biological functions. Ml macrophages are typically characterized by phenotypes in which the cells express TLR-2, TLR-4, CD80, CD86, iNOS, and MHC-II on the surface. These cells release various cytokines and chemokines e.g., TNF-a, IL-la, IL-ip, IL-6, IL-12, CXCL9, and CXCL10, and typically exhibit activation of transcription factors, such as NF-kB, STAT1, STAT5, IRF3, and IRF5 that regulate the expression of Ml genes. It is believed that NF-KB and STAT1 are the two major pathways involved in Ml macrophage polarization. The Ml phenotype is associated with microbicidal and tumoricidal functions of macrophages, exhibiting high phagocytic and inflammatory function. On the other hand, tumor associated macrophages subject to immunosuppressive environment become generally more M2 polarized. A myeloid can include a tumor infiltrating monocyte (TIM). A myeloid can include a tumor associated monocyte (TAM). A myeloid can include a myeloid derived suppressor cell (MDSC). A myeloid can include a tissue resident macrophage. AWSGR Docket No. 56371-768.601 myeloid can include a tumor associated DC (TADC). Accordingly, a myeloid cell may express one or more cell surface markers, for example, CDl lb, CD14, CD15, CD16, CD38, CCR5, CD66, Lox-1, CDl lc, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CDlc, CD83, CD141, CD209, MHC-II, CD 123, CD303, CD304, a SIGLEC family protein and a CLEC family protein. In some cases, a myeloid cell may be characterized by a high or a low expression of one or more of cell surface markers, for example, CDl lb, CD14, CD15, CD16, CD66, Lox-1, CDl lc, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CDlc, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304 or a combination thereof. In one embodiment, activating the Ml polarization of macrophages are desirable using the methods described herein.

[0091] The term “cancer” refers to any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one nonlimiting example of a neoplasia or cancer. The terms “cancer” or “tumor” or “hyperproliferative disorder” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell.

[0092] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0093] Nucleic acid molecules useful in the methods of the disclosure include, but are not limited to, any nucleic acid molecule with activity or that encodes a polypeptide. A nucleic acid can be a DNA or an RNA. When a nucleic acid sequence is provided with thymidine (T), it is not necessary to indicate that the nucleic acid is a DNA; the nucleic acid can also be an RNA with the counterpart uracil (U) substituted for T, and vice versa.

[0094] Polynucleotides having substantial identity to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridize” refers to when nucleic acid molecules pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399;Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration canWSGR Docket No. 56371-768.601 ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions can ordinarily include temperatures of at least about 30° C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In an exemplary embodiment, hybridization can occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization can occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps can include a temperature of at least about 25°C, of at least about 42°C, or at least about 68°C. In exemplary embodiments, wash steps can occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In other exemplary embodiments, wash steps can occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, wash steps can occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.WSGR Docket No. 56371-768.601Engineered Chimeric Fusion Protein

[0095] In some aspects, the present disclosure describes an engineered chimeric fusion protein (CFP) and a nucleic acid sequence encoding the engineered chimeric fusion protein. In some cases, the nucleic acid sequence encoding the engineered CFP is codon optimized.

[0096] A CFP can comprise an extracellular domain (ECD) comprising an antigen binding domain that binds to an antigen of a target cell. The extracellular domain can be fused to a hinge domain or an extracellular domain derived from a receptor, such as CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor or an integrin receptor. The CFP can further comprise a transmembrane domain, such as a transmembrane domain derived from CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor or an integrin receptor. In some embodiments, a CFP further comprises an intracellular domain comprising an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor or an integrin receptor. For example, the intracellular domain can comprise one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor or an integrin receptor. For example, the intracellular domain can comprise one or more intracellular signaling domains that promote phagocytic activity, inflammatory response, nitric oxide production, integrin activation, enhanced effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and / or enhanced cross presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, a CFP encoded by the recombinant nucleic acid further comprises an intracellular domain comprising a recruitment domain. For example, the intracellular domain can comprise one or more PI3K recruitment domains, caspase recruitment domains or caspase activation and recruitment domains (CARDs).

[0097] In some embodiments, the CFP comprises (i) a transmembrane domain, (ii) an intracellular domain, and (iii) an extracellular domain comprising an antigen binding domain specific to an antigen, e.g., an antigen of or presented on a target cell; wherein the transmembrane domain and the antigen binding domain are operatively linked such that antigen binding to the target by the antigen binding domain of the fused receptor activated in the intracellular signaling domain of the intracellular domain.

[0098] In some embodiments, the transmembrane domain comprises a transmembrane domain from CD 16a, CD64, CD68 or CD89.

[0099] In some embodiments, the intracellular domain comprises an intracellular domain derived from a phagocytic receptor. In some embodiments, the intracellular domain comprises anWSGR Docket No. 56371-768.601 intracellular domain derived from a T cell receptor, such as a CD3 molecule, e.g., CD3(^. In some embodiments, the intracellular domain comprises an intracellular domain derived from a phagocytic receptor other than a phagocytic receptor selected from Megfl 0, MerTk, FcRa, or Bail . In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a receptor selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor Al (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3(^, CR3, CR4, Tim-1, Tim-4 and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular domain does not comprise a PI3K recruitment domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a scavenger receptor. In some embodiments, the intracellular domain comprises a CD47 inhibition domain. In some embodiments, the intracellular domain comprises a Rac inhibition domain, a Cdc42 inhibition domain or a GTPase inhibition domain. In some embodiments, the Rac inhibition domain, the Cdc42 inhibition domain or the GTPase inhibition domain inhibits Rac, Cdc42 or GTPase at a phagocytic cup of a cell expressing the PFP. In some embodiments, the intracellular domain comprises an F-actin disassembly activation domain, a ARHGAP12 activation domain, a ARHGAP25 activation domain or a SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibition domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibition domain. In some embodiments, the intracellular domain comprises at least one ITAM domain. In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain comprises at least one ITAM domain select from an ITAM domain of CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the at least one ITAM domain comprises a Src-family kinase phosphorylation site. In some embodiments, the at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprisesWSGR Docket No. 56371-768.601 an F-actin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0100] In some embodiments, the protein comprising the CFP further comprises a signal peptide, for example, a GMCSF signal peptide having a sequence of MWLQSLLLLGTVACSIS (SEQ ID NO: 1)

[0101] Additional exemplary CFPs and components thereof can be found in PCT / US2023 / 03352, filed on September 22, 2023, which is incorporated by reference in its entirety.TROP2-CFP

[0102] In some embodiments, the target protein is TROP2. In some embodiments, the extracellular domain of the CFP comprises an antigen binding domain capable of specifically binding a TROP2 antigen. In some embodiments, the CFP comprises an extracellular domain comprising an anti-TROP2 scFv, a CD89 transmembrane domain, and a CD89 intracellular domain.

[0103] In some cases, the CFP comprises an amino acid sequence ofMWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQA PGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSI TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPE DFAVYYCQQHYITPLTFGAGTKVEIKRGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAI LVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 2).

[0104] In some cases, the CFP comprises an amino acid sequence of MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQA PGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSI TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPE DFAVYYCQQHYITPLTFGAGTKVEIKRSGGGGAAAGSDSIHQDYTTQNLIRMAVAGLVL VALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 39).

[0105] In some cases, the CFP comprises an amino acid sequence of QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGWSGR Docket No. 56371-768.601AGTKVEIKRGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASA DVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 3).

[0106] In some cases, the CFP comprises an amino acid sequence ofQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSS (SEQ ID NO: 175)

[0107] In some cases, the CFP comprises an amino acid sequence ofDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPD RFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKR (SEQ ID NO: 176).

[0108] In some cases, the CFP comprises an amino acid sequence ofQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFG AGTKVEIKR (SEQ ID NO: 177).

[0109] In some cases, the CFP comprises an amino acid sequence ofDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQ PGLTFARTPSVCK (SEQ ID NO: 178).

[0110] In some cases, the CFP comprises a sequence ofQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFG AGTKVEIKRSGGGGAAAGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTAL NKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 179).[OHl] Additional exemplary TROP-CFPs and components thereof can be found in PCT / US2023 / 03352, filed on September 22, 2023, which is incorporated by reference in its entirety.Nucleic Acid Sequences Encoding TROP2-CFP

[0112] Disclosed herein are nucleic acids encoding the TROP2 targeting CFP. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a codon-optimized coding sequence for the TROP2 targeting CFP. In some embodiment, the nucleic acid encoding the TROP2 targeting CFP comprises a 5’ UTR. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR.WSGR Docket No. 56371-768.601

[0113] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a coding sequence of AUGUGGCUGCAGUCUCUGCUGCUGCUGGGAACAGUGGCCUGUAGCAUCUCUCAAG UGCAGCUGCAGCAGAGCGGCAGCGAGCUGAAAAAGCCCGGAGCCAGCGUGAAAGU GUCCUGCAAGGCUUCUGGCUACACAUUCACCAAUUACGGCAUGAACUGGGUCAAG CAGGCCCCUGGACAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCG AACCUACAUACACAGAUGACUUCAAGGGCAGAUUCGCCUUCAGCCUGGACACCAG CGUGUCCACCGCUUAUCUGCAGAUCAGCAGCCUGAAGGCCGACGAUACCGCCGUG UACUUUUGUGCCCGGGGCGGAUUUGGCUCUAGCUACUGGUACUUCGACGUGUGGG GCCAGGGCAGCCUGGUGACCGUGUCUAGCGGAGGCGGAGGAUCAGGUGGCGGUGG AUCUGGCGGUGGUGGCUCUGACAUCCAGCUGACACAGAGCCCAUCUAGCCUGAGC GCUAGCGUGGGCGACAGAGUGUCUAUUACCUGUAAAGCUUCUCAGGACGUGUCCA UCGCCGUCGCCUGGUAUCAGCAGAAGCCCGGCAAGGCCCCUAAGCUGCUGAUCUA CAGCGCCUCCUACAGAUACACCGGCGUGCCCGAUAGAUUCAGCGGAAGCGGCAGC GGAACAGAUUUUACCCUGACAAUCAGCAGCCUGCAGCCUGAGGACUUCGCCGUGU ACUACUGCCAGCAACACUACAUCACCCCUCUGACCUUCGGCGCCGGCACCAAGGU GGAAAUCAAGCGGUCAGGCGGCGGAGGAgcggccGCaGGCAGUGACUCCAUUCAUCA GGAUUAUACCACACAGAACCUCAUUCGGAUGGCAGUAGCAGGAUUGGUGCUGGU UGCAUUGCUCGCCAUACUCGUUGAGAACUGGCAUUCACACACCGCGCUGAAUAAG GAGGCCAGCGCCGAUGUGGCCGAACCUUCAUGGUCCCAACAAAUGUGUCAGCCCG GUCUUACUUUUGCGAGAACACCAUCAGUAUGCAAGUGA (SEQ ID NO: 4).

[0114] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity having an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, or about 85% to about 90% sequence identity to SEQ ID NO: 4.WSGR Docket No. 56371-768.601

[0115] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of AUGUGGCUGCAGUCUCUGCUGCUGCUGGGCACCGUGGCCUGCAGCAUCAGCCAGG UGCAGCUGCAGCAGAGCGGCUCCGAGCUGAAGAAGCCCGGAGCCAGCGUGAAGGU GAGCUGCAAGGCCAGCGGCUACACCUUCACCAACUACGGCAUGAACUGGGUGAAG CAGGCCCCUGGGCAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCG AGCCUACCUACACCGACGACUUCAAGGGCAGGUUCGCCUUCAGCCUGGACACCAG CGUGUCCACCGCCUACCUGCAGAUCAGCAGCCUGAAGGCCGACGACACCGCCGUG UACUUCUGCGCCAGGGGCGGCUUCGGCUCCUCCUACUGGUACUUCGAUGUGUGGG GCCAGGGCAGCCUGGUGACCGUGUCCUCCGGCGGAGGCGGCUCAGGGGGGGGGGG CUCUGGGGGCGGCGGGAGCGACAUCCAGCUGACCCAGAGCCCCUCCUCCCUGAGC GCCUCCGUGGGGGACAGGGUGUCCAUCACAUGCAAGGCCAGCCAGGACGUGAGCA UCGCCGUGGCCUGGUACCAGCAGAAGCCCGGCAAGGCCCCCAAGCUGCUGAUCUA CAGCGCCAGCUACCGCUACACCGGCGUGCCCGACCGGUUUAGCGGCAGCGGCUCC GGCACCGACUUCACCCUGACCAUCAGCAGCCUGCAGCCCGAGGACUUUGCCGUGU ACUACUGCCAGCAGCACUACAUCACCCCCCUGACCUUCGGCGCUGGCACCAAGGU GGAGAUCAAGCGGAGCGGCGGCGGCGGCGCCGCCGCCGGCUCCGACAGCAUCCAC CAGGACUACACCACACAGAACCUGAUCCGGAUGGCCGUGGCCGGGCUGGUGCUGG UGGCCCUGCUGGCCAUCCUGGUGGAGAACUGGCACUCCCACACCGCCCUGAACAA GGAGGCCUCCGCCGACGUGGCCGAGCCCAGCUGGAGCCAGCAGAUGUGCCAGCCC GGCCUGACCUUCGCCCGGACCCCCUCUGUGUGCAAGUGA (SEQ ID NO: 5).

[0116] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity having an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 5.WSGR Docket No. 56371-768.601

[0117] The nucleic acid encoding the TROP2 targeting CFP can comprise a 5’ UTR. In some embodiments, the 5’ UTR comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a sequence of GCCACC (SEQ ID NO: 6). In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 5’ UTR comprising a sequence of GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACC (SEQ ID NO: 7). In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 5’ UTR comprising a sequence with at least 90% or at least 95% sequence identity to SEQ ID NO: 7.

[0118] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 5’ UTR comprising a sequence of AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACC (SEQ ID NO: 8). In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 5’ UTR comprising a sequence with at least 90% or at least 95% sequence identity to SEQ ID NO: 8.

[0119] In some embodiments, the 5’UTR comprises a Cap 1.

[0120] The nucleic acid encoding the TROP2 targeting CFP can comprise a 3’ UTR. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR comprising a sequence of CUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUA GUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAGG UGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUG AGUAGGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAG GAUUGGGAAGACAAUAGC (SEQ ID NO: 9). In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR comprising a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9.

[0121] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR comprising a sequence of CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG UCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 10). In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR comprising a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10.

[0122] In some embodiments, the 3’ UTR comprises a microRNA binding site. In some embodiments, the 3’ UTR comprises at least two microRNA binding sites. In some embodiments, the 3’ UTR comprises two, three, four, or five microRNA binding sites. In someWSGR Docket No. 56371-768.601 embodiments, the microRNA binding sites on the 3’ UTR are the same sequence. In some embodiments, the microRNA binding sites on the 3’ UTR are different sequences.

[0123] In some embodiments, the microRNA binding site comprises a sequence for specifically binding to miR-27b. In some embodiments, the exemplary human microRNA is miR-27b (NR_029665) 97 bp is:ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGUUUCC GCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGAAGGUG (SEQ ID NO: 11). In some embodiments, the exemplary human microRNA miR-lOa (NR 029608) 110 bp RNA is: GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAAUUUGUGUAAGGAAUUU UGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAACACUCCGCU CU (SEQ ID NO: 25). In some embodiments, the miR-27b binding site comprises a sequence of UCACCAAUCAGCUAAGCUCU (SEQ ID NO: 12). In some embodiments, the 3’ UTR comprises two, three, four, or five copies of SEQ ID NO: 12.

[0124] In some embodiments, the miR-27b binding site comprises a sequence of UUCACCAAUCAGCUAAGCUCU (SEQ ID NO: 37). In some embodiments, the 3’ UTR comprises two, three, four, or five copies of SEQ ID NO: 37.

[0125] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a 3’ UTR comprising a sequence of CUCGAGUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUUCACCAAUCAGCUAAGCUCUCG AUUUCACCAAUCAGCUAAGCUCUUCUAGAUUCACCAAUCAGCUAAGCUCUUCACU UCACCAAUCAGCUAAGCUCUGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAA GACAAUAGC (miR-27b binding site underlined) (SEQ ID NO: 13).

[0126] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCAUGUGGCUGCAG UCUCUGCUGCUGCUGGGAACAGUGGCCUGUAGCAUCUCUCAAGUGCAGCUGCAGC AGAGCGGCAGCGAGCUGAAAAAGCCCGGAGCCAGCGUGAAAGUGUCCUGCAAGGC UUCUGGCUACACAUUCACCAAUUACGGCAUGAACUGGGUCAAGCAGGCCCCUGGA CAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCGAACCUACAUACA CAGAUGACUUCAAGGGCAGAUUCGCCUUCAGCCUGGACACCAGCGUGUCCACCGC UUAUCUGCAGAUCAGCAGCCUGAAGGCCGACGAUACCGCCGUGUACUUUUGUGCC CGGGGCGGAUUUGGCUCUAGCUACUGGUACUUCGACGUGUGGGGCCAGGGCAGCC UGGUGACCGUGUCUAGCGGAGGCGGAGGAUCAGGUGGCGGUGGAUCUGGCGGUG GUGGCUCUGACAUCCAGCUGACACAGAGCCCAUCUAGCCUGAGCGCUAGCGUGGGWSGR Docket No. 56371-768.601CGACAGAGUGUCUAUUACCUGUAAAGCUUCUCAGGACGUGUCCAUCGCCGUCGCC UGGUAUCAGCAGAAGCCCGGCAAGGCCCCUAAGCUGCUGAUCUACAGCGCCUCCU ACAGAUACACCGGCGUGCCCGAUAGAUUCAGCGGAAGCGGCAGCGGAACAGAUUU UACCCUGACAAUCAGCAGCCUGCAGCCUGAGGACUUCGCCGUGUACUACUGCCAG CAACACUACAUCACCCCUCUGACCUUCGGCGCCGGCACCAAGGUGGAAAUCAAGC GGUCAGGCGGCGGAGGAgcggccGCaGGCAGUGACUCCAUUCAUCAGGAUUAUACCA CACAGAACCUCAUUCGGAUGGCAGUAGCAGGAUUGGUGCUGGUUGCAUUGCUCGC CAUACUCGUUGAGAACUGGCAUUCACACACCGCGCUGAAUAAGGAGGCCAGCGCC GAUGUGGCCGAACCUUCAUGGUCCCAACAAAUGUGUCAGCCCGGUCUUACUUUUG CGAGAACACCAUCAGUAUGCAAGUGACUCGAGUCUAGAGGGCCCGUUUAAACCCG CUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCC CCCGUGCCUUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAA AUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCUGGGGGGUG GGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (SEQ ID NO: 14)

[0127] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 14.

[0128] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCAUGUGGCUGCAG UCUCUGCUGCUGCUGGGCACCGUGGCCUGCAGCAUCAGCCAGGUGCAGCUGCAGC AGAGCGGCUCCGAGCUGAAGAAGCCCGGAGCCAGCGUGAAGGUGAGCUGCAAGGC CAGCGGCUACACCUUCACCAACUACGGCAUGAACUGGGUGAAGCAGGCCCCUGGG CAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCGAGCCUACCUACAWSGR Docket No. 56371-768.601CCGACGACUUCAAGGGCAGGUUCGCCUUCAGCCUGGACACCAGCGUGUCCACCGC CUACCUGCAGAUCAGCAGCCUGAAGGCCGACGACACCGCCGUGUACUUCUGCGCC AGGGGCGGCUUCGGCUCCUCCUACUGGUACUUCGAUGUGUGGGGCCAGGGCAGCC UGGUGACCGUGUCCUCCGGCGGAGGCGGCUCAGGGGGGGGGGGCUCUGGGGGCGG CGGGAGCGACAUCCAGCUGACCCAGAGCCCCUCCUCCCUGAGCGCCUCCGUGGGG GACAGGGUGUCCAUCACAUGCAAGGCCAGCCAGGACGUGAGCAUCGCCGUGGCCU GGUACCAGCAGAAGCCCGGCAAGGCCCCCAAGCUGCUGAUCUACAGCGCCAGCUA CCGCUACACCGGCGUGCCCGACCGGUUUAGCGGCAGCGGCUCCGGCACCGACUUC ACCCUGACCAUCAGCAGCCUGCAGCCCGAGGACUUUGCCGUGUACUACUGCCAGC AGCACUACAUCACCCCCCUGACCUUCGGCGCUGGCACCAAGGUGGAGAUCAAGCG GAGCGGCGGCGGCGGCGCCGCCGCCGGCUCCGACAGCAUCCACCAGGACUACACC ACACAGAACCUGAUCCGGAUGGCCGUGGCCGGGCUGGUGCUGGUGGCCCUGCUGG CCAUCCUGGUGGAGAACUGGCACUCCCACACCGCCCUGAACAAGGAGGCCUCCGC CGACGUGGCCGAGCCCAGCUGGAGCCAGCAGAUGUGCCAGCCCGGCCUGACCUUC GCCCGGACCCCCUCUGUGUGCAAGUGACUCGAGUGAUAAUAGGCUGGAGCCUCGG UGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCAC CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 15)

[0129] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 15.

[0130] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCAUGUGGCUGCAG UCUCUGCUGCUGCUGGGCACCGUGGCCUGCAGCAUCAGCCAGGUGCAGCUGCAGCWSGR Docket No. 56371-768.601AGAGCGGCUCCGAGCUGAAGAAGCCCGGAGCCAGCGUGAAGGUGAGCUGCAAGGC CAGCGGCUACACCUUCACCAACUACGGCAUGAACUGGGUGAAGCAGGCCCCUGGG CAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCGAGCCUACCUACA CCGACGACUUCAAGGGCAGGUUCGCCUUCAGCCUGGACACCAGCGUGUCCACCGC CUACCUGCAGAUCAGCAGCCUGAAGGCCGACGACACCGCCGUGUACUUCUGCGCC AGGGGCGGCUUCGGCUCCUCCUACUGGUACUUCGAUGUGUGGGGCCAGGGCAGCC UGGUGACCGUGUCCUCCGGCGGAGGCGGCUCAGGGGGGGGGGGCUCUGGGGGCGG CGGGAGCGACAUCCAGCUGACCCAGAGCCCCUCCUCCCUGAGCGCCUCCGUGGGG GACAGGGUGUCCAUCACAUGCAAGGCCAGCCAGGACGUGAGCAUCGCCGUGGCCU GGUACCAGCAGAAGCCCGGCAAGGCCCCCAAGCUGCUGAUCUACAGCGCCAGCUA CCGCUACACCGGCGUGCCCGACCGGUUUAGCGGCAGCGGCUCCGGCACCGACUUC ACCCUGACCAUCAGCAGCCUGCAGCCCGAGGACUUUGCCGUGUACUACUGCCAGC AGCACUACAUCACCCCCCUGACCUUCGGCGCUGGCACCAAGGUGGAGAUCAAGCG GAGCGGCGGCGGCGGCGCCGCCGCCGGCUCCGACAGCAUCCACCAGGACUACACC ACACAGAACCUGAUCCGGAUGGCCGUGGCCGGGCUGGUGCUGGUGGCCCUGCUGG CCAUCCUGGUGGAGAACUGGCACUCCCACACCGCCCUGAACAAGGAGGCCUCCGC CGACGUGGCCGAGCCCAGCUGGAGCCAGCAGAUGUGCCAGCCCGGCCUGACCUUC GCCCGGACCCCCUCUGUGUGCAAGUGACUCGAGUAGGCUGGAGCCUCGGUGGCCA UGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUAC CCCCUUCACCAAUCAGCUAAGCUCUCGAUUUCACCAAUCAGCUAAGCUCUUCUAG AUUCACCAAUCAGCUAAGCUCUUCACUUCACCAAUCAGCUAAGCUCUGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 16).

[0131] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 16.WSGR Docket No. 56371-768.601

[0132] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCAUGUGGCUGCAG AGCCUGCUGCUGCUGGGCACCGUGGCCUGCAGCAUCAGCCAGGUGCAGCUGCAGC AGAGCGGCAGCGAGCUGAAGAAGCCCGGCGCCAGCGUGAAGGUGAGCUGCAAGGC CAGCGGCUACACCUUCACCAACUACGGCAUGAACUGGGUGAAGCAGGCCCCCGGC CAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCGAGCCCACCUACA CCGACGACUUCAAGGGCAGGUUCGCCUUCAGCCUGGACACCAGCGUGAGCACCGC CUACCUGCAGAUCAGCAGCCUGAAGGCCGACGACACCGCCGUGUACUUCUGCGCC AGGGGCGGCUUCGGCAGCAGCUACUGGUACUUCGACGUGUGGGGCCAGGGCAGCC UGGUGACCGUGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGACAUCCAGCUGACCCAGAGCCCCAGCAGCCUGAGCGCCAGCGUGGGC GACAGGGUGAGCAUCACCUGCAAGGCCAGCCAGGACGUGAGCAUCGCCGUGGCCU GGUACCAGCAGAAGCCCGGCAAGGCCCCCAAGCUGCUGAUCUACAGCGCCAGCUA CAGGUACACCGGCGUGCCCGACAGGUUCAGCGGCAGCGGCAGCGGCACCGACUUC ACCCUGACCAUCAGCAGCCUGCAGCCCGAGGACUUCGCCGUGUACUACUGCCAGC AGCACUACAUCACCCCCCUGACCUUCGGCGCCGGCACCAAGGUGGAGAUCAAGAG GAGCGGCGGCGGCGGCGCCGCCGCCGGCAGCGACAGCAUCCACCAGGACUACACC ACCCAGAACCUGAUCAGGAUGGCCGUGGCCGGCCUGGUGCUGGUGGCCCUGCUGG CCAUCCUGGUGGAGAACUGGCACAGCCACACCGCCCUGAACAAGGAGGCCAGCGC CGACGUGGCCGAGCCCAGCUGGAGCCAGCAGAUGUGCCAGCCCGGCCUGACCUUC GCCAGGACCCCCAGCGUGUGCAAGUGACUCGAGUGAUAAUAGGCUGGAGCCUCGG UGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCAC CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC(SEQ ID NO: 33)

[0133] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, theWSGR Docket No. 56371-768.601 nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 33.

[0134] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCAUGUGGCUGCAG AGCCUGCUGCUGCUGGGCACCGUGGCCUGCAGCAUCAGCCAGGUGCAGCUGCAGC AGAGCGGCAGCGAGCUGAAGAAGCCCGGCGCCAGCGUGAAGGUGAGCUGCAAGGC CAGCGGCUACACCUUCACCAACUACGGCAUGAACUGGGUGAAGCAGGCCCCCGGCCAGGGCCUGAAGUGGAUGGGCUGGAUCAACACCUACACCGGCGAGCCCACCUACA CCGACGACUUCAAGGGCAGGUUCGCCUUCAGCCUGGACACCAGCGUGAGCACCGC CUACCUGCAGAUCAGCAGCCUGAAGGCCGACGACACCGCCGUGUACUUCUGCGCC AGGGGCGGCUUCGGCAGCAGCUACUGGUACUUCGACGUGUGGGGCCAGGGCAGCC UGGUGACCGUGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGACAUCCAGCUGACCCAGAGCCCCAGCAGCCUGAGCGCCAGCGUGGGCGACAGGGUGAGCAUCACCUGCAAGGCCAGCCAGGACGUGAGCAUCGCCGUGGCCU GGUACCAGCAGAAGCCCGGCAAGGCCCCCAAGCUGCUGAUCUACAGCGCCAGCUA CAGGUACACCGGCGUGCCCGACAGGUUCAGCGGCAGCGGCAGCGGCACCGACUUC ACCCUGACCAUCAGCAGCCUGCAGCCCGAGGACUUCGCCGUGUACUACUGCCAGC AGCACUACAUCACCCCCCUGACCUUCGGCGCCGGCACCAAGGUGGAGAUCAAGAG GAGCGGCGGCGGCGGCGCCGCCGCCGGCAGCGACAGCAUCCACCAGGACUACACCACCCAGAACCUGAUCAGGAUGGCCGUGGCCGGCCUGGUGCUGGUGGCCCUGCUGG CCAUCCUGGUGGAGAACUGGCACAGCCACACCGCCCUGAACAAGGAGGCCAGCGC CGACGUGGCCGAGCCCAGCUGGAGCCAGCAGAUGUGCCAGCCCGGCCUGACCUUC GCCAGGACCCCCAGCGUGUGCAAGUGACUCGAGUAGGCUGGAGCCUCGGUGGCCA UGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUAC CCCCUUCACCAAUCAGCUAAGCUCUCGAUUUCACCAAUCAGCUAAGCUCUUCUAGAUUCACCAAUCAGCUAAGCUCUUCACUUCACCAAUCAGCUAAGCUCUGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 36).

[0135] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, atWSGR Docket No. 56371-768.601 most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 36. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, or about 95% to about 100% sequence identity to SEQ ID NO: 36.

[0136] Additional Exemplary TROP2 targeting CFPs are summarized in Tables 16 and 17. In some embodiments, the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16. In some embodiments, the TROP2 targeting CFP comprises a sequence having at most 80%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16. In some embodiments, the TROP2 targeting CFP comprises a sequence of any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16.

[0137] In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence having at most 80%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17. In some embodiments, the nucleic acid encoding the TROP2 targeting CFP comprises a sequence of any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17.WSGR Docket No. 56371-768.601TABLE 16. Exemplary TROP-2 Chimeric Fusion Protein (CFP) SequencesWSGR Docket No. 56371-768.601TABLE 17. Exemplary TROP-2 CEP nucleic acid sequencesWSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601

[0138] DNA, mRNA and Circular RNA: In some embodiments, naked DNA or messenger RNA (mRNA) may be used to introduce the nucleic acid inside the cell. In some embodiments, DNA or mRNA encoding the CFP is introduced into the phagocytic cell by lipid nanoparticle (LNP) encapsulation. mRNA is single stranded and may be codon optimized. In some embodiments the mRNA may comprise one or more modified or unnatural bases such as 5 ’-Methylcytosine, orWSGR Docket No. 56371-768.601Pseudouridine. mRNA may be 50-10,000 bases long. In one aspect the transgene is delivered as an mRNA. The mRNA may comprise greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases long. In some embodiments, the mRNA may be about 11,000 bases long. In some embodiments, the mRNA may be about 12,000 bases long. In some embodiments, the mRNA comprises a transgene sequence that encodes a fusion protein. LNP encapsulated DNA or RNA can be used for transfecting myeloid cells, such as macrophages, or can be administered directly to a subject.

[0139] In some embodiments, circular RNA (circRNAs) encoding the CFP is used. In circular RNAs (circRNAs) the 3' and 5' ends are covalently linked, constitute a class of RNA. CircRNA may be delivered inside a cell or a subject using LNPs.Lipid Nanoparticle (LNP) Delivery Vehicles

[0140] In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle (LNP) delivery vehicle encapsulating the nucleic acid such as the RNA. In some embodiments, the lipid nanoparticle delivery vehicle or LNP comprises a cationic lipid, a non-cationic lipid, a neutral lipid, a PEGylated lipid, or a combination thereof. In some embodiments, the lipid nanoparticle is 34-75 nm in diameter.

[0141] In some embodiments, the expression of a component of the lipid nanoparticle delivery vehicle is not persistent in detectable levels in vivo after 7 days of infusion.

[0142] In some embodiments, the LNP comprises a cationic lipid, l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, and PEG lipid components. In some embodiments, the proportion (mol%) of cationic lipid in the LNP is from about 30% to about 60%, from about 35% to about 55%, from about 40% to about 50%. In some embodiments, the proportion (mol%) of cationic lipid in the LNP is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. In some embodiments, the proportion (mol%) of cationic lipid in the LNP is about 35%. In some embodiments, the proportion (mol%) of cationic lipid in the LNP is about 45%. In some embodiments, the proportion (mol%) of DSPC in the LNP is from about 5% to about 20%, or from about 10% to about 15%. In some embodiments, the proportion (mol%) of DSPC in the LNP is about 5%, about 10%, about 15%, or about 20%. In some embodiments, the proportion (mol%) of DSPC in the LNP is about 10%. In some embodiments, the proportion (mol%) of cholesterol in the LNP is from about 25% to about 55%, from about 30% to about 50%, from about 35% to about 40%, or from about 40% to about 50%. In some embodiments, the proportion (mol%) of cholesterol in the LNP is about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, the proportion (mol%) of cholesterol inWSGR Docket No. 56371-768.601 the LNP is about 40%. In some embodiments, the proportion (mol%) of the PEG lipid in the LNP is from about 1.0% to about 5.0%, from about 1.5% to about 4.5%, from about 2.0% to about 4.0%, from about 2.5% to about 3.5%, or from about 2.0% to about 3.5%. In some embodiments, the proportion (mol%) of the PEG lipid in the LNP is about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, or about 5.0%. In some embodiments, the proportion (mol%) of the PEG lipid in the LNP is about 2.5%.

[0143] In some embodiments, the cationic lipid comprises 9-[9-(2-butyloctanoyloxy)nonyl-(3- hydroxypropyl)amino]nonyl 2-butyloctanoate. In some embodiments, the cationic lipid is an ionizable lipid. In some embodiments, the cationic lipid comprises the chemical formula (Formula I):

[0144] In some embodiments, the ratio of mRNA to cationic lipid (N / P) is from about 2.0 to about 10.0, from about 3.0 to about 9.0, from about 4.0 to about 8.0, from 5.0 to about 8.0, or from 5.0 to about 7.0. In some embodiments, the ratio of mRNA to cationic lipid (N / P) is 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0. In some embodiments, the ratio of mRNA to ionizable lipid (N / P) is from about 2.0 to about 10.0, from about 3.0 to about 9.0, from about 4.0 to about 8.0, from 5.0 to about 8.0, or from 5.0 to about 7.0. In some embodiments, the ratio of mRNA to ionizable lipid (N / P) is 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0.

[0145] In some embodiments, the PEG lipid comprises 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide. In some embodiments, the PEG- lipid comprises the chemical formula (Formula II):wherein, n is an integer between 46 and 52.

[0146] In some embodiments, the DSPC comprises the chemical formula (Formula III):WSGR Docket No. 56371-768.601

[0147] In some embodiments, the LNP further comprises a cholesterol. In some embodiments, the cholesterol comprises the following structure (Formula IV):

[0148] In some embodiments, the LNP alone does not induce an immune cell. When an immune cell is induced, it releases cytokines. In some embodiments, the cytokines act as markers for immune cell induction or activation. In some embodiments, the activation marker for an immune cell is a detectable induction of cytokines such as TNF-alpha, IFN-gamma, IL-1 or IL2. In some embodiments, for example, induction of a myeloid cell causes activation of the myeloid cell to release a cytokine, e.g., TNF-alpha. In some embodiments, induction of a myeloid cell causes activation of the myeloid cell to release of IFN-gamma. In some embodiments, activation of the myeloid cell causes release of CD86. CD86 is a surface molecule on macrophages, for example, that is a marker for macrophage activation and acts as a costimulatory signal for T cell activation and survival. In some embodiments, when a monocyte or macrophage is incubated with empty LNP use herein in vitro, the monocyte / macrophage does not induce CD86. For example, when two identical cultures of monocyte / macrophages are incubated with (i) empty LNP in the first set of the two identical cultures, and (ii) in the second set of the two identical cultures, an LNP comprising an RNA encoding an antigen is added. It is observed that only the second set, incubated with the LNP comprising the RNA encoding the antigen may induce CD86, but not the set incubated with the empty LNP. In some embodiments, the LNP is a next generation LNP which is does not induce any inflammatory reaction in cells that uptake them. In some embodiments, the empty LNP does not induce TNF alpha in immune cells that take up the empty LNP.

[0149] In some embodiments, uptake of an empty LNP as described herein does not cause induction of interferon-gamma or IL- lb in a monocyte or macrophage that takes up the empty LNP, but when the LNP encapsulates and delivers an inflammatory agent to the cell, it responds adequately to the agent. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, PEG and cholesterol as described herein.GPC3-CFP

[0150] In some embodiments, the target protein is GPC3. In some embodiments, the extracellular domain of the CFP comprises an antigen binding domain capable of specifically binding a GPC3 antigen. In some embodiments, the CFP comprises an extracellular domain comprising an anti- GPC3 scFv, a CD89 transmembrane domain, and a CD89 intracellular domain.WSGR Docket No. 56371-768.601

[0151] In some cases, the CFP comprises an amino acid sequence ofMWLQSLLLLGTVACSISQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQA PGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQ SLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCSQNTHVPPTFGQGTKLEIKGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAIL VENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 40).In some embodiments, the GPC3 targeting CFP comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 841%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40. In some embodiments, the GPC3 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 40. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with a range of sequence identity having an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, or about 85% to about 90% sequence identity to SEQ ID NO: 40. Nucleic Acid Sequences Encoding GPC3-CFP

[0152] Disclosed herein are nucleic acids encoding the GPC3 targeting CFP. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a codon-optimized coding sequence for the GPC3 targeting CFP. In some embodiment, the nucleic acid encoding the GPC3 targeting CFP comprises a 5’ UTR. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 3’ UTR.

[0153] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a coding sequence ofAUGUGGCUGCAGUCUCUGCUGCUGCUGGGGACCGUGGCCUGCAGCAUCUCCCAGG UGCAGCUGGUGCAGAGCGGCGCCGAGGUGAAGAAGCCCGGCGCCAGCGUGAAGGU GAGCUGCAAGGCCAGCGGCUACACCUUCACCGACUACGAGAUGCACUGGGUGCGG CAGGCCCCCGGCCAGGGCCUGGAGUGGAUGGGCGCCCUGGACCCCAAGACCGGCG ACACCGCCUACAGCCAGAAGUUCAAGGGCAGGGUGACCCUGACUGCCGACAAGAG CACCUCCACCGCCUACAUGGAGCUGUCCUCCCUGACCAGCGAGGACACCGCCGUG UACUACUGCACCAGGUUCUACAGCUACACCUACUGGGGCCAGGGCACCCUGGUGA CAGUGAGCUCCGGCGGCGGCGGGAGCGGCGGGGGCGGCAGCGGGGGCGGGGGCUCWSGR Docket No. 56371-768.601UGACGUGGUGAUGACCCAGAGCCCCCUGUCCCUGCCUGUGACCCCCGGCGAGCCC GCCAGCAUCAGCUGCCGGAGCUCCCAGAGCCUGGUGCACAGCAACCGGAACACCU ACCUGCACUGGUACCUGCAGAAGCCCGGCCAGUCCCCCCAGCUGCUGAUCUACAA GGUGAGCAAUCGGUUCAGCGGCGUGCCAGACCGGUUCUCCGGCUCUGGCAGCGGC ACCGACUUCACCCUGAAGAUCAGCAGGGUGGAGGCCGAGGACGUGGGGGUGUACU ACUGCAGUCAGAACACCCACGUGCCCCCCACCUUCGGCCAGGGCACCAAGCUGGA GAUCAAGGGGUCCGGCGGCAGCGACUCCAUCCACCAGGACUACACCACCCAGAAC CUGAUCCGGAUGGCCGUGGCCGGCCUGGUGCUGGUGGCCCUGCUGGCCAUCCUGG UGGAGAACUGGCACAGCCACACCGCCCUGAACAAGGAGGCCAGCGCCGAUGUGGC UGAGCCCUCCUGGAGCCAGCAGAUGUGCCAGCCCGGCCUGACCUUCGCCAGGACC CCCAGCGUGUGCAAGUGA (SEQ ID NO: 41).

[0154] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 41. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with a range of sequence identity having an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, or about 85% to about 90% sequence identity to SEQ ID NO: 41.

[0155] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 5’ UTR. In some embodiments, the 5’ UTR comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a sequence of GCCACC (SEQ ID NO: 6). In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 5’ UTR comprising a sequence of AGGAGACCCAAGCUGGCUAGCGCCACC (SEQ ID NO: 42). In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 5’ UTR comprising a sequence with at least 90% or at least 95% sequence identity to SEQ ID NO: 42.

[0156] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 3’ UTR comprising a sequence of CUCGAGUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCGAUUUCUAGAUUCACUGUGGUWSGR Docket No. 56371-768.601CUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 43). In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 3’ UTR comprising a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43.

[0157] In some embodiments, the 3’ UTR comprises a microRNA binding site. In some embodiments, the 3’ UTR comprises at least two microRNA binding sites. In some embodiments, the 3’ UTR comprises two, three, four, or five microRNA binding sites. In some embodiments, the microRNA binding sites on the 3’ UTR are the same sequence. In some embodiments, the microRNA binding sites on the 3’ UTR are different sequences.

[0158] In some embodiments, the microRNA binding site comprises a sequence for specifically binding to miR-27b. In some embodiments, the exemplary human microRNA is miR-27b (NR_029665) 97 bp is: ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGUUUCC GCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGAAGGUG (SEQ ID NO: 11) In some embodiments, the miR-27b binding site comprises a sequence of UCACCAAUCAGCUAAGCUCU (SEQ ID NO: 12). In some embodiments, the 3’ UTR comprises two, three, four, or five copies of SEQ ID NO: 12.

[0159] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a 3’ UTR comprising a sequence of CUCGAGUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUUCACCAAUCAGCUAAGCUCUCG AUUUCACCAAUCAGCUAAGCUCUUCUAGAUUCACCAAUCAGCUAAGCUCUUCACU UCACCAAUCAGCUAAGCUCUGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAA GACAAUAGC (miR-27b binding site underlined) (SEQ ID NO: 13).

[0160] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence of AGGAGACCCAAGCUGGCUAGCGCCACCAUGUGGCUGCAGUCUCUGCUGCUGCUGG GGACCGUGGCCUGCAGCAUCUCCCAGGUGCAGCUGGUGCAGAGCGGCGCCGAGGU GAAGAAGCCCGGCGCCAGCGUGAAGGUGAGCUGCAAGGCCAGCGGCUACACCUUC ACCGACUACGAGAUGCACUGGGUGCGGCAGGCCCCCGGCCAGGGCCUGGAGUGGA UGGGCGCCCUGGACCCCAAGACCGGCGACACCGCCUACAGCCAGAAGUUCAAGGG CAGGGUGACCCUGACUGCCGACAAGAGCACCUCCACCGCCUACAUGGAGCUGUCC UCCCUGACCAGCGAGGACACCGCCGUGUACUACUGCACCAGGUUCUACAGCUACA CCUACUGGGGCCAGGGCACCCUGGUGACAGUGAGCUCCGGCGGCGGCGGGAGCGG CGGGGGCGGCAGCGGGGGCGGGGGCUCUGACGUGGUGAUGACCCAGAGCCCCCUGWSGR Docket No. 56371-768.601UCCCUGCCUGUGACCCCCGGCGAGCCCGCCAGCAUCAGCUGCCGGAGCUCCCAGAG CCUGGUGCACAGCAACCGGAACACCUACCUGCACUGGUACCUGCAGAAGCCCGGC CAGUCCCCCCAGCUGCUGAUCUACAAGGUGAGCAAUCGGUUCAGCGGCGUGCCAG ACCGGUUCUCCGGCUCUGGCAGCGGCACCGACUUCACCCUGAAGAUCAGCAGGGU GGAGGCCGAGGACGUGGGGGUGUACUACUGCAGUCAGAACACCCACGUGCCCCCC ACCUUCGGCCAGGGCACCAAGCUGGAGAUCAAGGGGUCCGGCGGCAGCGACUCCA UCCACCAGGACUACACCACCCAGAACCUGAUCCGGAUGGCCGUGGCCGGCCUGGU GCUGGUGGCCCUGCUGGCCAUCCUGGUGGAGAACUGGCACAGCCACACCGCCCUG AACAAGGAGGCCAGCGCCGAUGUGGCUGAGCCCUCCUGGAGCCAGCAGAUGUGCC AGCCCGGCCUGACCUUCGCCAGGACCCCCAGCGUGUGCAAGUGACUCGAGUAGGC UGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCC CCUUCCUGCACCCGUACCCCCUUCACCAAUCAGCUAAGCUCUCGAUUUCACCAAUC AGCUAAGCUCUUCUAGAUUCACCAAUCAGCUAAGCUCUUCACUUCACCAAUCAGC UAAGCUCUGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 44).

[0161] In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 944%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 944%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 44. In some embodiments, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence with a range of sequence identity having an upper limit as mentioned earlier and a lower limit as mentioned earlier. For example, the nucleic acid encoding the GPC3 targeting CFP comprises a sequence having a sequence identity of about 80% to about 99%, about 85% to about 95%, or about 85% to about 90% sequence identity to SEQ ID NO: 44.Modified Cytokine Receptors

[0162] Also provided herein are modified cytokine receptors and nucleic acids encoding modified cytokine receptors. In some embodiments, the modified cytokine receptor comprises an extracellular binding domain of a first cytokine receptor and an intracellular signaling domain of a second cytokine receptor, wherein the modified cytokine receptor comprising the extracellular binding domain of the first cytokine receptor and the intracellular signaling domain of the second cytokine receptor does not exist naturally. In some embodiments, the first cytokine receptor is aWSGR Docket No. 56371-768.601 proinflammatory cytokine receptor. In some embodiments, the first cytokine receptor is an antiinflammatory cytokine receptor. In some embodiments, the second cytokine receptor is a proinflammatory cytokine receptor. In some embodiments, the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is a proinflammatory cytokine receptor and the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is an anti-inflammatory cytokine receptor and the second cytokine receptor is a proinflammatory cytokine receptor. In some embodiments, the anti-inflammatory cytokine receptor is selected from the group consisting of an IL10RA, TGFpRII, TGFpRI, IFNGR1, and IL17RA. In some embodiments, the proinflammatory cytokine receptor is selected from the group consisting of IFNXR1, IFNAR1, IFNAR2, IL10RA, CD30, IL28R, TREM2, MerTK, and CSF3R. In some embodiments, the intracellular domain is derived from a receptor selected from the group consisting of TGF-PRI, TGF-PRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3z, 4-IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5R, IL-7R, IL-7Ra, and BTLA. In some embodiments, the intracellular domain is from CD40. In some embodiments, the intracellular domain is from MyD88. In some embodiments, the first cytokine receptor is associated with an Ml phenotype. In some embodiments, the first cytokine receptor is associated with an M2 phenotype. In some embodiments, the second cytokine receptor is associated with an Ml phenotype. In some embodiments, the second cytokine receptor is associated with an M2 phenotype. Exemplary cytokine receptor domain sequences are provided in Tables 4-7. In some embodiments, the modified cytokine receptor comprises one or more of the sequences selected from the sequences provided in Tables 4-7. In some embodiments, the modified cytokine receptor comprises one or more sequences having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more sequences selected from the sequences provided in Tables 4-7. In some embodiments, cytokine receptor domains are operably linked by a GSS linker sequence. In some embodiments, the modified cytokine receptor comprises a P2A cleavage site. In some embodiments, the P2A cleavage site comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 45).WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601Table 5. Full Length Modified Cytokine Receptor SequencesWSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601Table 6. Exemplary Cytokine Receptor Extracellular DomainsWSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601Table 7. Exemplary Cytokine Receptor Intracellular DomainsWSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601

[0163] In some embodiments, a cell expressing any one of the CFP constructs described herein is engineered to express a modified cytokine receptor disclosed herein. In some embodiments, a cell expressing any one of the modified cytokine receptors described herein is engineered to express any one of the CFP constructs disclosed herein. In some embodiments, one or moreWSGR Docket No. 56371-768.601 nucleic acids encoding (1) any one of the CFP constructs described herein and (2) a modified cytokine receptor disclosed herein are expressed in a cell.Dominant Negative TGF0R.II

[0164] Also provided herein is a dominant negative TGFP receptor type II (DN-TGFpRII) and nucleic acids encoding the DN-TGFpRII. Also provided herein is one or more nucleic acids encoding (1) any one CFP described herein and (2) a DN-TGFpRII described herein. In some embodiments, DN-TGFpRII comprises a sequence of MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS TCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASP KCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAIS VIIIFYCYRVNRQQKLSS (SEQ ID NO: 38).

[0165] In some embodiments, the DN-TGFpRII comprises a sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38. In some embodiments, the DN-TGFpRII comprises a sequence with at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the DN-TGFpRII comprises a sequence with a range of sequence identity with an upper limit as mentioned earlier and a lower limit as mentioned earlier. In some embodiments, a cell expressing any one of the CFP constructs described herein is engineered to express the DN- TGFpRII disclosed herein. In some embodiments, a cell is engineered to express any one of the CFP constructs, any one of the modified cytokine receptors disclosed herein, and any one of the DN-TGFpRII disclosed herein.Delivery of nucleic acids into a cell

[0166] Nucleic acids encoding the CFP as described herein may be introduced to a cell, e.g., a myeloid cell, via different delivery approaches. An engineered nucleic acid as described herein may be introduced to a cell in vitro, ex vivo or in vivo. In some embodiments, a nucleic acid is introduced into a myeloid cell in the form of a plasmid or a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an expression vector, for example, a vector comprising one or more promoters, and other regulatory components, including enhancer binding sequence, initiation and terminal codons, a 5’ UTR, a 3’ UTR comprising a transcript stabilization element, optional conserved regulatory protein binding sequences and others. In some embodiments, the vector is a phage, a cosmid, or an artificial chromosome.WSGR Docket No. 56371-768.601

[0167] In some embodiments, a vector is introduced or incorporated in the cell by known methods of transfection, such as using lipofectamine, or calcium phosphate, or via physical means such as electroporation or nucleofection. In some embodiments the vector is introduced or incorporated in the cell by infection, a process commonly known as viral transduction.

[0168] In some embodiments, the vector for expression of the CFP is of a viral origin. In some embodiments, the engineered nucleic acid is encoded by a viral vector capable of replicating in non-dividing cells. In some embodiments, the nucleic acid encoding the engineered nucleic acid is encoded by a lentiviral vector, e.g. HIV and FIV-based vectors. In some embodiments the lentiviral vector is prepared in-house and manufactured in large scale for the purpose. In some embodiments, commercially available lentiviral vectors are utilized, as is known to one of skill in the art. In some embodiments, the engineered nucleic acid is encoded by a herpes simplex virus vector, a vaccinia virus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.

[0169] In some embodiments, a stable integration of transgenes into myeloid cells, such as macrophages, and other phagocytic cells may be accomplished via the use of a transposase and transposable elements, in particular, mRNA-encoded transposase. In one embodiment, Long Interspersed Element- 1 (LI) RNAs may be contemplated for retrotransposition of the transgene and stable integration into myeloid cells, such as macrophages or phagocytic cells.Retrotransposon may be used for stable integration of an engineered nucleic acid encoding the CFP.

[0170] In some embodiments, the myeloid cell may be modified by expressing a transgene via incorporation of the transgene in a transient expression vector. In some embodiments expression of the transgene may be temporally regulated by a regulator from outside the cell. Examples include the Tet-on Tet-off system, where the expression of the transgene is regulated via presence or absence of tetracycline.

[0171] In some embodiments, the engineered nucleic acid described herein is a circular RNA (circRNA). A circular RNA comprises an RNA molecule where the 5’ end and the 3’ end of the RNA molecule are joined together. Without wishing to be bound by any theory, circRNAs have no free ends and may have longer half-life as compared to some other forms of RNAs or nucleic acid and may be resistant to digestion with RNase R exonuclease and turn over more slowly than its counterpart linear RNA in vivo. In some embodiments, the half-life of a circRNA is more than 20 hours. In some embodiments, the half-life of a circRNA is more than 30 hours. In some embodiments, the half-life of a circRNA is more than 40 hours. In some embodiments, the halflife of a circRNA is more than 48 hours. In certain embodiments, a circRNA comprises an internal ribosome entry site (IRES) element that engages a eukaryotic ribosome and an RNAWSGR Docket No. 56371-768.601 sequence element encoding a polypeptide operatively linked to the IRES for insertion into cells in order to produce a polypeptide of interest.

[0172] CircRNAs can be prepared by methods known to those skilled in the art. For example, circRNAs may be chemically synthesized and / or enzymatically synthesized, for example by enzymatically synthesis of the RNA followed by chemical joining of the ends of the RNA to form the circRNA. In some embodiments, a linear primary construct or linear mRNA may be cyclized, or concatemerized to create a circRNA. The mechanism of cyclization or concatemerization may occur through methods such as, but not limited to, chemical, enzymatic, or ribozyme catalyzed methods. The newly formed 5 '- / 3 '-linkage may be an intramolecular linkage or an intermolecular linkage. In some embodiments, a linear primary construct or linear mRNA may be cyclized, or concatemerized using the chemical method to form a circRNA. In the chemical method, the 5 '-end and the 3 '-end of the nucleic acid (e.g., linear primary construct or linear mRNA) contain chemically reactive groups that, when close together, form a new covalent linkage between the 5 '-end and the 3 '-end of the molecule. The 5 '-end may contain a NHS-ester reactive group and the 3 '-end may contain a 3 '-amino-terminated nucleotide such that in an organic solvent the 3 '-amino-terminated nucleotide on the 3 '-end of a linear RNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5 '- / 3 '-amide bond. In some embodiments, a DNA or RNA ligase, e.g. a T4 ligase, may be used to enzymatically link a 5 '-phosphorylated nucleic acid molecule (e.g., a linear primary construct or linear mRNA) to the 3 '-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In some embodiments, a linear primary construct or linear mRNA may be cyclized or concatemerized by using at least one non-nucleic acid moiety. For example, the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and / or near the 3' terminus of the linear primary construct or linear mRNA in order to cyclize or concatemerize the linear primary construct or linear mRNA. In some embodiments, a linear primary construct or linear mRNA may be cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecules surfaces at, near or linked to the 5' and 3' ends of the linear primary construct or linear mRNA. For example, a linear primary construct or linear mRNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces. In some embodiments, a linear primary construct or linear mRNA may comprise a ribozyme RNA sequence near the 5' terminus and near the 3' terminus. In some embodiments, a circRNA may be synthesized by inserting DNA fragments into a plasmid containing sequences having the capability of spontaneous cleavage and selfcircularization. In some embodiments, a circRNA is produced by making a DNA construct encoding an RNA cyclase ribozyme, expressing the DNA construct as an RNA, and thenWSGR Docket No. 56371-768.601 allowing the RNA to self-splice, which produces a circRNA free from intron in vitro. In some embodiments, a circRNA is produced by synthesizing a linear polynucleotide, combining the linear nucleotide with a complementary linking oligonucleotide under hybridization conditions, and ligating the linear polynucleotide.

[0173] In some embodiments, the circRNA is administered directly to tissues of a subject. Additional description of circRNAs in U.S. Patent No. s 5,766,903, 5,580,859, 5,773,244, 6,210,931, PCT publication No. W01992001813, Hsu et al., Nature (1979) 280:339-340, Harland & Misher, Development (1988) 102:837-852, Memczak et al. Nature (2013) 495:333- 338, Jeck et al., and RNA (2013) 19: 141-157, each of which is incorporated herein by reference in its entirety.

[0174] In some embodiments, a nucleic acid is introduced into a myeloid cell with a nanoparticle (NP). A nanoparticle may be of various shapes or sizes and may harbor the nucleic acid encoding the CFP or PFP. In some embodiments, the NP is a lipid nanoparticle (LNP). In some embodiments, the NP comprises poly(amino acids), polysaccharides and poly(alpha-hydroxy acids), gold, silver, carbon, iron, silica, or any combination thereof. In some embodiments, the NP comprises a polylactide-co-glycolide (PGLA) particle. In some embodiments, the nucleic acid is encapsulated in the NP, for example, via water / oil emulsion or water-oil-water emulsion. In some embodiments, the nucleic acid is conjugated to the NP.

[0175] Nanoparticles (NPs) may be delivered to a cell in vitro, ex vivo or in vivo. In some embodiments, a NP is delivered to a phagocytic cell ex vivo. In some embodiments, a NP is delivered to a phagocytic cell in vivo. In some embodiments, the NP is less than lOOnm in diameter. In some embodiments, the NP is more than lOOnm in diameter. In some embodiments, the NP is a rod-shaped NP. In some embodiments, the NP is a spherical NP. In particular embodiments, the NP is a spherical NP for delivery to a phagocytic cell. In additional embodiments, the NP is at least lOOnm in diameter and does not trigger or triggers reduced toxicity when delivered to a cell.

[0176] In some embodiments, the NP is positively charged. In some embodiments, the NP is negatively charged. In some embodiments, the NP is neutral. In some embodiments, the NP is a cationic NP that is delivered and taken up by a myeloid cell ex vivo or in vivo.

[0177] In some embodiments, the NP encapsulates the nucleic acid wherein the nucleic acid is a naked DNA molecule. In some embodiments, the NP encapsulates the nucleic acid wherein the nucleic acid is an mRNA molecule. In some embodiments, the NP encapsulates the nucleic acid wherein the nucleic acid is a circular RNA (circRNA) molecule. In some embodiments, the NP encapsulates the nucleic acid wherein the nucleic acid is a vector, a plasmid, or a portion or fragment thereof.WSGR Docket No. 56371-768.601

[0178] In some embodiments, the NP is a Lipid nanoparticle (LNP). LNPs may comprise a polar and or a nonpolar lipid. In some embodiments cholesterol is present in the LNPs for efficient delivery. LNPs are 100-300 nm in diameter provide efficient means of mRNA delivery to various cell types, including myeloid cells, such as macrophages. In some embodiments, LNP may be used to introduce the nucleic acids into a cell in in vitro cell culture. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is a naked DNA molecule. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is an mRNA molecule. In some embodiment described herein, lipid nanoparticles are formed associating or encapsulating the full length recombinant (engineered) mRNA. In some embodiments, the number of mRNA molecules per LNP is regulated for optimum delivery of the mRNA inside the cell. In some embodiments, the LNP is used to deliver mRNA systemically, that may be taken up by myeloid cells in vivo. In some embodiments, the LNP may comprise target moieties.

[0179] In some embodiments, the LNP does not comprise myeloid cell-targeting moieties on the surface, but the mRNA is designed for myeloid cell-specific expression. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is inserted in a vector, such as a plasmid vector. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is a circRNA molecule.

[0180] In some embodiments, mRNA can be encapsidated within mammalian retro-viral like PEG10 packages that deliver the mRNA inside a cell. Specific fusogens may be used for cell targeting with PEG10 delivery to organ, tissue or cells, e.g., myeloid cells. PEG10 is known to bind to its own mRNA and deliver it inside a cell. PEG10 UTR regions may be incorporated flanking the coding region of the mRNA, to facilitate PEG10 encapsidation. (Segel et al., Science (2021), 373: 6557, p882-889).

[0181] In some embodiments, the LNP is used to deliver the nucleic acid into the subject. LNP can be used to deliver nucleic acid systemically in a subject. It can be delivered by injection. In some embodiments, the LNP comprising the nucleic acid is injected by intravenous route. In some embodiments the LNP is injected subcutaneously. In some embodiments the LNP is injected intramuscularly.Pharmaceutical Composition

[0182] Provided herein is a pharmaceutical composition, comprising engineered myeloid cells, such as macrophages, comprising an engineered nucleic acid encoding the CFP and a pharmaceutically acceptable excipient.

[0183] Also provided herein is a pharmaceutical composition, comprising a nucleic acid encoding the CFP and a pharmaceutically acceptable excipient. The pharmaceutical compositionWSGR Docket No. 56371-768.601 may comprise DNA, mRNA or circRNA or an LNP or a liposomal composition comprising any one of these.

[0184] Also provided herein is a pharmaceutical composition comprising a vector comprising the engineered nucleic acid encoding the CFP and a pharmaceutically acceptable excipient. The pharmaceutical composition may comprise DNA, mRNA or circRNA inserted in a plasmid vector or a viral vector.

[0185] Also provided herein is A pharmaceutical composition, comprising: (I) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR3 sequence comprises a sequence of GGFGSSYWYFDV (SEQ ID NO: 182); and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR3 sequence comprises a sequence of QQHYITPLT (SEQ ID NO: 191); (b) a CD89 transmembrane domain operatively linked to the extracellular domain; and (II) a lipid nanoparticle delivery vehicle encapsulating the RNA in (I).

[0186] In some embodiments, the CFP comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 99%, or 100% identity to SEQ ID NO: 39 or 179. In some embodiments, the RNA encoding the CFP comprises a sequence with at least 60%, 70%, 80%, 90%, 99%, or 100% identity to any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36. In some embodiments, the RNA encoding the CFP comprises a sequence of any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36. In some embodiments, the CFP has an amino acid sequence of SEQ ID NO: 39 or 179. In some embodiments, the LNP comprises an ionizable lipid, wherein the ionizable lipid is ALC-0366. In some embodiments, the LNP comprises an ionizable lipid, wherein the ionizable lipid comprisesthe chemical formula: . In some embodiments, the ionizable lipid is present in the LNP at a mol% of about 47.5%. In some embodiments, the LNP comprises a PEG lipid. In some embodiments, the PEG-lipid is ALC- 0159. In some embodiments, the PEG lipid comprises the chemical formula:WSGR Docket No. 56371-768.601In someembodiments, n is an integer between 46 and 52. In some embodiments, the PEG-lipid is present in the LNP at a mol% of about 2.5%. In some embodiments, the LNP comprises distearoylphosphatidylcholine (DSPC). In some embodiments, the DSPC is present in the LNP at mol% of about 10%. In some embodiments, the LNP comprises cholesterol. In some embodiments, the cholesterol is present in the LNP at a mol% of about 40%.

[0187] In some embodiments the engineered myeloid cells, such as macrophages, are grown in cell culture sufficient for a therapeutic administration dose, and washed, and resuspended into a pharmaceutical composition.

[0188] In some embodiments the excipient comprises a sterile buffer, (e.g. HEPES or PBS) at neutral pH. In some embodiment, the pH of the pharmaceutical composition is at 7.5. In some embodiments, the pH may vary within an acceptable range. In some embodiments, the engineered cells may be comprised in sterile enriched cell suspension medium comprising complement deactivated or synthetic serum. In some embodiments the pharmaceutic composition further comprises cytokines, chemokines or growth factors for cell preservation and function. In some embodiments, the pharmaceutical composition is lyophilizable. In some embodiments, the composition has a stable shelf life of at least 6 months.

[0189] In some embodiments, the concentration of the RNA in the pharmaceutical composition is between about 1.5 to about 0.5 mg / mL or between about 1.3 to about 0.7 mg / mL when stored in a container. In some embodiments, the container is a single-use or multi-use vial.

[0190] In some embodiments, the pharmaceutical composition may comprise additional therapeutic agents, co-administered with the engineered cells or nucleic acids disclosed herein. Kits

[0191] Disclosed herein are kits for carrying out a method as disclosed herein. In some embodiments, the kit comprises any one composition or pharmaceutical composition disclosed herein. A kit can include a first therapeutic agent and at least one additional therapeutic agent. In some embodiments, the kit includes a first therapeutic that comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain, and at least one additional therapeutic agent that comprises (i) a prophylactic agent or (ii) a chemotherapeutic agent and at least one of (a) a tumor target drug or (b) an immune checkpoint inhibitor. In some embodiments, the kit comprises a nucleic acid comprising a sequence encoding a chimeric fusionWSGR Docket No. 56371-768.601 protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain, and at least one additional therapeutic agent that comprises a chemotherapeutic agent and at least one of (a) a tumor target drug or (b) an immune checkpoint inhibitor. In some embodiments, the kit comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain, and at least one additional therapeutic agent that comprises a chemotherapeutic agent and at least one of a tumor target drug. In some embodiments, the kit comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain, and at least one additional therapeutic agent that comprises a chemotherapeutic agent and at least one of an immune checkpoint inhibitor.

[0192] In some embodiments, the chemotherapeutic agent is selected from the group consisting of capecitabine, oxaliplatin, fluoropyrimidine, cisplatin, and carboplatin. In some embodiments, the chemotherapeutic agent comprises capecitabine and oxaliplatin (Capox). In some embodiments, the chemotherapeutic agent comprises Capox and fluoropyrimidine. In some embodiments, the chemotherapeutic agent comprises Capox and cisplatin. In some embodiments, the chemotherapeutic agent comprises Capox and carboplatin. In some embodiments, the chemotherapeutic agent comprises Capox, fluoropyrimidine and cisplatin. In some embodiments, the chemotherapeutic agent comprises Capox, fluoropyrimidine and carboplatin. In some embodiments, the chemotherapeutic agent comprises Capox, cisplatin, and carboplatin. In some embodiments, the chemotherapeutic agent comprises Capox, fluoropyrimidine, cisplatin, and carboplatin. In some embodiments, the kit comprises Capox and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1. In some embodiments, the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1. In some embodiments, the immune checkpoint inhibitor comprises an antibody specifically binds to PD-L1. In some embodiments, the antibody is pembrolizumab or nivolumab. In some embodiments, the antibody is pembrolizumab. In some embodiments, the antibody is nivolumab. In some embodiments, the kit comprises Capox and pembrolizumab. In some embodiments, the kit comprises Capox and nivolumab.

[0193] In some embodiments, the tumor target drug is an antibody specifically binds to a tumor antigen. In some embodiments, the tumor antigen comprises HER2, CD 19, CD20, CD22, KappaWSGR Docket No. 56371-768.601 or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3 / 4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL- 13 receptor a 2, IL- 11 receptor a, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSC1, folate receptor-a, CD44v7 / 8, 8H9, NCAM, VEGF receptors, 514, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, or viral-associated antigens expressed by the tumor. In some embodiments, the tumor antigen is HER-2. In some embodiments, the antibody comprises trastuzumab or pertuzumab. In some embodiments, the antibody is trastuzumab. In some embodiments, the antibody is pertuzumab. In some embodiments, the tumor target drug is lapatinib. In some embodiments, the tumor target drug is trastuzumab emtansine. In some embodiments, the kit comprises Capox and trastuzumab. In some embodiments, the kit further comprises fluoropyrimidine. In some embodiments, the kit further comprises cisplatin. In some embodiments, the kit further comprises fluoropyrimidine and cisplatin. In some embodiments, the kit further comprises nivolumab. In some embodiments, the kit further comprises pembrolizumab.

[0194] In one aspect, provided herein is a combination therapeutic kit for treating a PD-L1- positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; capecitabine and oxaliplatin; and nivolumab.

[0195] In one aspect, provided herein is a combination therapeutic kit for treating a PD-L1- positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; capecitabine and oxaliplatin; and pembrolizumab.

[0196] In one aspect, provided herein is a combination therapeutic kit for treating a PD-L1- positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; fluoropyrimidine and cisplatin, and pembrolizumab.

[0197] In one aspect, provided herein is a combination therapeutic kit for treating a PD-L1- positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; fluoropyrimidine and cisplatin, and nivolumab.

[0198] In one aspect, provided herein is a combination therapeutic kit for treating a HER2- positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; capecitabine and oxaliplatin; and trastuzumab.

[0199] In one aspect, provided herein is a combination therapeutic kit for treating a HER-2 positive cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; capecitabine and oxaliplatin; and trastuzumab.

[0200] In some embodiments, the cancer is esophageal, gastroesophageal junction (GEJ), or gastric adenocarcinoma.

[0201] In some embodiments, the cancer is metastatic esophagogastric cancer.WSGR Docket No. 56371-768.601

[0202] In one aspect, provided herein is a combination therapeutic kit for treating a cancer, comprising: an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; tocilizumab and anakinra.Treatment Methods

[0203] Provided herein are methods for treating cancer in a subject using a pharmaceutical composition comprising engineered myeloid cells or engineered nucleic acid encoding a CFP as disclosed herein, to target, attack and kill cancer cells directly or indirectly.

[0204] Cancers include, but are not limited to T cell lymphoma, cutaneous lymphoma, B cell cancer (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), the heavy chain diseases (such as, for example, alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chainWSGR Docket No. 56371-768.601 disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers can be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, or undifferentiated. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases. Cancers to be treated also include, but are not limited to, cervical cancer, colorectal cancer, esophageal, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, and urothelial cancer

[0205] In some aspects, any gene of interest can be expressed in a myeloid cell, such that the cell can be used to treat a disease that requires, for example an active phagocytic cell, such as an infection, where the myeloid cell may be specifically engineered to target, engulf and destroy the pathogen.

[0206] In general, cellular immunotherapy comprises providing the patient a medicament comprising live cells. In some aspects a patient or a subject having cancer, is treated with autologous cells, the method comprising, isolation of PBMC-derived myeloid cells, such as macrophages, modifying the cells ex vivo to generate phagocytic myeloid cells capable of tumor lysis by introducing into the cells a engineered nucleic acid encoding a CFP, and administering the modified myeloid cells into the subject.

[0207] In some embodiment, the subject is administered a pharmaceutical composition comprising the DNA, or the mRNA or the circRNA in a vector, or in a pharmaceutically acceptable excipient described above.

[0208] In some embodiments the administration of the off the shelf cellular products may be instantaneous, or may require 1 day, 2 days or 3 days or 4 days or 5 days or 6 days or 7 days or more prior to administration. The pharmaceutical composition comprising cell, or nucleic acid may be preserved over time from preparation until use in frozen condition. In some embodiments, the pharmaceutical composition may be thawed once. In some embodiments, the pharmaceutical composition may be thawed more than once. In some embodiments, the pharmaceutical composition is stabilized after a freeze-thaw cycle prior administering to theWSGR Docket No. 56371-768.601 subject. In some embodiments the pharmaceutical composition is tested for final quality control after thawing prior administration.

[0209] In some embodiments, a composition comprising 10A6 engineered cells are administered per administration dose. In some embodiments, a composition comprising 10A7 engineered cells are administered per administration dose. In some embodiments, a composition comprising 5X 10A7 engineered cells are administered per administration dose. In some embodiments, a composition comprising 10A8 engineered cells are administered per administration dose. In some embodiments, a composition comprising 2xlOA8 engineered cells are administered per administration dose. In some embodiments, a composition comprising 5xl0A8 engineered cells are administered per administration dose. In some embodiments, a composition comprising 10A9 engineered cells are administered per administration dose. In some embodiments, a composition comprising 10Al 0 engineered cells are administered per administration dose.

[0210] Also provided herein is a method of treating a cancer in a human in need thereof, the method comprising administering to the subject a combination of: (a) a first component comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2; (b) at least one prophylactic agent; and (c) at least one anti -cancer agent selected from: (i) a chemotherapeutic agent (ii) a tumor target drug or (iii) an immune checkpoint inhibitor.

[0211] Also provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering the subject: (a) a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain, and a CD89 intracellular domain; and an anti-cancer agent comprising: a chemotherapeutic agent; and (i) a tumor target drug, or (ii) an immune checkpoint inhibitor.

[0212] In some embodiments, the at least one anti-cancer agent comprises an anti -neoplastic drug. In some embodiments, the anti -neoplastic drug is administered before, simultaneously with, or after the administration of the first therapeutic agent. In some embodiments, the first therapeutic agent and the at least one additional therapeutic agent (e.g., the anti-neoplastic drug or the anti-cancer drug, used interchangeably herein) may each be administered multiple times. In some embodiments, either the first therapeutic agent or the at least one additional therapeutic agent may be administered only for a finite period. In some embodiments, the finite period of each may or may not overlap with the finite period of the other. The method may comprise a dose regimen of the first therapeutic agent, the dose regimen of the at least one additional therapeutic agent. In some embodiments, the method comprises a series of doses of the first therapeutic agent. In some embodiments, the method comprises a series of doses of the at leastWSGR Docket No. 56371-768.601 one additional therapeutic agent. In some embodiments, the method comprises a series of doses of the first therapeutic agent and a series of doses of the at least one additional therapeutic agent. In some embodiments, the series of doses of the first therapeutic agent and the series of doses of the at least one additional therapeutic agent may or may not overlap. In some embodiments, the series of doses of the first therapeutic agent and the series of doses of the at least one additional therapeutic agent may be interspersed with each other. A dose referred herein can be a single administration of a therapeutic agent. A therapeutic regimen or dosing regimen may also be referred to a dosing schedule, or schedule or any variants thereof.

[0213] In one embodiment, the engineered nucleic acid encoding TROP2 targeting CFP is the first therapeutic agent and is administered in combination with the at least one additional therapeutic agent that may be delivered simultaneously or independent of each other to a subject in need thereof. In some embodiments, the at least one additional therapeutic agent comprises an anti -neoplastic drug. In some embodiments, the at least one additional therapeutic agent comprises a chemotherapy agent. In some embodiments, the at least one additional therapeutic agent comprises an immune checkpoint inhibitor. In some embodiments, the at least one additional therapeutic agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof comprises an antibody or antigen binding fragment specifically binding to an immune checkpoint. In some embodiments, the immune checkpoint is programmed cell death protein (PD-1). In some embodiments, the at least one additional therapeutic agent comprises nivolumab or pembrolizumab. In some embodiments, the antibody or antigen binding fragment thereof comprises an antibody or antigen binding fragment specifically binding to a cancer antigen. In some embodiments, the cancer antigen is HER2. In some embodiments, at least one additional therapeutic agent comprises trastuzumab, pertuzumab, or margetuximab. In some embodiments, the at least one additional therapeutic agent comprises capecitabine and oxaliplatin (i.e., “CapOx” or “Capox”). In some embodiments, the at least one additional therapeutic agent comprises capecitabine, oxaliplatin, nivolumab, or trastuzumab, or any combination thereof.

[0214] Capecitabine is a nucleoside metabolic inhibitor with antineoplastic activity indicated for the treatment of adjuvant colon cancer, metastatic colorectal cancer, and metastatic breast cancer. For example, in the EU, capecitabine is a standard of care agent for the first-line treatment of first-line treatment of gastroesophageal cancer in a platinum-based regimen. Oxaliplatin is a platinum-based drug used in combination with a fluoropyrimidine, that is indicated for the treatment of advanced colorectal cancer or for adjuvant treatment of stage III colon cancer in patients who have undergone complete resection of the primary tumor. Oxaliplatin is a standard of care agent for the first-line treatment of gastroesophageal cancer in combination with aWSGR Docket No. 56371-768.601 fluoropyrimidine. Nivolumab is a PD-1 blocking antibody which releases PD-1 pathway- mediated inhibition of the immune response, including the antitumor response. Nivolumab is indicated for the treatment of various types of cancers, including locally advanced or metastatic gastroesophageal cancer expressing PD-L1. Trastuzumab is a monoclonal antibody that targets HER2. It is indicated for treatment of advanced HER2 overexpressing gastroesophageal cancers in combination with platinum and fluoropyrimidine based regimens.

[0215] In some embodiments, the at least one additional therapeutic agent is CapOx. CapOx is a combination therapeutic of capecitabine and oxaliplatin. In some embodiments, a single dose (a dose) of oxaliplatin may be 130 mg / m2 of a subject, administering intravenously (IV) within a span of 24 hours. In some embodiments, a single dose (a dose) of capecitabine in the combination with oxaliplatin as indicated above may comprise capecitabine at 1000 mg / m2 (m2 referring to body surface area) of a subject, administering orally; and is administered 2 doses per day (twice daily), wherein a single dose of capecitabine 1000 mg / m2 in the morning and a single dose of capecitabine 1000 mg / m2 in the evening. In some embodiments the above dose and / or the above dose regimen may be considered standard of care for the specific components, or for the specific therapeutic molecules. Variations of the above are encompassed within the scope of this disclosure, comprising about 2%, about 5%, about 10%, about 20%, or about 50% variation and any range within, for example, of the single dose of oxaliplatin, combined with the given dose of, or a variation of a dose of capecitabine comprising about 2%, about 5%, about 10%, about 20% variation and any range within, of the indicated dose of capecitabine.

[0216] In some embodiments, the at least one additional therapeutic agent comprises an anti- PDL1 therapy. In some embodiments, the anti-PDLl therapy is nivolumab. A single dose of nivolumab may comprise 240 mg / kg (kg referring to body weight) of a subject, administered as IV. A single dose of nivolumab may comprise 360 mg / kg (kg referring to body weight) of a subject, administered as IV, once every 3 weeks. In some embodiments the above dose and / or the above dose regimen may be considered standard of care for nivolumab. Variations of the above are encompassed within the scope of this disclosure, comprising about 2%, about 5%, about 10%, about 20%, or about 50% variation and any range within for a dose of nivolumab. Nivolumab may be administered at a dose regimen of a single dose, once every 3 weeks. Nivolumab may be administered at a dose regimen of once every 2 weeks. Nivolumab may be administered at a dose regimen of once every week. Nivolumab may be administered at a dose regimen of once every 15 days, once every 13 days, once every 12 days, once every 11 days, once every 10 days, once every 9 days, once every 8 days, once every 6 days, once every 5 days, once every 4 days, once every 3 days, once every 2 days, once every day, or any time intervals ranging in between. In some embodiments, the anti-PDLl therapy is pembrolizumab (Keytruda). PembrolizumabWSGR Docket No. 56371-768.601(KEYTRUDA®) in combination with chemotherapy (typically CAPOX or fluoropyrimidine + cisplatin) is approved for first-line treatment of PD-L1 -positive advanced or metastatic gastric and gastroesophageal junction (GEJ) cancers.

[0217] In some embodiments, the at least one additional therapeutic agent is trastuzumab. A single dose of trastuzumab may comprise about 6 mg / kg administered as IV. A single dose of trastuzumab may comprise about 6 mg / kg administered as IV.

[0218] Other drugs and combinations may be administered to the subject, which may comprise an anti-emetic, analgesic, anti-pyretic, anti -diarrheal, anti-inflammatory drug. In some embodiments, the additional drug comprises an anti-emetic drug, and wherein the anti-emetic drug comprises olanzapine, 5-HT3 receptor antagonist, or NK-1 receptor antagonist. In some embodiments, the additional drug further comprises a steroid. In some embodiments, the steroid comprises methylprednisolone. In some embodiments, the 5-HT3 receptor antagonist is ondansetron or palonosetron. In some embodiments, the NK-1 receptor antagonist is aprepitant or fosaprepitant.

[0219] In some embodiment, one or more additional drugs may be administered to the subject prior to treatment with the pharmaceutical composition comprising the engineered nucleic acid encoding a CFP, e.g., the anti-TROP2 CFP, wherein the one or more additional drug is a prophylactic drug. In some embodiments, the prophylactic drug is a drug to prevent or treat cytokine release syndrome (CRS). In some embodiments, the prophylactic agent is an antiinterleukin (IL) agent. In some embodiments, the anti-IL agent is an IL receptor inhibitor. In some embodiments, the IL receptor inhibitor is an antibody. In some embodiments, the IL receptor inhibitor is a small molecule. In some embodiments, the IL receptor inhibitor comprises an IL-1 receptor inhibitor. In some embodiments, the IL receptor inhibitor comprises an IL-6 receptor inhibitor. In some embodiments, the prophylactic drug comprises tocilizumab. In some embodiments, the prophylactic drug comprises anakinra. In some embodiments, the prophylactic drug comprises tocilizumab and anakinra. In some embodiments, administration of a prophylactic drug may reduce inflammation. In some embodiments, administration of a prophylactic drug may reduce risk of inflammation. In some embodiments, administration of a prophylactic drug may reduce risk of cytokine release syndrome (CRS). In some embodiments, administration of a prophylactic drug such as anakinra and / or tocilizumab may mitigate the risk of CRS. In some embodiments, prophylactic administration of anakinra may mitigate the risk of CRS or other inflammatory syndrome. In some embodiments, prophylactic administration of tocilizumab may mitigate the risk of CRS or other inflammatory syndrome. CRS is a form of systemic inflammatory response syndrome that arises as a complication of some diseases or infections, and is also an adverse effect of some treatment such as adoptive immune cellWSGR Docket No. 56371-768.601 therapies. Any of the methods described herein may involve monitoring a patient for CRS, e.g., a CRS event following commencement of any of the methods described above. CRS can be associated with elevations in a wide array of cytokines, including marked elevations in IL-1, IFN-y, IL-6, and TNF-a levels. CRS can be associated with IL-6 levels. IL-6 is a proinflammatory, multi-functional cytokine produced by a variety of cell types, which has been shown to be involved in a diverse array of physiological processes, including T cell activation. In some embodiments, the reduction of CRS can be measured based on the cytokine level changes before and after the treatment. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IFN-y. In some embodiments, the cytokine is TNF-a. In some embodiments, the cytokine is IL-1. In some embodiments, the reduction of CRS can be assessed by the ASTCT grading system.

[0220] In one embodiment, anti-emetic treatments are administered to the subject prior to during and after the anti-TROP2 CFP drug therapy. For example, in some embodiments, premedication prior to oxaliplatin infusion may include standard doses of olanzapine, a 5-HT3 receptor antagonist and an NK-1 receptor antagonist, or per alternative institutional guidelines. After initial treatments, steroids (e.g., methylprednisolone or equivalent) or other anti-emetics may be added according to the SmPC.

[0221] Olanzapine may be frequently used in combination with other antiemetics, such as 5-HT3 antagonists (e.g., ondansetron) and NKl receptor antagonists (e.g., aprepitant), although olanzapine itself can be effective when used alone in many cases. In some embodiments, a potential steroid-sparing triple-combination regimen for managing chemotherapy -induced nausea and vomiting (CINV) can include: Olanzapine: 5-10 mg of olanzapine can be administered orally the evening before chemotherapy and then continued daily for up to 4 days after chemotherapy, based on efficacy and tolerability.

[0222] 5 -HT3 Receptor Antagonist (e.g., ondansetron, palonosetron) may be administered on the day of chemotherapy to prevent acute CINV. Palonosetron (given once, as it has a longer half-life) is often preferred for single-day dosing, while ondansetron may be administered on multiple days. NK-1 Receptor Antagonist (e.g., aprepitant or fosaprepitant): NK-1 antagonist can be added for help against delayed nausea and vomiting, especially with highly emetogenic chemotherapy. A single dose of fosaprepitant or a 3-day regimen of aprepitant can be given in combination with olanzapine and a 5-HT3 antagonist.

[0223] In one aspect, the treatment objective is to reduce or ameliorate at least one symptom associated with the disease. In some embodiments, a reduction of at least one symptom typically associated with the disease is considered as an effect of the treatment. The reduction in the symptom may be evident over a continuous period of time in order to be considered as aWSGR Docket No. 56371-768.601 reduction consequent to the treatment, for example, depending on the symptom, the reduction may last or be evident continuously over a period of 2, 3, 4, 5, 6 or more months. In one embodiment, the at least one symptom associated with cancer may be a reduction in tumor volume. In some embodiments, a reduction in tumor volume may be 10% reduction compared to the tumor volume prior to the commencement of the therapeutic. In some embodiments, a reduction in tumor volume may be 11% reduction, 12% reduction, 13% reduction, 14% reduction, 15% reduction, 16% reduction, 17% reduction, 18% reduction, 19% reduction, 20% reduction, 25% reduction, 30% reduction, 35% reduction, 40% reduction, 50% reduction, 60% reduction, 70% reduction, 80% reduction, 90% reduction, 95% reduction, 99% reduction or 100% reduction, and any % within the range. In some embodiments, an effect of the therapy described herein may be reduction in tumor volume compared to the volume of tumor prior to commencement of the therapy by about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the therapeutic is considered effective if there is a reduction in tumor volume compared to the volume of tumor by about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the therapeutic is considered effective if there is a reduction in tumor volume compared to the volume of tumor by about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% that is persistent over a period of time of at least 2 months or as indicated elsewhere in the disclosure.

[0224] In some embodiments, the combination treatment method results in more reduction of symptoms as compared to treatment without the additional therapeutic agent. In someWSGR Docket No. 56371-768.601 embodiments, the combination treatment method results in more reduction of tumor volume as compared to treatment without the additional therapeutic agent. In some embodiments, a reduction in tumor volume may be 10% reduction compared to the tumor volume treated without the additional therapeutic agent. In some embodiments, a reduction in tumor volume may be 11% reduction, 12% reduction, 13% reduction, 14% reduction, 15% reduction, 16% reduction, 17% reduction, 18% reduction, 19% reduction, 20% reduction, 25% reduction, 30% reduction, 35% reduction, 40% reduction, 50% reduction, 60% reduction, 70% reduction, 80% reduction, 90% reduction, 95% reduction, 99% reduction or 100% reduction, and any % within the range. In some embodiments, an effect of the therapy described herein may be reduction in tumor volume compared to the volume of tumor treated without the additional therapeutic agent by about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0225] It is an objective of the instant application that a therapeutic described herein, alone or in combination of one or more therapeutic components stalls a tumor (or lesion) growth, and halts progressive disease (PD) in a subject to whom the therapeutic is administered. Provided herein is a therapeutic, comprising one or more components, at an effective amount and dosing regimen such that after administration of the therapeutic in the dosing regimen, the progression of a tumor or a lesion may be halted or stabilized, and may result in at least a stable disease in the subject. An effective amount or dose of a pharmaceutical composition may be the amount per single administration that is well tolerated, (e.g., with minimum adverse effects), and a stabilization or reduction of a tumor volume may be noted. Provided herein is a therapeutic, comprising one or more components, at an effective amount and dosing regimen such that after administration of the therapeutic in the dosing regimen, a reduction in a tumor or a lesion may be noted that continues over a period of 2, 3, 4, 5, 6, 12, 18 or more months. Provided herein is a therapeutic, comprising one or more components, at an effective amount and dosing regimen such that after administration of the therapeutic in the dosing regimen, an amelioration or abrogation of a tumor or a lesion may be noted without observed resurgence for more than 2, 3, 4, 5, 6, 12, 18 or more months.

[0226] In some embodiments, the pharmaceutical composition is administered to the subject about once a week (QW), about once every 2 weeks (Q2W), about once every 3 weeks (Q3W), about once every 4 weeks (Q4W), about once every 5 weeks (Q5W), about once every 6 weeksWSGR Docket No. 56371-768.601(Q6W), about once every 7 weeks (Q7W), about once every 8 weeks (Q8W), about once every 9 weeks (Q9W), about once every 10 weeks (Q10W), about once every 11 weeks (QI 1W), or about once every 12 weeks (Q12W) for reach of the cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject QW or Q2W or Q4W for each of the cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject with the same frequency in each of the treatment cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject with different frequencies for at least two of the treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject QW or Q2W in the first treatment cycle.

[0227] In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 times. In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject at least 1 to at least 3, at least 3 to at least 6, at least 6 to at least 9, or at least 9 to at least 12 times. In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 times. In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject at most 1 to at most 3, at most 3 to at most 6, at most 6 to at most 9, or at most 9 to at most 12 times. In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject between 1 to 12 times.

[0228] In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times.

[0229] In some embodiments, the first cycle comprises administering the pharmaceutical composition to the subject 3 times.

[0230] In some embodiments, a second cycle follows the first cycle. In some embodiments, the method further comprises administering the pharmaceutical composition to the subject during the second cycle.

[0231] In some embodiments, the pharmaceutical composition is administered to the subject about QW, about Q2W, about Q3W, about Q4W, about Q5W, about Q6W, about Q7W, about Q8W, about Q9W, about Q10W, about QI 1W, or about Q12W during the second cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject Q4W during the second cycle.WSGR Docket No. 56371-768.601

[0232] In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 times. In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject at least 1 to at least 3, at least 3 to at least 6, at least 6 to at least 9, or at least 9 to at least 12 times. In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 times. In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject at most 1 to at most 3, at most 3 to at most 6, at most 6 to at most 9, or at most 9 to at most 12 times. In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject between 1 to 12 times. In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times.

[0233] In some embodiments, the second cycle comprises administering the pharmaceutical composition to the subject 3 times.

[0234] In some embodiments, the method comprises administering the pharmaceutical composition to the subject for three treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject at the same dose for each of the three treatment cycles.

[0235] In some embodiments, the method comprises administering the pharmaceutical composition to the subject for four treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject every 2 weeks in the first cycle and every 4 weeks in the second, third and fourth cycle. In some embodiments, the method comprising administering the pharmaceutical composition at a dose from about 0.0050 to about 0.05 mg / kg or from about 0.0075 to about 0.03 mg / kg. In some embodiments, the method comprises administering the pharmaceutical composition to the subject once a week in the first cycle and once every 4 weeks in the second, third, and fourth cycle.

[0236] In some embodiments, the method comprises administering an effective amount of the pharmaceutical composition to the subject.

[0237] In some embodiments, the effective amount of the pharmaceutical composition ranges from about 0.001 to about 0.0015, about 0.0015 to about 0.002, about 0.002 to about 0.0025, about 0.0025 to about 0.003, about 0.003 to about 0.0035, about 0.0035 to about 0.004, about 0.004 to about 0.0045, about 0.0045 to about 0.005, about 0.005 to about 0.0055, about 0.0055 to about 0.006, about 0.006 to about 0.0065, about 0.0065 to about 0.007, about 0.007 to aboutWSGR Docket No. 56371-768.6010.0075, about 0.0075 to about 0.008, about 0.008 to about 0.0085, about 0.0085 to about 0.009, about 0.009 to about 0.0095, or about 0.0095 to about 0.01 mg / kg of the RNA.

[0238] In some embodiments, the effective amount of the pharmaceutical composition ranges from about 0.01 to about 0.015, about 0.015 to about 0.02, about 0.02 to about 0.025, about 0.025 to about 0.03, about 0.03 to about 0.035, about 0.035 to about 0.04, about 0.04 to about 0.045, about 0.045 to about 0.05, about 0.05 to about 0.055, about 0.055 to about 0.06, about 0.06 to about 0.065, about 0.065 to about 0.07, about 0.07 to about 0.075, about 0.075 to about 0.08, about 0.08 to about 0.085, about 0.085 to about 0.09, about 0.09 to about 0.095, or about 0.095 to about 0.1 mg / kg of the RNA.

[0239] In some embodiments, the effective amount of the pharmaceutical composition comprises about 0.001, about 0.0015, about 0.002, about 0.0025, about 0.003, about 0.0035, about 0.004, about 0.0045, about 0.005, about 0.0055, about 0.006, about 0.0065, about 0.007, about 0.0075, about 0.008, about 0.0085, about 0.009, about 0.0095, or about 0.01 mg / kg of the RNA.

[0240] In some embodiments, the effective amount of the pharmaceutical composition comprises about 0.01, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035, about 0.04, about 0.045, about 0.05, about 0.055, about 0.06, about 0.065, about 0.07, about 0.075, about 0.08, about 0.085, about 0.09, about 0.095, or about 0.1 mg / kg of the RNA.

[0241] In some embodiments, the effective amount of the pharmaceutical composition is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IV infusion doses.Exemplary Embodiment Set 1

[0242] Embodiment 1. A pharmaceutical formulation, comprising: (I) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR3 sequence comprises a sequence of GGFGSSYWYFDV; and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR3 sequence comprises a sequence of QQHYITPLT; (b) a CD89 transmembrane domain operatively linked to the extracellular domain; and (II) a lipid nanoparticle delivery vehicle encapsulating the RNA in (I).

[0243] Embodiment 2. The pharmaceutical formulation of Embodiment 1, wherein the pharmaceutical formulation is an aqueous formulation.

[0244] Embodiment 3. The pharmaceutical formulation of Embodiment 1 or 2, wherein the TROP-2 binding domain comprises an scFV.WSGR Docket No. 56371-768.601

[0245] Embodiment 4. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the HCDR1 comprises a sequence of WINTYTGEPTYTDDFKG, and the HCDR2 comprises a sequence of GGFGSSYWYFDV.

[0246] Embodiment 5. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the LCDR1 comprises a sequence of KASQDVSIAVA and the LCDR2 comprises a sequence of SASYRYT.

[0247] Embodiment 6. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the TROP-2 binding domain comprises a sequence that is at least 90% identical to a sequence of QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSS or a sequence that is at least 90% identical to a sequence of DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPD RF SGSGSGTDFTLTIS SLQPEDF AVYYCQQHYITPLTFGAGTKVEIKR.

[0248] Embodiment 7. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the TROP-2 binding domain comprises: a heavy chain variable domain, comprising (i) an HCDR1, comprising a sequence of NYGMN; (ii) an HCDR2, comprising a sequence of WINTYTGEPTYTDDFKG; and (iii) an HCDR3, comprising a sequence of GGFGSSYWYFDV; and a light chain variable domain, comprising (i) an LCDR1, comprising a sequence of KASQDVSIAVA; (ii) an LCDR2, comprising a sequence of SASYRYT; and (iii) an LCDR3, comprising a sequence of QQHYITPLT.

[0249] Embodiment 8. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the TROP-2 binding domain comprises a sequence that is at least 90% identical to a sequence of QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTG EPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGS LVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFG AGTKVEIKR.

[0250] Embodiment 9. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs 4, 5, 7, 8, 9, 13, 14, 36, 34, 36, and 194.

[0251] Embodiment 10. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs 14, 33, 36 and 194.WSGR Docket No. 56371-768.601

[0252] Embodiment 11. The pharmaceutical formulation of any one of the preceding Embodiments, wherein RNA a sequence is the sequence set forth in SEQ ID NO: 14, 33, 36 and 194; or a sequence having at least 90% identity with a sequence set forth in SEQ ID NOs: 14, 33, 36 and 194.

[0253] Embodiment 12. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the RNA comprises a 5’UTR sequence or a 3’ UTR sequence or both.

[0254] Embodiment 13. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the RNA comprises a 5’UTR sequence with at least 90% identity of (i) GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACC, or (ii) AGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACC.

[0255] Embodiment 14. The pharmaceutical formulation of Embodiment 11 or 13, wherein the RNA further comprises a sequence encoding a pro-inflammatory polypeptide.

[0256] Embodiment 15. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the CD89 transmembrane domain comprises a sequence that is at least 90% identical to a sequence of LIRMAVAGLVLVALLAILV.

[0257] Embodiment 16. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the TROP-2 binding domain is connected to the CD89 transmembrane domain by an extracellular linker sequence or a hinge domain.

[0258] Embodiment 17. The pharmaceutical formulation of Embodiment 16, wherein the extracellular linker sequence or the hinge domain comprises a sequence that is at least 90% identical to a sequence of SGGGGAAAGS or GSGGSDSH4QDYTTQN.

[0259] Embodiment 18. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the extracellular domain comprises a CD89 extracellular domain.

[0260] Embodiment 19. The pharmaceutical formulation of Embodiment 18, wherein the CD89 extracellular domain comprises a sequence that is at least 90% identical to a sequence of DSH4QDYTTQN.

[0261] Embodiment 20. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the CFP further comprises a CD89 intracellular domain.

[0262] Embodiment 21. The pharmaceutical formulation of Embodiment 20, wherein the CD89 intracellular domain comprises a sequence that is at least 90% identical to a sequence of ENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK.

[0263] Embodiment 22. The pharmaceutical formulation of Embodiment 20 or 21, wherein the CD89 intracellular domain is linked to the CD89 transmembrane domain.WSGR Docket No. 56371-768.601

[0264] Embodiment 23. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the lipid nanoparticle delivery vehicle comprises a cationic lipid, which is an ionizable lipid.

[0265] Embodiment 24. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the lipid nanoparticle delivery vehicle comprises a PEG-lipid that comprises the chemical formulawherein, n is an integer between 46 and 52.

[0266] Embodiment 25. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the ratio of mRNA to ionizable lipid (N / P) is 6.0.

[0267] Embodiment 26. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the lipid nanoparticle is 34-75 nm in diameter.

[0268] Embodiment 27. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the formulation is lyophilizeable.

[0269] Embodiment 28. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the formulation has a stable shelf life of at least 6 months.

[0270] Embodiment 29. The pharmaceutical formulation of any one of the precedingEmbodiments, wherein the concentration of the RNA is between about 1.5 to about 0.5 mg / mL or between about 1.3 to about 0.7 mg / mL when stored in a container.

[0271] Embodiment 30. The pharmaceutical formulation of Embodiment 29, wherein the container is a single-use or multi-use vial.

[0272] Embodiment 31. The pharmaceutical formulation of any one of the preceding Embodiments, wherein the RNA is mRNA.

[0273] Embodiment 32. A lyophilized composition of the pharmaceutical formulation of any one of the preceding Embodiments, further comprising a lyoprotectant.

[0274] Embodiment 33. The lyophilized composition of Embodiment 32, wherein the lyoprotectant is sucrose.

[0275] Embodiment 34. The lyophilized composition of Embodiment 32 or 33, which has a stable shelf life of at least 6 months.

[0276] Embodiment 35. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical formulation of any one of the preceding Embodiments.

[0277] Embodiment 36. The method of Embodiment 35, wherein the subject is human.WSGR Docket No. 56371-768.601

[0278] Embodiment 37. The method of Embodiment 35 or 36, wherein the cancer comprises epithelial cancer.

[0279] Embodiment 38. The method of any one of Embodiments 35-37, wherein the cancer is advanced or metastatic.

[0280] Embodiment 39. The method of any one of Embodiments 35-38, wherein the cancer comprises a tumor.

[0281] Embodiment 40. The method of any one of Embodiments 35-39, wherein the cancer is selected from the group consisting of cervical cancer, colorectal cancer, esophageal, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, and urothelial cancer.

[0282] Embodiment 41. The method of any one of Embodiments 35-40, wherein the administering is associated with a reduction of, or amelioration of the tumor in the subject.

[0283] Embodiment 42. The method of any one of Embodiments 35-41, wherein treating comprises reducing the tumor by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or at least 10% after the treatment.

[0284] Embodiment 43. The method of any one of Embodiments 35-42, wherein treating comprises alleviating at least one of the symptoms associated with cancer.

[0285] Embodiment 44. The method of any one of Embodiments 35-43, wherein administering comprises administering to the subject intravenously.

[0286] Embodiment 45. The method of any one of Embodiments 35-44, wherein the RNA is expressed in vascularized tissues of the subject after infusion.

[0287] Embodiment 46. The method of any one of Embodiments 35-45, wherein the RNA is expressed predominantly in myeloid cells in vivo.

[0288] Embodiment 47. The method of any one of Embodiments 35-46, wherein the expression of a sequence encoded by the RNA is detectable in a cell within the subject for about 72 hours post infusion.

[0289] Embodiment 48. The method of any one of Embodiments 35-47, wherein the administration of the pharmaceutical formulation does not generate a cytokine response.

[0290] Embodiment 49. The method of any one of Embodiments 35-48, wherein the expression of a component of the lipid nanoparticle delivery vehicle under (ii) is not persistent in detectable levels in vivo after 7 days of infusion.

[0291] Embodiment 50. The method of any one of Embodiments 35-49, wherein the method comprises administering the pharmaceutical formulation to the subject for at least one treatment cycle.WSGR Docket No. 56371-768.601

[0292] Embodiment 51. The method of any one of Embodiments 35-50, wherein the method comprises administering the pharmaceutical formulation to the subject for at least 2, 3, 4, or 5 treatment cycles.

[0293] Embodiment 52. The method of Embodiment 50 or 51, wherein the method comprises administering the pharmaceutical formulation to the subject about once a week, about once every2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about or once every 9 weeks in each of the treatment cycle.

[0294] Embodiment 53. The method of Embodiment 51 or 52, wherein the method comprises administering the pharmaceutical formulation to the subject once a week or once every 2 weeks during the first treatment cycle.

[0295] Embodiment 54. The method of any one of Embodiments 50-53, wherein the method comprises administering the pharmaceutical formulation to the subject at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 times for each treatment cycle.

[0296] Embodiment 55. The method of any one of Embodiments 50-54, wherein the method comprises administering the pharmaceutical formulation to the subject about 1 to about 3, about3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle.

[0297] Embodiment 56. The method of any one of Embodiments 50-55, wherein the method comprises administering the pharmaceutical formulation to the subject at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 times for each treatment cycle.

[0298] Embodiment 57. The method of any one of Embodiments 50-56, wherein the method comprises administering the pharmaceutical formulation to the subject between 1 to 12 times for each treatment cycle.

[0299] Embodiment 58. The method of any one of Embodiments 49-56, wherein the method comprises administering the pharmaceutical formulation to the subject between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle.

[0300] Embodiment 59. The method of any one of Embodiments 50-58, wherein the method comprises administering the pharmaceutical formulation to the subject 3 times in a first cycle.

[0301] Embodiment 60. The method of any one of Embodiments 50-59, wherein a second cycle follows a first cycle.

[0302] Embodiment 61. The method of any one of Embodiments 50-60, wherein the method further comprises administering the pharmaceutical formulation to the subject during the second cycle.WSGR Docket No. 56371-768.601

[0303] Embodiment 62. The method of Embodiment 61, wherein the pharmaceutical formulation is administered to the subject about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, or about once every 9 weeks during the second cycle.

[0304] Embodiment 63. The method of Embodiment 62, wherein the method comprises administering the pharmaceutical formulation to the subject once every 4 weeks during the second cycle.

[0305] Embodiment 64. The method of any one of Embodiments 61-63, wherein the method comprises administering the pharmaceutical formulation to the subject at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 times in the second cycle.

[0306] Embodiment 65. The method of any one of Embodiments 61-64, wherein the method comprises administering the pharmaceutical formulation to the subject from about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times in the second cycle.

[0307] Embodiment 66. The method of any one of Embodiments 61-65, wherein the method comprises administering the pharmaceutical formulation to the subject at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 times in the second cycle.

[0308] Embodiment 67. The method of any one of Embodiments 61-66, wherein the method comprises administering the pharmaceutical formulation to the subject between 1 to 12 times in the second cycle.

[0309] Embodiment 68. The method of any one of Embodiments 61-67, wherein the method comprises administering the pharmaceutical formulation to the subject between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times in the second cycle.

[0310] Embodiment 69. The method of any one of Embodiments 61-68, wherein the method comprises administering the pharmaceutical formulation to the subject 3 times in the second cycle.

[0311] Embodiment 70. The method of any one of Embodiments 61-69, wherein an effective amount of the pharmaceutical formulation ranges from 0.01 mg / kg / dose to 3.0 mg / kg / dose.

[0312] Embodiment 71. The method of any one of Embodiments 61-70, wherein an effective amount of the pharmaceutical formulation ranges from 0.05 mg / kg / dose to 2.5 mg / kg / dose.

[0313] Embodiment 72. The method of any one of Embodiments 61-71, wherein an effective amount of the pharmaceutical formulation ranges from 0.1 mg / kg / dose to 1.0 mg / kg / dose.WSGR Docket No. 56371-768.601

[0314] Embodiment 73. The method of any one of Embodiments 61-72, wherein an effective amount of the pharmaceutical formulation comprises about 0.1, 0.15, 0.2, 0.22, 0.25, 0.275, 0.3, 0.32, 0.33, 0.34, 0.35, 0.36, 0.38, 0.4, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.7, 0.8, 0.9 or 1.0 mg / kg / dose.

[0315] Embodiment 74. The method of any one of Embodiments 61-73, wherein an effective amount of the pharmaceutical formulation ranges from about 0.001 to about 0.0015, about 0.0015 to about 0.002, about 0.002 to about 0.0025, about 0.0025 to about 0.003, about 0.003 to about 0.0035, about 0.0035 to about 0.004, about 0.004 to about 0.0045, about 0.0045 to about 0.005, about 0.005 to about 0.0055, about 0.0055 to about 0.006, about 0.006 to about 0.0065, about 0.0065 to about 0.007, about 0.007 to about 0.0075, about 0.0075 to about 0.008, about 0.008 to about 0.0085, about 0.0085 to about 0.009, about 0.009 to about 0.0095, or about 0.0095 to about 0.01 mg / kg of the RNA.

[0316] Embodiment 75. The method of any one of Embodiments 61-74, wherein an effective amount of the pharmaceutical formulation ranges from about 0.01 to about 0.015, about 0.015 to about 0.02, about 0.02 to about 0.025, about 0.025 to about 0.03, about 0.03 to about 0.035, about 0.035 to about 0.04, about 0.04 to about 0.045, about 0.045 to about 0.05, about 0.05 to about 0.055, about 0.055 to about 0.06, about 0.06 to about 0.065, about 0.065 to about 0.07, about 0.07 to about 0.075, about 0.075 to about 0.08, about 0.08 to about 0.085, about 0.085 to about 0.09, about 0.09 to about 0.095, or about 0.095 to about 0.1 mg / kg of the RNA.

[0317] Embodiment 76. The method of any one of Embodiments 61-75, wherein an effective amount of the pharmaceutical formulation comprises about 0.001, about 0.0015, about 0.002, about 0.0025, about 0.003, about 0.0035, about 0.004, about 0.0045, about 0.005, about 0.0055, about 0.006, about 0.0065, about 0.007, about 0.0075, about 0.008, about 0.0085, about 0.009, about 0.0095, or about 0.01 mg / kg of the RNA.

[0318] Embodiment 77. The method of any one of Embodiments 61-76, wherein an effective amount of the pharmaceutical formulation comprises about 0.01, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035, about 0.04, about 0.045, about 0.05, about 0.055, about 0.06, about 0.065, about 0.07, about 0.075, about 0.08, about 0.085, about 0.09, about 0.095, or about 0.1 mg / kg of the RNA.

[0319] Embodiment 78. The method of any one of Embodiments 61-77, wherein the effective amount of the pharmaceutical formulation is administered in a 60-minute intravenous (IV) infusion per dose.

[0320] Embodiment 79. The method of any one of Embodiments 61-78, wherein the effective amount of the pharmaceutical formulation is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IV infusion doses.WSGR Docket No. 56371-768.601

[0321] Embodiment 80. The method of any one of Embodiments 61-79, wherein the effective amount of the pharmaceutical formulation is administered at an interval of once in a week.

[0322] Embodiment 81. The method of any one of Embodiments 61-80, wherein the effective amount of the pharmaceutical formulation is administered at an interval of once every 10 days.

[0323] Embodiment 82. The method of any one of Embodiments 61-81, wherein the effective amount of the pharmaceutical formulation is administered at an interval of once every 2 weeks.

[0324] Embodiment 83. The method of any one of Embodiments 61-82, wherein the effective amount of the pharmaceutical formulation is administered for at least 5 doses.

[0325] Embodiment 84. The method of any one of Embodiments 35-83, wherein the subject exhibited progressive disease at baseline, or a refractory disease or a relapse in response to standard of care.Exemplary Embodiment Set 2

[0326] Embodiment 1. A composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding TROP2, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5, 33, or 34.

[0327] Embodiment 2. The composition of Embodiment 1, wherein the nucleic acid comprises a sequence having at least 95%sequence identity to SEQ ID NO: 5, 33, or 34.

[0328] Embodiment 3. The composition of Embodiment 1 or 2, wherein the nucleic acid comprises a sequence of SEQ ID NO:5.

[0329] Embodiment 4. The composition of Embodiment 1 or 2, wherein the nucleic acid comprises a sequence of SEQ ID NO:33.

[0330] Embodiment 5. The composition of Embodiment 1 or 2, wherein the nucleic acid comprises a sequence of SEQ ID NO:34.

[0331] Embodiment 6. The composition of any one of Embodiments 1-5, wherein the nucleic acid further comprises a 5’ UTR.

[0332] Embodiment 7. The composition of Embodiment 6, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 7.

[0333] Embodiment 8. The composition of Embodiment 6, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 8.

[0334] Embodiment 9. The composition of any one of Embodiments 1-8, wherein the nucleic acid further comprises a 3’ UTR.

[0335] Embodiment 10. The composition of Embodiment 9, wherein the 3’ UTR is a bGH 3’ UTR.WSGR Docket No. 56371-768.601

[0336] Embodiment 11. The composition of Embodiment 10, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 9.

[0337] Embodiment 12. The composition of Embodiment 9, wherein the 3’ UTR is a a globin 3’ UTR.

[0338] Embodiment 13. The composition of Embodiment 12, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 10.

[0339] Embodiment 14. The composition of Embodiment 9, wherein the 3’ UTR comprises one or more microRNA binding site.

[0340] Embodiment 15. The composition of Embodiment 14, wherein the 3’ UTR comprises two, three, four, or five microRNA binding sites.

[0341] Embodiment 16. The composition of Embodiment 15, wherein the 3’ UTR comprises four microRNA binding sites.

[0342] Embodiment 17. The composition of any one of Embodiments 14-16, wherein the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p.

[0343] Embodiment 18. The composition of Embodiment 17, wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11.

[0344] Embodiment 19. The composition of Embodiment 17 or 18, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 12.

[0345] Embodiment 20. The composition of Embodiment 17 or 18, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 24.

[0346] Embodiment 21. The composition of any one of Embodiments 9-19, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 13.

[0347] Embodiment 22. The composition of any one of Embodiments 1-19, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 16.

[0348] Embodiment 23. The composition of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 16.

[0349] Embodiment 24. The composition of any one of Embodiments 1-23, wherein the nucleic acid is an RNA.

[0350] Embodiment 25. The composition of Embodiment 24, wherein the RNA is an mRNA.

[0351] Embodiment 26. The composition of any one of Embodiments 1-25, wherein the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 33.

[0352] Embodiment 27. The composition of any one of Embodiments 1-25, wherein the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 36.WSGR Docket No. 56371-768.601

[0353] Embodiment 28. The composition of any one of Embodiments 1-27, wherein the composition further comprises a nucleic sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO:38.

[0354] Embodiment 29. The composition of any one of Embodiments 1-28, wherein the composition further comprises a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7.

[0355] Embodiment 30. A composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding GPC3, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 41.

[0356] Embodiment 31. The composition of Embodiment 30, wherein the nucleic acid comprises a sequence having at least 95% sequence identity to SEQ ID NO: 41.

[0357] Embodiment 32. The composition of Embodiment 30 or 31, wherein the nucleic acid comprises a sequence of SEQ ID NO: 41.

[0358] Embodiment 33. The composition of any one of Embodiments 30-32, wherein the nucleic acid further comprises a 5’ UTR.

[0359] Embodiment 34. The composition of Embodiment 33, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 42.

[0360] Embodiment 35. The composition of any one of Embodiments 30-34, wherein the nucleic acid further comprises a 3’ UTR.

[0361] Embodiment 36. The composition of Embodiment 35, wherein the 3’ UTR is a a globin 3’ UTR.

[0362] Embodiment 37. The composition of Embodiment 36, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 43.

[0363] Embodiment 38. The composition of Embodiment 36, wherein the 3’ UTR comprises one or more microRNA binding site.

[0364] Embodiment 39. The composition of Embodiment 38, wherein the 3’ UTR comprises two, three, four, or five microRNA binding sites.

[0365] Embodiment 40. The composition of Embodiment 39, wherein the 3’ UTR comprises four microRNA binding sites.

[0366] Embodiment 41. The composition of any one of Embodiments 38-40, wherein the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p.

[0367] Embodiment 42. The composition of Embodiment 41, wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11.

[0368] Embodiment 43. The composition of Embodiment 41 or 42, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 12.WSGR Docket No. 56371-768.601

[0369] Embodiment 44. The composition of any one of Embodiments 9-19, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 13.

[0370] Embodiment 45. The composition of any one of Embodiments 35-44, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 44.

[0371] Embodiment 46. The composition of Embodiment 45, wherein the nucleic acid comprises a sequence of SEQ ID NO: 44.

[0372] Embodiment 47. The composition of any one of Embodiments 30-46, wherein the nucleic acid is an RNA.

[0373] Embodiment 48. The composition of Embodiment 47, wherein the RNA is an mRNA.

[0374] Embodiment 49. The composition of any one of Embodiments 30-48, wherein the composition further comprises a nucleic acid sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38.

[0375] Embodiment 50. The composition of any one of Embodiments 30-49, wherein the composition further comprises a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7.

[0376] Embodiment 51. A pharmaceutical composition, comprising the composition of any one of Embodiments 1-50, and a pharmaceutical acceptable excipient.

[0377] Embodiment 52. The pharmaceutical composition of Embodiment 51, wherein the pharmaceutical composition further comprises a lipid nanoparticle, wherein the nucleic acid is encapsulated in the lipid nanoparticle.

[0378] Embodiment 53. A cell comprising the composition of any one of Embodiments 1-50.

[0379] Embodiment 54. The cell of Embodiment 53, wherein the cell is an immune cell.

[0380] Embodiment 55. The cell of Embodiment 53, wherein the cell is a myeloid cell, a lymphoid cell, a precursor cell, a stem cell or an induced pluripotent cell.

[0381] Embodiment 56. The cell of Embodiment 53, wherein the cell is CD14+ CD16- cell.

[0382] Embodiment 57. A method for treating a patient in need thereof, comprising administering the pharmaceutical composition of Embodiment 51 or 52 to the patient.

[0383] Embodiment 58. A method for treating a patient in need thereof, comprising administering the cell of any one of Embodiments 53-56 to the patient.

[0384] Embodiment 59. The method of Embodiment 57 or 58, wherein the patient has a cancer.

[0385] Embodiment 60. The method of Embodiment 59, wherein the cancer is metastatic.

[0386] Embodiment 61. The method of Embodiment 59 or 60, wherein the cancer is an epithelial cancer.

[0387] Embodiment 62. The method of Embodiment 59 or 60, wherein the cancer is selected from Urothelial, Cervical, Ovarian epithelial, Triple-negative breast, HR+ / HER2- breast,WSGR Docket No. 56371-768.601Pancreatic ductal adenocarcinoma, Gastric adenocarcinoma, Esophageal carcinoma, non-small cell lung and Colorectal cancer.Exemplary Embodiment Set 3

[0388] Embodiment 1. A method of treating a cancer in a subject in need thereof, comprising administering to the subject (i) a composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain operatively linked to the extracellular domain, and a CD89 intracellular domain operatively linked to the transmembrane domain; and (ii) a prophylactic agent for preventing or treating cytokine release syndrome (CRS).

[0389] Embodiment 2. The method of Embodiment 1, wherein the cancer is a breast cancer.

[0390] Embodiment 3. The method of Embodiment 1, wherein the breast cancer is HER2- positive.

[0391] Embodiment 4. Them method of Embodiment 1 or 2, wherein the breast cancer is hormone receptor (HR)-positive.

[0392] Embodiment 5. The method of any one of Embodiments 1-4, wherein the breast cancer is metastatic.

[0393] Embodiment 6. The method of any one of Embodiments 1-5, wherein the breast cancer is advanced.

[0394] Embodiment 7. The method of any one of Embodiments 1-6, wherein the breast cancer is CAI 5.3 -positive or CEA-positive.

[0395] Embodiment 8. The method of Embodiment 1, wherein the cancer is ovarian cancer.

[0396] Embodiment 9. The method of Embodiment 8, wherein the ovarian cancer is CA125- positive.

[0397] Embodiment 10. The method of Embodiment 1, wherein the cancer is gastrointestinal cancer.

[0398] Embodiment 11. The method of Embodiment 10, wherein the gastrointestinal cancer is CEA-positive.

[0399] Embodiment 12. The method of Embodiment 1, wherein the cancer is pancreatic cancer.

[0400] Embodiment 13. The method of Embodiment 12, wherein the pancreatic cancer is CA15.3-positive.

[0401] Embodiment 14. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2.WSGR Docket No. 56371-768.601

[0402] Embodiment 15. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3.

[0403] Embodiment 16. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 39.

[0404] Embodiment 17. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 175.

[0405] Embodiment 18. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 176.

[0406] Embodiment 19. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 177.

[0407] Embodiment 20. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 178.

[0408] Embodiment 21. The method of any one of Embodiments 1-13, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 179.

[0409] Embodiment 22. The method of any one of Embodiments 1-21, wherein the nucleic acid is RNA.

[0410] Embodiment 23. The method of Embodiment 22, wherein the nucleic acid comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 4, 5, 14-16, 33, or 36.

[0411] Embodiment 24. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 4.

[0412] Embodiment 25. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 5.

[0413] Embodiment 26. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 14.

[0414] Embodiment 27. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 15.WSGR Docket No. 56371-768.601

[0415] Embodiment 28. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 16.

[0416] Embodiment 29. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 33.

[0417] Embodiment 30. The method of Embodiment 22, wherein the nucleic acid comprises a sequence of SEQ ID NO: 36.

[0418] Embodiment 31. The method of any one of Embodiments 22-30, wherein the nucleic acid comprises a 5’ UTR, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 7, or a sequence of SEQ ID NO: 8.

[0419] Embodiment 32. The method of any one of Embodiments 22-31, wherein the nucleic acid comprises a 3’ UTR comprises a sequence of SEQ ID NO: 13.

[0420] Embodiment 33. The method of any one of Embodiments 1-32, wherein the prophylactic agent is administered before, after, or simultaneously with the composition comprising the nucleic acid.

[0421] Embodiment 34. The method of any one of Embodiments 1-33, wherein the prophylactic agent is an anti-interleukin (IL) agent.

[0422] Embodiment 35. The method of Embodiment 34, wherein the anti-IL agent is an IL receptor inhibitor.

[0423] Embodiment 36. The method of Embodiment 35, wherein the IL receptor inhibitor comprises tocilizumab or anakinra.

[0424] Embodiment 37. The method of Embodiment 35, wherein the IL receptor inhibitor comprises tocilizumab and anakinra.

[0425] Embodiment 38. The method of Embodiment 36 or 37, wherein the tocilizumab is administered at least one hour before the administration of the composition comprising the nucleic acid.

[0426] Embodiment 39. The method of Embodiment 38, wherein the tocilizumab is administered about one hour before the administration of the composition comprising the nucleic acid.

[0427] Embodiment 40. The method of Embodiment 38 or 39, wherein the tocilizumab is administered at dose of 8 mg / kg (max 800 mg).

[0428] Embodiment 41. The method of any one of Embodiments 36-40, wherein the tocilizumab is administered via IV over 60 minutes.

[0429] Embodiment 42. The method of Embodiment 36 or 37, wherein the anakinra is administered before the administration of the composition comprising the nucleic acid.

[0430] Embodiment 43. The method of Embodiment 33, wherein the anakinra is administered at the time of the administration of the composition comprising the nucleic acid.WSGR Docket No. 56371-768.601

[0431] Embodiment 44. The method of Embodiment 42 or 43, wherein the anakinra is administered subcutaneously.

[0432] Embodiment 45. The method of any one of Embodiments 42-44, wherein the anakinra is administered once daily.

[0433] Embodiment 46. The method of any one of Embodiments 42-45, wherein the anakinra is administered every 12 hours.

[0434] Embodiment 47. The method of Embodiment 45 or 46, wherein the amount of the anakinra administered is 100 mg.

[0435] Embodiment 48. The method of any one of Embodiments 42-47, wherein the anakinra is administered for at least 3 days.

[0436] Embodiment 49. The method of any one of Embodiments 42-47, wherein the anakinra is administered for at least 5 days.

[0437] Embodiment 50. The method of any one of Embodiments 42-47, wherein the anakinra is administered for from about 3 to about 5 days.

[0438] Embodiment 51. The method of any one of Embodiments 42-47, wherein the anakinra is administered intravenously.

[0439] Embodiment 52. The method of Embodiment 51, wherein the amount of the anakinra administered is 2 mg / kg.

[0440] Embodiment 53. The method of Embodiment 51 or 52, wherein the anakinra is administered every 6 hours.

[0441] Embodiment 54. The method of any one of Embodiments 1-53, wherein the composition is administered at a dose of 0.01-1.0 mg / kg of body weight of the subject.

[0442] Embodiment 55. The method of Embodiment 54, wherein the composition is administered at a dose of 0.05-0.15 mg / kg of body weight of the subject.

[0443] Embodiment 56. The method of Embodiment 54 or 55, wherein the composition is administered every 7 days.

[0444] Embodiment 57. The method of Embodiment 54 or 55, wherein the composition is administered every 14 days.

[0445] Embodiment 58. The method of Embodiment 54 or 55, wherein the composition is administered every 28 days.

[0446] Embodiment 59. The method of any one of Embodiments 54-58, wherein the composition is administered for at least 3 doses.

[0447] Embodiment 60. The method of any one of Embodiments 54-59, wherein the composition is administered at a dose of at least 0.005, 0.015, 0.03, 0.06, 0.10, or 0.15 mg / kg.WSGR Docket No. 56371-768.601

[0448] Embodiment 61. The method of any one of Embodiments 54-60, wherein the composition is administered at a dose of at most 0.015, 0.03, 0.06, 0.10, 0.15, 0.18, 0.20, 0.22, 0.25, 0.28, or 0.30 mg / kg.

[0449] Embodiment 62. The method of any one of Embodiments 54-61, wherein the composition is administered for at least 4 cycles.

[0450] Embodiment 63. The method of Embodiment 62, wherein the composition is administered every 14 days for cycle 1, and every 28 days for cycle 2 and afterwards.

[0451] Embodiment 64. The method of Embodiment 63, wherein the composition is administered a dose of 0.005 to 0.03 mg / kg.

[0452] Embodiment 65. The method of Embodiment 64, wherein the composition is administered every 7 days for cycle 1, and every 28 days for cycle 2 and afterwards.

[0453] Embodiment 66. The method of Embodiment 65, wherein the composition is administered at a dose of 0.03 mg / kg.

[0454] Embodiment 67. The method of Embodiment 62, wherein the composition is administered every 14 days for each cycle.

[0455] Embodiment 68. The method of Embodiment 67, wherein the composition is administered at a dose of 0.06 to 0.30 mg / kg.

[0456] Embodiment 69. The method of Embodiment 67, wherein the composition is administered at a dose of 0.06 to 0.20 mg / kg.

[0457] Embodiment 70. The method of any one of Embodiments 1-69, wherein the CRS is reduced as compared to a control group lacking the administration of the prophylactic agent.

[0458] Embodiment 71. The method of Embodiment 70, wherein the CRS comprises elevated liver enzymes, D-dimer, ferritin, or C-reactive protein (CRP).

[0459] Embodiment 72. The method of Embodiment 70 or 71, wherein the CRP is measured according to ASTCT CRS grading.

[0460] Embodiment 73. A method of treating a cancer in a subject in need thereof, the method comprising administering the subject: (i) a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain, and a CD89 intracellular domain; and (ii) an anti-cancer agent comprising: a chemotherapeutic agent; and (a) a tumor target drug, or (b) an immune checkpoint inhibitor.

[0461] Embodiment 74. The method of Embodiment 73, wherein the nucleic acid and the anticancer agent are administered at separate times.WSGR Docket No. 56371-768.601

[0462] Embodiment 75. The method of Embodiment 73 or 74, wherein administering the nucleic acid is administered after administering the anti-cancer agent.

[0463] Embodiment 76. The method of any one of Embodiments 73-75, wherein the nucleic acid is administered before administering the anti-cancer agent.

[0464] Embodiment 77. The method of any one of Embodiments 73-76, wherein the nucleic acid and the anti-cancer agent overlaps for at least one administration.

[0465] Embodiment 78. The method of any one of Embodiments 73-77, wherein the cancer is metastatic or advanced.

[0466] Embodiment 79. The method of any one of Embodiments 73-78, wherein the cancer is an epithelial cancer.

[0467] Embodiment 80. The method of any one of Embodiments 73-78, wherein the cancer is selected from the group consisting of urothelial, cervical, ovarian, Triple-negative breast cancer, HR+ / HER2 negative breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, and non-small cell lung cancer.

[0468] Embodiment 81. The method of any one of Embodiments 73-80, wherein the cancer is a refractory cancer.

[0469] Embodiment 82. The method of any one of Embodiments 73-81, wherein the cancer is a relapsed cancer.

[0470] Embodiment 83. The method of any one of Embodiments 73-82, wherein the cancer is esophagogastric cancer.

[0471] Embodiment 84. The method of any one of Embodiments 73-83, wherein the method comprises administering to the nucleic acid and the anti-cancer agent at a dose level and a time interval suitable for inducing a reduction in at least one symptom of the cancer after a period of time.

[0472] Embodiment 85. The method of Embodiment 84, wherein the period of time comprises at least the time for completion of one cycle of administering each of the nucleic acid and the anticancer agent.

[0473] Embodiment 86. The method of Embodiment 84, wherein the period of time comprises the time for completion of more than one cycle of administering.

[0474] Embodiment 87. The method of Embodiment 84, wherein the reduction in the at least one symptom of the cancer comprises a reduction in tumor volume compared to an initial volume at a time immediately prior to onset of the treatment.

[0475] Embodiment 88. The method of Embodiment 87, wherein the method results in a reduction in tumor volume by at least 5%, at least 10%, at least 15% or at least 20% compared to the initial volume prior to onset of the treatment.WSGR Docket No. 56371-768.601

[0476] Embodiment 89. The method of Embodiment 84, wherein the reduction in at least one symptom of the cancer comprises a reduction in number of lesions compared to the number of lesions at a time prior to onset of the treatment.

[0477] Embodiment 90. The method of Embodiment 84, wherein the reduction in at least one symptom of the cancer comprises a response to the treatment.

[0478] Embodiment 91. The method of Embodiment 90, wherein the response is stable.

[0479] Embodiment 92. The method of Embodiment 90, wherein the response is partial or complete.

[0480] Embodiment 93. The method of any one of Embodiments 73-92, wherein the cancer is hormone receptor (HR)-positive.

[0481] Embodiment 94. The method of any one of Embodiments 73-93, wherein the cancer is HER2-positive.

[0482] Embodiment 95. The method of any one of Embodiments 73-93, wherein the cancer is HER-negative.

[0483] Embodiment 96. The method of any one of Embodiments 73-95, wherein the cancer is esophageal, gastroesophageal junction (GEJ), or gastric adenocarcinoma.

[0484] Embodiment 97. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2.

[0485] Embodiment 98. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3.

[0486] Embodiment 99. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 39.

[0487] Embodiment 100. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 175.

[0488] Embodiment 101. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 176.

[0489] Embodiment 102. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 177.WSGR Docket No. 56371-768.601

[0490] Embodiment 103. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 178.

[0491] Embodiment 104. The method of any one of Embodiments 73-96, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 179.

[0492] Embodiment 105. The method of any one of Embodiments 73-104, wherein the nucleic acid is RNA.

[0493] Embodiment 106. The method of Embodiment 105, wherein the nucleic acid comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 4, 5, 14-16, 33, or 36.

[0494] Embodiment 107. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 4.

[0495] Embodiment 108. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 5.

[0496] Embodiment 109. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 14.

[0497] Embodiment 110. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 15.

[0498] Embodiment 111. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 16.

[0499] Embodiment 112. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 33.

[0500] Embodiment 113. The method of Embodiment 105, wherein the nucleic acid comprises a sequence of SEQ ID NO: 36.

[0501] Embodiment 114. The method of any one of Embodiments 73-113, wherein the chemotherapeutic agent is selected from the group consisting of capecitabine, oxaliplatin, fluoropyrimidine, cisplatin, and carboplatin.

[0502] Embodiment 115. The method of any one of Embodiments 73-114, wherein the chemotherapeutic agent comprises capecitabine and oxaliplatin (Capox).

[0503] Embodiment 116. The method of any one of Embodiments 73-115, wherein the anticancer agent comprises an immune checkpoint inhibitor.

[0504] Embodiment 117. The method of Embodiment 116, wherein the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1.WSGR Docket No. 56371-768.601

[0505] Embodiment 118. The method of Embodiment 117, wherein the antibody is pembrolizumab or nivolumab.

[0506] Embodiment 119. The method of any one of Embodiments 73-113, wherein the anticancer agent comprises a tumor target drug.

[0507] Embodiment 120. The method of Embodiment 119, wherein the tumor target drug is an antibody specifically binds to a tumor antigen.

[0508] Embodiment 121. The method of Embodiment 120, wherein the tumor antigen is ITER-2.

[0509] Embodiment 122. The method of Embodiment 121, wherein the antibody is trastuzumab.

[0510] Embodiment 123. The method of any one of Embodiments 73-115, wherein the chemotherapeutic agent is capecitabine and oxaliplatin (Capox), and wherein the oxaliplatin is administered at a dose of 130 mg / mA2.

[0511] Embodiment 124. The method of Embodiment 123, wherein the oxaliplatin is administered intravenously (IV).

[0512] Embodiment 125. The method of any one of Embodiments 123-124, wherein the oxaliplatin is administered on Day 1 of each cycle.

[0513] Embodiment 126. The method of any one of Embodiments 123-125, wherein the capecitabine is administered at a dose of 1000 mg / mA2.

[0514] Embodiment 127. The method of Embodiment 126, wherein the capecitabine is administered orally.

[0515] Embodiment 128. The method of Embodiment 126 or 127, wherein the capecitabine is administered twice daily on Days 1-14 of each cycle, every three weeks.

[0516] Embodiment 129. The method of any one of Embodiments 117-128, wherein the cancer is PD-L1 -positive, and the anti-cancer agent comprises the immune checkpoint inhibitor.

[0517] Embodiment 130. The method of Embodiment 129, wherein the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1.

[0518] Embodiment 131. The method of Embodiment 130, wherein the tumor target drug is nivolumab or pembrolizumab.

[0519] Embodiment 132. The method of Embodiment 131, wherein the tumor target drug is nivolumab and wherein the nivolumab is administered at a dose of 360 mg / kg.

[0520] Embodiment 133. The method of Embodiment 131 or 132, wherein the tumor target drug is administered IV.

[0521] Embodiment 134. The method of any one of Embodiments 131-133, wherein the tumor target drug is administered every 3 weeks.WSGR Docket No. 56371-768.601

[0522] Embodiment 135. The method of any one of Embodiments 131-134, wherein the cancer is HER2-positive, and the anti-cancer agent comprises the tumor target drug that specifically binds to HER2.

[0523] Embodiment 136. The method of Embodiment 135, wherein the tumor target drug is trastuzumab.

[0524] Embodiment 137. The method of Embodiment 136, wherein the trastuzumab is administered at a dose of 6 mg / kg.

[0525] Embodiment 138. The method of Embodiment 136 or 137, wherein the trastuzumab is administered IV.

[0526] Embodiment 139. The method of any one of Embodiments 136-138, wherein the trastuzumab is administered every 3 weeks.

[0527] Embodiment 140. The method of any one of Embodiments 136-139, wherein the trastuzumab is administered with a loading dose.

[0528] Embodiment 141. The method of Embodiment 140, wherein the loading dose is 8 mg / kg.

[0529] Embodiment 142. The method of any one of Embodiments 136-141, wherein the nucleic acid is administered on the same day of the anti-cancer agent and is administered every 3 weeks.

[0530] Embodiment 143. The method of Embodiment 142, wherein the nucleic acid is administered before the anti-cancer agent on the same day.

[0531] Embodiment 144. The method of any one of Embodiments 136-143, wherein the nucleic acid is administered at a dose of at least 0.006, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.15, 0.18, 0.20, 0.25, or 0.30 mg / kg.

[0532] Embodiment 145. The method of any one of Embodiments 136-144, wherein the nucleic acid is administered at a dose of at most 0.006, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.15, 0.18, 0.20, 0.25, or 0.30 mg / kg.

[0533] Embodiment 146. The method of any one of Embodiments 136-145, wherein the nucleic acid is administered at a dose of about 0.006, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.15, 0.18, 0.20, 0.25, or 0.30 mg / kg.

[0534] Embodiment 147. The method of any one of Embodiments 136-146, wherein the method comprises at least 3 cycles.

[0535] Embodiment 148. The method of Embodiment 147, wherein each of the at least 3 cycles comprises 3 weeks.

[0536] Embodiment 149. The method of Embodiment 147 or 148, wherein the nucleic acid is administered on Day 1 and Day 8 of Cycle 1, Day 1 of Cycle 2 and afterwards.

[0537] Embodiment 150. The method of Embodiment 149, wherein the oxaliplatin is administered on Day 8 of Cycle 1, and on Day 1 of Cycle 2 and afterwards.WSGR Docket No. 56371-768.601

[0538] Embodiment 151. The method of Embodiment 149 or 150, wherein the capecitabine is administered on Day 8 and Day 15 of Cycle 1, and Day 1 and Day 8 of Cycle 2 and afterwards.

[0539] Embodiment 152. The method of Embodiment 151, wherein the nivolumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards.

[0540] Embodiment 153. The method of Embodiment 149, wherein the trastuzumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards.

[0541] Embodiment 154. The method of Embodiment 149 or 150, wherein the capecitabine is administered on Day 8 and Day 15 of Cycle 1, and Day 1 and Day 8 of Cycle 2 and afterwards.

[0542] Embodiment 155. The method of any one of Embodiments 136-154, further comprising administering an additional drug, wherein the additional drug comprises an anti-emetic, analgesic, anti-pyretic, anti -diarrheal, anti-inflammatory drug.

[0543] Embodiment 156. The method of Embodiment 155, wherein the additional drug comprises an anti-emetic drug, and wherein the anti-emetic drug comprises olanzapine, 5-HT3 receptor antagonist, or NK-1 receptor antagonist.

[0544] Embodiment 157. The method of Embodiment 156, wherein the additional drug further comprises a steroid.

[0545] Embodiment 158. The method of Embodiment 157, wherein the steroid comprises methylprednisolone.

[0546] Embodiment 159. The method of Embodiment 156, wherein the 5-HT3 receptor antagonist is ondansetron or palonosetron.

[0547] Embodiment 160. The method of Embodiment 156, wherein the NK-1 receptor antagonist is aprepitant or fosaprepitant.

[0548] Embodiment 161. A combination therapeutic kit for treating a cancer in a subject in need thereof, comprising a first therapeutic agent and at least one additional therapeutic agent, wherein the first therapeutic agent comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain; and wherein the at least one additional therapeutic agent comprises (i) a prophylactic agent or (ii) a chemotherapeutic agent and at least one of (a) a tumor target drug or (b) an immune checkpoint inhibitor.

[0549] Embodiment 162. The combination therapeutic kit of Embodiment 161, wherein the at least one additional therapeutic agent comprises the chemotherapeutic agent and at least one of (a) the tumor target drug or (b) the immune checkpoint inhibitor.WSGR Docket No. 56371-768.601

[0550] Embodiment 163. The combination therapeutic kit of Embodiment 161 or 162, wherein the chemotherapeutic agent is selected from the group consisting of capecitabine, oxaliplatin, fluoropyrimidine, cisplatin, and carboplatin.

[0551] Embodiment 164. The combination therapeutic kit of Embodiment 161 or 162, wherein the chemotherapeutic agent comprises capecitabine and oxaliplatin (Capox).

[0552] Embodiment 165. The combination therapeutic kit of any one of Embodiments 162-164, wherein the at least one additional therapeutic agent comprises the chemotherapeutic agent and the immune checkpoint inhibitor.

[0553] Embodiment 166. The combination therapeutic kit of Embodiment 165, wherein the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1.

[0554] Embodiment 167. The combination therapeutic kit of Embodiment 166, wherein the antibody is pembrolizumab or nivolumab.

[0555] Embodiment 168. The combination therapeutic kit of any one of Embodiments 161-167, wherein the at least one additional therapeutic agent comprises the chemotherapeutic agent and the tumor target drug.

[0556] Embodiment 169. The combination therapeutic kit of Embodiment 168, wherein the tumor target drug is an antibody specifically binds to a tumor antigen.

[0557] Embodiment 170. The combination therapeutic kit of Embodiment 169, wherein the tumor antigen is ITER-2.

[0558] Embodiment 171. The combination of Embodiment 170, wherein the antibody is trastuzumab.

[0559] Embodiment 172. The combination therapeutic kit of any one of Embodiments 161-171, wherein the combination therapeutic kit comprises capecitabine and oxaliplatin (Capox) and trastuzumab.

[0560] Embodiment 173. The combination therapeutic kit of Embodiment 172, wherein the kit further comprises fluoropyrimidine or cisplatin.

[0561] Embodiment 174. The combination therapeutic kit of Embodiment 172 or 173, wherein the kit further comprises nivolumab or pembrolizumab.

[0562] Embodiment 175. The combination therapeutic kit of any one of Embodiments 161-174, wherein the first therapeutic agent and the at least one additional therapeutic agent are present in therapeutically effective doses for treating an epithelial cancer a subject in need thereof.

[0563] Embodiment 176. The combination therapeutic kit of any one of Embodiments 161-175, wherein the first therapeutic agent and the at least one additional therapeutic agent are formulated in different compositions.WSGR Docket No. 56371-768.601

[0564] Embodiment 177. The combination therapeutic kit of Embodiment 176, wherein the antigen binding domain comprises an anti-TROP2 scFv.

[0565] Embodiment 178. The combination therapeutic kit of any one of Embodiments 161-177, wherein the CFP further comprises an intracellular domain operably linked to the transmembrane domain.

[0566] Embodiment 179. The combination therapeutic kit of Embodiment 178, wherein the intracellular domain comprises a CD89 intracellular domain.

[0567] Embodiment 180. The combination therapeutic kit of any one of Embodiments 161-179, wherein the extracellular domain further comprises at least a portion of a CD89 extracellular domain.

[0568] Embodiment 181. The combination therapeutic kit of any one of Embodiments 161-180, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 175.

[0569] Embodiment 182. The combination therapeutic kit of any one of Embodiments 161-181, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 176.

[0570] Embodiment 183. The combination therapeutic kit of any one of Embodiments 161-182, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 177.

[0571] Embodiment 184. The combination therapeutic kit of any one of Embodiments 161-183, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 178.

[0572] Embodiment 185. The combination therapeutic kit of any one of Embodiments 161-184, wherein the CFP comprises an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 2, 3, 39, or 179.

[0573] Embodiment 186. The combination therapeutic kit of Embodiment 185, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 39.

[0574] Embodiment 187. The combination therapeutic kit of Embodiment 185, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 179.

[0575] Embodiment 188. The combination therapeutic kit of Embodiment 185, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 2.

[0576] Embodiment 189. The combination therapeutic kit of Embodiment 185, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 3.

[0577] Embodiment 190. The combination therapeutic kit of any one of Embodiments 161-189, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO: 4, 5, 14-16, 33, or 36.

[0578] Embodiment 191. The combination therapeutic kit of any one of Embodiments 161-190, wherein the nucleic acid comprises a sequence having at least 95% sequence identity to any one of SEQ ID NO: 4, 5, 14-16, 33, or 36.WSGR Docket No. 56371-768.601

[0579] Embodiment 192. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 4.

[0580] Embodiment 193. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 5.

[0581] Embodiment 194. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 14.

[0582] Embodiment 195. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 15.

[0583] Embodiment 196. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 16.

[0584] Embodiment 197. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 33.

[0585] Embodiment 198. The combination therapeutic kit of any one of Embodiments 161-191, wherein the nucleic acid comprises a sequence of SEQ ID NO: 36.

[0586] Embodiment 199. The combination therapeutic kit of any one of Embodiments 161-198, wherein the nucleic acid is RNA.

[0587] Embodiment 200. The combination therapeutic kit of any one of Embodiments 199, wherein the RNA is mRNA.

[0588] Embodiment 201. The combination therapeutic kit of any one of Embodiments 161-200, wherein the nucleic acid is formulated for systemic delivery.

[0589] Embodiment 202. The combination therapeutic kit of any one of Embodiments 161-201, wherein the nucleic acid is formulated for cell specific expression in vivo.

[0590] Embodiment 203. The combination therapeutic kit of any one of Embodiments 161-202, wherein the nucleic acid expresses in myeloid cells in vivo, and does not substantially express on a T cell.

[0591] Embodiment 204. The combination therapeutic kit of any one of Embodiments 161-203, wherein the nucleic acid does not specifically express on an epithelial cell in vivo.

[0592] Embodiment 205. The combination therapeutic kit of any one of Embodiments 161-204, wherein the nucleic acid further comprises a 5’ UTR.

[0593] Embodiment 206. The combination therapeutic kit of Embodiment 205, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 7.

[0594] Embodiment 207. The combination therapeutic kit of Embodiment 205, wherein the 5’ UTR comprises a sequence of SEQ ID NO: 8.

[0595] Embodiment 208. The combination therapeutic kit of any one of Embodiments 161-207, wherein the nucleic acid further comprises a 3’ UTR.WSGR Docket No. 56371-768.601

[0596] Embodiment 209. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR is a bGH 3’ UTR.

[0597] Embodiment 210. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 9.

[0598] Embodiment 211. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR is a a globin 3’ UTR.

[0599] Embodiment 212. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 10.

[0600] Embodiment 213. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR comprises one or more microRNA binding site.

[0601] Embodiment 214. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR comprises two, three, four, or five microRNA binding sites.

[0602] Embodiment 215. The combination therapeutic kit of Embodiment 208, wherein the 3’ UTR comprises four microRNA binding sites.

[0603] Embodiment 216. The combination therapeutic kit of any one of Embodiments 208-215, wherein the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p.

[0604] Embodiment 217. The combination therapeutic kit of Embodiment 216, wherein the miR- 27b-5p comprises a sequence of SEQ ID NO: 11.

[0605] Embodiment 218. The combination therapeutic kit of Embodiment 216, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 12.

[0606] Embodiment 219. The combination therapeutic kit of Embodiment 216, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 24.

[0607] Embodiment 220. The combination therapeutic kit of any one of Embodiments 208-219, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 13.

[0608] Embodiment 221. The combination therapeutic kit of any one of Embodiments 161-220, wherein the kit further comprises a nucleic sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38.

[0609] Embodiment 222. The combination therapeutic kit of any one of Embodiments 161-221, wherein the kit further comprises a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 1 or 3.

[0610] Embodiment 223. The combination therapeutic kit of any one of Embodiments 161-222, wherein the first therapeutic agent is formulated for IV delivery.WSGR Docket No. 56371-768.601

[0611] Embodiment 224. The combination therapeutic kit of any one of Embodiments 161-223, wherein the first therapeutic agent is present at a concentration of 0.5 -2.5 mg / ml injectable composition in single use vial.

[0612] Embodiment 225. The combination therapeutic kit of any one of Embodiments 161-224, wherein the first therapeutic agent is present at a concentration of 1.0 -2.0 mg / ml injectable composition in single use vial.

[0613] Embodiment 226. The combination therapeutic kit of any one of Embodiments 161-225, wherein the first therapeutic agent is present at a concentration of 1.0±0.3 mg / ml injectable composition in 2.0 ml single use vial.

[0614] Embodiment 227. The combination therapeutic kit of any one of Embodiments 161-226, wherein the at least one additional agent is capecitabine and oxaliplatin (Capox), and wherein the capecitabine is formulated as an oral administration composition and oxaliplatin is formulated for IV delivery.

[0615] Embodiment 228. The combination therapeutic kit of any one of Embodiments 161-227, wherein the prophylactic agent is an agent that prevents or treats cytokine release syndrome.

[0616] Embodiment 229. The combination therapeutic kit of Embodiment 228, wherein the prophylactic agent is an anti-interleukin (IL) agent.

[0617] Embodiment 230. The combination therapeutic kit of Embodiment 229, wherein the anti- IL agent is an IL receptor inhibitor.

[0618] Embodiment 231. The combination therapeutic kit of Embodiment 230, wherein the IL receptor inhibitor comprises tocilizumab or anakinra.

[0619] Embodiment 232. The combination therapeutic kit of Embodiment 231, wherein the IL receptor inhibitor comprises tocilizumab and anakinra.

[0620] Embodiment 233. The combination therapeutic kit of any one of Embodiments 161-232, wherein the cancer is an epithelial cancer.

[0621] Embodiment 234. The combination therapeutic kit of any one of Embodiments 161-232, wherein the cancer is selected from the group consisting of gastric cancer, advanced gastric cancer, gastric carcinoma, gastric adenocarcinoma, metastatic gastric cancer, unresectable gastric cancer, unresectable advanced gastric cancer, unresectable metastatic gastric cancer, esophageal cancer, esophageal carcinoma, esophageal adenocarcinoma, advanced esophageal cancer, metastatic esophageal cancer, unresectable esophageal cancer, unresectable esophageal advanced cancer, unresectable metastatic esophageal cancer, gastroesophageal junction (GEJ) cancer, advanced gastroesophageal junction (GEJ) cancer, metastatic gastroesophageal junction (GEJ) cancer, unresectable gastroesophageal junction (GEJ) cancer, unresectable advanced gastroesophageal junction (GEJ) cancer, unresectable metastatic gastroesophageal junction (GEJ)WSGR Docket No. 56371-768.601 cancer, advanced or metastatic gastric gastroesophageal junction cancer and advanced or metastatic gastric esophageal carcinoma.

[0622] Embodiment 235. The combination therapeutic kit of any one of Embodiments 161-232, wherein the cancer is selected from the group consisting of urothelial, cervical, ovarian, Triplenegative breast cancer, HR+ / HER2 negative breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, and non-small cell lung cancer.

[0623] Embodiment 236. The combination therapeutic kit of any one of Embodiments 161-232, wherein the cancer is esophageal, gastroesophageal junction (GEJ), or gastric adenocarcinoma.

[0624] Embodiment 237. The combination therapeutic kit of any one of Embodiments 161-236, wherein the cancer is advanced or metastatic.

[0625] Embodiment 238. The combination therapeutic kit of any one of Embodiments 161-237, wherein the cancer is PD-L1 -positive.

[0626] Embodiment 239. The combination therapeutic kit of any one of Embodiments 161-238, wherein the cancer is HER2-positive.

[0627] Embodiment 240. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) nivolumab.

[0628] Embodiment 241. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) pembrolizumab.

[0629] Embodiment 242. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) fluoropyrimidine and cisplatin, and (iii) pembrolizumab.

[0630] Embodiment 243. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) fluoropyrimidine and cisplatin, and (iii) nivolumab.

[0631] Embodiment 244. A combination therapeutic kit for treating a HER2 -positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) trastuzumab.

[0632] Embodiment 245. A combination therapeutic kit for treating a HER-2 positive cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) capecitabine and oxaliplatin; and (iii) trastuzumab.

[0633] Embodiment 246. The combination therapeutic kit of any one of Embodiments 243-245, wherein the cancer is esophageal, gastroesophageal junction (GEJ), or gastric adenocarcinoma.WSGR Docket No. 56371-768.601

[0634] Embodiment 247. The combination therapeutic kit of any one of Embodiments 243-246, wherein the cancer is metastatic esophagogastric cancer.

[0635] Embodiment 248. A combination therapeutic kit for treating a cancer, comprising: (i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; (ii) tocilizumab and anakinra.

[0636] Embodiment 249. The combination therapeutic kit of Embodiment 248, wherein the cancer is an epithelial cancer.

[0637] Embodiment 250. A method of treating a cancer in a human in need thereof, the method comprising administering to the subject a combination of: (i) a first component comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2; (ii) at least one prophylactic agent; and (iii) at least one additional therapeutic selected from: (a) a chemotherapeutic agent (b) a tumor target drug or (c) an immune checkpoint inhibitor.

[0638] Embodiment 251. The method of Embodiment 250, wherein, administering each of (i), (ii) and (iii) is in an amount and a dosing interval sufficient to reduce or ameliorate at least one condition associated with the cancer.

[0639] Embodiment 252. The method of Embodiment 251, wherein the amount and the dosing interval is in accordance to any one of Embodiments 73-160.

[0640] Embodiment 253. The method of any one of Embodiments 251-252, wherein the at least one condition associated with the cancer is tumor volume.

[0641] Embodiment 254. The method of any one of Embodiments 250-253, wherein the administering reduces tumor volume or ameliorates tumor growth in the subject compared to an initial volume at a time immediately prior to onset of treatment.

[0642] Embodiment 255. The method of Embodiment 254, wherein the administering stably reduces tumor volume by at least 10% compared to an initial volume at a time immediately prior to onset of the treatment.EXAMPLES

[0643] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited herein.

[0644] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.WSGR Docket No. 56371-768.601Example 1. Chimeric antigen receptor protein constructs with intracellular interferon activating domains

[0645] In this example, amino acid and nucleic acid sequences of the various CFP constructs described herein are disclosed. Nucleic acid sequences as detailed below can be easily interpreted by one of skill in the art for DNA and mRNA sequences in order to provide guidance in making and using a suitable constructs or variants therefrom using commonly used molecular cloning techniques. FIG. 1 illustrates the general principle of the chimeric fusion protein mode of expression and function. The transmembrane domain (TMD) selected for construction of the fusion proteins are those that can dimerize or multimerize with an endogenous myeloid cell transmembrane protein for successful expression and function of the chimeric fusion protein. In the exemplary constructs such a TM domain is CD89 TMD. By making use of a transmembrane domain that multimerizes with an endogenous Fcgamma mediated myeloid cell targeted expression of the chimeric fusion protein illustrated in FIG. 1, shown in FIG. 2 are the different constructs that have been prepared using a TROP2 binder domain in the extracellular region, and with the transmembrane domain from CD89 TMD, and the intracellular signaling domains as shown in FIG. 2. In some embodiments, the CFPs comprising the CD89 TMD are designed to comprise an intracellular domain of CD89 or a fragment thereof, with no additional intracellular signaling domain; the idea being that the mature CFP will express in the cell membrane and associate with an endogenous FcR gamma, and the associate will ensure the stabilization of the CFP in the cell, and trigger intracellular signaling via the FcR gamma intracellular domains, once the extracellular domain of the CFP binds to a target antigen on a cancer cell. In some embodiments, the constructs comprising CD89 transmembrane domain may be designed to comprise an FcR intracellular domain, e.g., an intracellular signaling domain. In some embodiments, the CFP may comprise a CD40 intracellular domain.Example 2. Functional Assays for testing CFP constructs

[0646] The targeted constructs are tested for functional properties. THP-1 cells or CD14+ / CD16- monocytes isolated from leukapheresis samples are transfected with the polynucleotide constructs encoding respecting CFPs.

[0647] Method for cell transfection and detection of transfection efficiency: THP-1 cells are harvested and washed once with MaxCyte Electroporation buffer. The cells are resuspended at 10 million / ml density and added to lOOug of ATAK-receptor RNA in an Eppendorf tube, mixed twice and loaded into a MaxCyte processing assembly (OC-25x3). The cells are electroporated using the THP-1 program on MaxCyte. After the electroporation, cells are incubated at 37°C for 10 mins to recover in the processing assembly and then transferred to plates containing prewarmed media at 0.5 million cells / ml density.WSGR Docket No. 56371-768.601

[0648] After overnight incubation, the expression of ATAK-receptor in electroporated monocytes is assessed by Flow cytometry. Anti -Fab -Alexa Fluor-647 antibody (1 :50 dilution) is used to detect the expression of scFv of ATAK receptor. The stained samples are acquired on Cytek Northern lights cytometer and percent of binder positive cells calculated based on increase in Anti-Fab intensity over mock-transfected control.

[0649] Method for phagocytosis assay: Target tumor cells (SKOV3) are labeled with pHrodo- Red dye (700ng / ml final concentration) following the Sartorius Incucyte pHrodo-Red labeling kit protocol. After labeling, the SKOV3 cells are resuspended at 0.5 million cells / ml density using the culture medium. CD14-positive monocytes are isolated from donor leukopak and electroporated with lOOug / ml of ATAK-receptor RNA using MaxCyte. After electroporation, cells are recovered overnight at 2 million cells / mL density in culture medium at 37°C. Next day, cells are counted using NC-200 and are resuspended at 2.5 million cells / ml density using the culture medium. In a low adhesion U-bottom 96-well plate, 50uL of tumor cells (50,000 cells total) are added to 50uL of ATAK-receptor transfected monocytes (125,000 cells total) at 5: 1 E:T ratio. The cells are mixed and incubated at 37°C overnight.

[0650] The following day, cells are stained with CD45-Alexa Fluor 700 which labels monocytes specifically. The samples are then acquired on Cytek Northern lights to detect pHrodo-Red and CD45 signal intensities. Phagocytosis is measured as percent phagocytosis index as well as specific increase in pHrodo-Red intensity in monocytes. The phagocytosis index is calculated as the percent of monocytes with high pHrodo-red signal normalized to total number of monocytes. Phagocytic activity of ATAK-monocytes is compared with the mock-transfected control to determine the efficacy of ATAK-receptor.

[0651] Phagocytosis can be tested using labeled tumor cells.

[0652] Method for SKOV3 cell killing assay: The tumoricidal activity of ATAK receptor- transfected monocytes is tested using SKOV3 -Luciferase cells. CD14-positive monocytes are isolated from donor leukopak and electroporated with lOOug / ml of ATAK-receptor RNA using MaxCyte. After electroporation, cells are recovered for 2 hours in culture medium at 37°C. After recovery, cells are resuspended at 2.5 million cells / ml density using the culture medium.SKOV3 -Luciferase cells are harvested and resuspended at density of 0.25 million cells / mL and lOOul of cell suspension (total 25,000 cells per well) is added in a 96 well flat-bottom plate. IOOUL of ATAK-receptor transfected monocytes are added to the same well at E:T ratio of 1 : 1 and 10:1. The cells are mixed and incubated at 37°C for 3 days.

[0653] On day 3, supernatant is collected and frozen to measure the cytokines and chemokines secreted by the ATAK-monocytes. The cells are lysed, and SKOV3 luciferase levels are measured using luminescence plate reader. A decrease in Luciferase level for samples containingWSGR Docket No. 56371-768.601ATAK-monocytes, compared to mock transfected control, is indicative of SKOV3 killing activity of the ATAK cells.

[0654] Polarization potential of myeloid cells is tested using the following method. These effector myeloid cells electroporated with the polynucleic acid construct and frozen for later use and testing. Upon thawing, the cells were then subject to culture in polarizing stimuli, for example in separate aliquot cultures, with (i) GMCSF (ii) IL4, IL10, and TGFbeta (M2 stimuli), (iii) activated T cell conditioned media (TCM) and (iv) MCSF. Cells were analyzed at 24, 48 and 72 hours by flow cytometry, and cytokine analysis was performed by Luminex.

[0655] Method for detecting NF-KB and IFN pathway activation using THPl-Dual cells: THPl-Dual cells have NF-KB response elements upstream of secreted Alkaline phosphatase and IFN stimulated response elements upstream of secreted Luciferase. Measuring the levels of Alkaline phosphatase in the supernatant indicates the activation of NF-KB signaling pathway, while levels of Luciferase in the supernatant indicates IFN signaling pathway activation. THPl- Dual cells are electroporated with lOOug / ml of ATAK-receptor RNA using MaxCyte. After electroporation, cells are recovered for 2 hours in culture medium at 37°C. Post recovery, cells are resuspended at 0.5 million cells / ml density using the culture medium. SKOV3 cells are harvested and resuspended at density of 0.5 million cells / mL. In a 96-well plate, lOOul of SKOV3 cell suspension (total 50,000 cells per well) and lOOuL of ATAK-THPl-Dual cells (50,000 cells total) are added in a 96 well flat-bottom plate at E:T ratio of 1 : 1. The cells are mixed and incubated for 24 hours at 37°C. After 24 hours, the cells are centrifuged, and supernatant is collected.

[0656] To detect NF-KB pathway activation, QUANTLBlue solution (Invivogen) is added to the supernatant and incubated at 37°C for 2 hours, after which OD is measured using an absorbance plate reader. Increase in absorbance is indicative of activation of NF-KB signaling and the OD values for ATAK-transfected THPl-Dual cells can be compared to that of mock transfected controls to determine activity of ATAK-receptors.

[0657] To detect IFN pathway activation, QUANTI-Luc solution (Invivogen) is added to the supernatant and luciferase levels are measured using luminescence plate reader. Increase in luciferase levels implies activation of IFN signaling and can be compared between the ATAK- transfected THPl-Dual cells compared to mock transfected controls.

[0658] Expression of TROP2 binders exemplified in FIG. 2 in monocytic cell lines. FIG. 3 shows data indicating the successful expression of each of the constructs. The results indicate that greater than at least 50% cells express each of the constructs. The results also indicate that the addition of multiple intracellular signaling domains are well tolerated and the constructs express well.WSGR Docket No. 56371-768.601

[0659] Additionally, these second generation in vivo receptors showed a higher level of pro- inflammatory cytokine and chemokine production. A comparison of the first generation constructs (having the CD89 TMD lacking the multiple intracellular signaling domains) with the second generation constructs, (namely, the TROP2-CD89-FcR, TROP2-CD89-FcR-PI3K, TROP2-CD89-TRIF, TROP2-CD89-CD40, TROP2-CD89-CD40-FcR) for cytokine generation is shown in FIGs. 4A and 4B. The second generation constructs showed higher IL-12p70 and IFN beta production compared to the representative first generation construct (FIG. 4A). Additionally, similar results were found for IP- 10, TNF alpha, and IL-6 production (FIG. 4B). These results indicate that the second generation anti-TROP2 constructs express successfully in monocytes and are highly functional and release pro-inflammatory cytokines; this activating the monocytic cells that express the constructs and rendering them pro-inflammatory. These cells therefore are activated phagocytes against TROP2 expressing cancer cells.Example 3. Exemplary sequences for designing an anti-CD5-CAR encoding mRNA construct with selective cell-specific expression

[0660] In this section, exemplary designs are contemplated for selective expression of a construct in a myeloid cell, when the construct is delivered to a cell population comprising myeloid cells and non-myeloid cells. Expression of anti-CD5 CAR is described as an exemplary CAR with the UTR modifications as described below. However, the invention can be carried on by replacing anti-CD5 CAR with any other known coding sequences, e.g., an anti-HER2 CAR sequence, which can be performed without additional invention. The prospective exemplary construct is an mRNA construct, Construct #1, in which miRNA 192 target sequence (miR-192-5p) sequence (SEQ ID NO: 17), is incorporated in the 5’-UTR within the mRNA. SEQ. ID NO: 17 is reverse complement of nucleic acid residues 24-44 of SEQ ID NO: 18. Exemplary construct has a 5’UTR, a sequence encoding an anti-CD5-CAR and a 3’UTR. The 5’-UTR comprises a 5' methyl guanylate cap. The 5’-UTR is at least 45 nucleotides in length. The 3’-UTR is terminated by a sequence of 200 adenylate residues (poly A). The exemplary (hypothetical) construct #1 thus has the following structural elements from 5’-3’ orientation: 5'-methyl guanylate cap; miR-192p- target sequence SEQ ID NO: 17; anti-CD5-CAR coding sequence; 3’UTR; poly A sequence-3’. The mRNA construct may contain linker sequences in between any two adjacent structural elements (e.g., 5'-methyl guanylate cap and miR-192p-target sequence SEQ ID NO: 17).

[0661] In another exemplary design, Construct #2, an miRNA192b target sequence (miR-192- 3p) sequence (SEQ ID NO: 19), is incorporated in the 3’-UTR within the mRNA. The exemplary Construct #2 has the following structural elements from 5 ’-3’ orientation: 5'-methyl guanylate cap; anti-CD5-CAR coding sequence; 3’UTR comprising miR-192-3p-target sequence (SEQ ID NO: 19); poly A sequence-3’. As described previously, and can be applied to other examplesWSGR Docket No. 56371-768.601 following, unless otherwise mentioned, each of the mRNA constructs described herein may contain linker sequences in between any two adjacent structural elements. A linker may be 3-100 nucleotides long.

[0662] An exemplary Construct #3 has the following structural elements from 5 ’-3’ orientation: 5'-methyl guanylate cap; miR-27-5p-target sequence SEQ ID NO: 21 in 5’UTR; anti-CD5-CAR coding sequence; 3’UTR; poly A sequence-3’. The mRNA construct may contain linker sequences in between any two adjacent structural elements.

[0663] In another exemplary design, Construct #4, an miRNA27b target sequence (miR-27-3p) sequence (SEQ ID NO: 22), is incorporated in the 3’-UTR within the mRNA. The exemplary Construct #4 has the following structural elements from 5 ’-3’ orientation: 5'-methyl guanylate cap; anti-CD5-CAR coding sequence; 3’UTR comprising miR-27-3p-target sequence SEQ ID NO: 22; poly A sequence-3’.

[0664] In another exemplary design, Construct #5, an miRNAlOa target sequence (miR-10a-5p) sequence (SEQ ID NO: 23), is incorporated in the 5’-UTR within the mRNA. The exemplary Construct #5 has the following structural elements from 5 ’-3’ orientation: 5'-methyl guanylate cap; miR-10-5p-target sequence SEQ ID NO: 23 in 5’UTR; anti-CD5-CAR coding sequence; 3’UTR; poly A sequence-3’.

[0665] In another exemplary design, Construct #6, an miRNAlOa target sequence (miR-10a-3p) sequence (SEQ ID NO: 24), is incorporated in the 3’-UTR within the mRNA. The exemplary Construct #6 has the following structural elements from 5 ’-3’ orientation: 5'-methyl guanylate cap; anti-CD5-CAR coding sequence; 3’UTR comprising miR-27-3p-target sequence SEQ ID NO: 24; poly A sequence-3’.

[0666] Another exemplary design, an mRNA construct, Construct #7 is disclosed herein, in which an miRNA 192 target sequence (miR-192-5p) sequence (SEQ ID NO: 17), is incorporated in the 5’-UTR within the mRNA and the miRNA27b target sequence (miR-27-3p) sequence (SEQ ID NO: 22), is incorporated in the 3’-UTR within the mRNA. The exemplary construct #7 has the following structural elements from 5’-3’: 5'-methyl guanylate cap; miR-192p-target sequence SEQ ID NO: 17; anti-CD5-CAR coding sequence; 3’UTR comprising miR-27-3p- target sequence SEQ ID NO: 22; poly A sequence-3’.

[0667] Yet another exemplary design, an mRNA construct, Construct #8 is disclosed herein, in which an miRNA 192 target sequence (miR-192-5p) sequence (SEQ ID NO: 17), is incorporated in the 5’-UTR within the mRNA and the miRNA27b target sequence (miR-27-3p) sequence (SEQ ID NO: 22) and the miRNAlOa target sequence (miR-10a-3p) sequence (SEQ ID NO: 24), are incorporated in the 3’-UTR within the mRNA. The exemplary construct #8 has the following structural elements from 5’-3’: 5'-methyl guanylate cap; miR-192p-target sequence (SEQ ID NO:WSGR Docket No. 56371-768.60117); anti-CD5-CAR coding sequence; 3’UTR comprising miR-27-3p-target sequence SEQ ID NO: 22 and the miRNAlOa target sequence (miR-10a-3p) sequence (SEQ ID NO: 24); and the poly A sequence-3 ’ .

[0668] Yet another exemplary mRNA construct design is shown: comprising target sequences of two miR-sequences in the 5’UTR. Exemplary construct #9 has the following structural elements from 5’-3’: 5'-methyl guanylate cap; miR-192p-target sequence (SEQ ID NO: 17); miR-27-5p- target sequence (SEQ ID NO: 21) in 5’UTR; anti-CD5-CAR coding sequence; 3’UTR comprising the miRNAlOa target sequence (miR-10a-3p) sequence (SEQ ID NO: 24); and the poly A sequence-3 ’ .

[0669] Any combinations of the above, including further additions and replacements of elements described herein as may be easily conceived by a skilled artisan is contemplated within the disclosure. The miRNA target sequences discussed are provided in the table below.Table 1. miRNA Target Sequences and Respective miRNA Binding SitesExample 4. Formulation and delivery of mRNA constructs

[0670] mRNA construct designs described above are prepared and formulated for in vivo delivery. An exemplary construct for testing cell specific expression in several tissues of the experimental mouse model may be generated by tagging the anti-CD5-CAR coding region with a GFP encoding sequence without a stop codon in between anti-CD5-CAR sequence and the GFPWSGR Docket No. 56371-768.601 coding sequence. In vitro transcribed mRNA is purified by HPLC and encapsulated into a lipid nanoparticle. Methods and compositions for generating lipid nanoparticle encapsulated (LNP) mRNA are known and described previously. Briefly, the LNP composition comprises a cationic lipid, and at least one neutral lipid. LNPs have a diameter of less than 200 nm. Nanoparticles are delivered via intravenous injection.

[0671] Upon delivery to experimental mouse model, mRNA expression will be tested in cells of various tissues and organs, hepatocytes at different time periods. Mice cohorts will be tested upon euthanasia at 72 hours after delivery and between 4-10 days. For example, a mouse model can be established for testing the expression of the anti-CD5 CARs in hepatocytes at 48, 72, 96 hours post injection. Hepatocytes uptake most of foreign nucleic acids delivered in an organisms. Expression of GFP can be used as surrogate for the expression of the anti-CD5-CAR. If miR- 192b - which is copiously expressed in hepatocytes can bind to the miR-192-target sequences incorporated in the UTR regions, then the mRNA construct will not be translated, and / or the mRNA construct will be degraded by the action of the miR-192b. As per the rationale, expression of the anti-CD5-CAR will not be detectable in hepatocytes, or will be detected at a significantly lower amount than counterpart control mice, which are injected with a construct that lacks the miRNA target sequences but is otherwise identical to the test construct.

[0672] miR-192b is also expressed in high levels in epithelial cells. Expression of the anti-CD5- CAR will not be detectable in epithelial cells.

[0673] Constructs 1, 2, 7, 8 or 9, having miR-192b target sequences incorporated in the 5’- or 3’UTR - will be acted upon by miR-192b expressed in hepatocytes and epithelial cells will not express ant-CD5 CAR of the constructs.Example 5. Exploiting miRNA 122-5p binding sites in Globin mRNA UTRs for in vivo expression constructs

[0674] In this example, alpha-Globin 3’ UTR was used to prepare exemplary mRNA constructs comprising a sequence encoding GFP to test GFP protein expression in myeloid cells - wherein the a-globin (hereafter globin) UTR is further engineered to comprise multiple miR122-5p binding sequences. miRNA 122 is abundant in hepatocytes. To test whether miR122-5p binding sites within the 3’UTR of the constructs can suppress expression of the GFP -encoding mRNA in hepatocytes, an exemplary globin 3’UTR tested has the sequence: CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCcaaacaccauugucacacuccaCG AUcaaacaccauugucacacuccaUCUAGAcaaacaccauugucacacuccaUCACcaaacaccauugucacac uccaGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 26).WSGR Docket No. 56371-768.601

[0675] In this sequence, 4 (four) miR122-5p binding sites are inserted within the globin 3’UTR, and the miR122-5p binding sites are marked in bold and lower case alphabets.

[0676] When Huh-7 cells, a human male hepatoma cell line was electroporated with an mRNA construct encoding GFP and comprising the 3’UTR (SEQ ID NO: 26), expression of the GFP was completely suppressed compared to transfection with a construct comprising a construct with globin 3’UTR that was not engineered to incorporate the miR122-5p binding sites (FIG. 5A, left graph). In contrast, primary monocytes expressing the same construct showed significant expression of the encoded protein despite the presence of the miRNA binding sequence (FIG. 5A, right graph). These results were highly encouraging and offer great mechanism for controlling expression of a construct specifically in one cell by design, in such circumstances where the construct is added to a mixed population of cells containing a variety of cell types, such as in an in vivo condition.Example 6. Construction of mRNA encoding a CFP that binds to GPC3 antigen, with an engineered UTR having miRNA binding sequences.

[0677] Based on the encouraging data above, an anti-GPC3 binding CFP construct was generated, with different miRNA binding sites for suppression of mRNA expression in hepatocytes, as listed as follows in Table 2, and tested.Table 2. List of novel miRNA binding sites tested for suppression of mRNA expression in hepatocytes.

[0678] GC33-CD89 stands for an anti-GPC3 binding CFP that comprises a variable heavy chain and a variable light chain domains connected by a short linker sequence, e.g.GGGGSGGGGSGGGGS; and is fused with a CD89 hinge, CD89 transmembrane and a short CD89 intracellular domain. In some of these experiments, an anti-GPC3 binding CFP comprises an amino acid sequence:WSGR Docket No. 56371-768.601MWLQSLLLLGTVACSISQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYENffiWVRQA PGOGLEWMGALDPKTGDTAYSQKFKGKATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTYWGOGTLVTVSSGGGGSGGGGSGGGGSDVVMTOSPLSLPVTPGEPASISCRSSQ SLVHSNRNTYLHWYLQKPGOSPOLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCSONTHVPPTFGQGTKLEIKGSGGSDSIHQDYTTQNZ / AA^4E4GZFZE4ZZ^ / ZFE NWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 27), which contains the following domains, and VH-CDR1, CDR2 and CDR3 are the VL CDR1, 2 and 3 are marked by underlining respectively.

[0679] FIG. 5B demonstrates the effects of the miRNA binding site on its expression in hepatocytes (left graph) as well as primary monocytes (right graph). In these cases the mRNA was constructed with the engineered globin 3’UTR with miR192-5p binding sites. The data indicate a similar trend as with data in case of constructs with miRNA binding site for miR122- 5p in FIG. 5A, there was a higher suppression of expression of the mRNA encoded polypeptide in hepatocyte (left graph) compared to in primary monocytes.

[0680] However, no effect was evident on polypeptide expression in case of the 3’UTR constructs having globin 3’UTR with four miR192-3p binding sites (FIG. 5C) (sequence provided below).

[0681] CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCcugugaccuauggaauuggc aggCGAUcugugaccuauggaauuggcaggUCUAGAcugugaccuauggaauuggcaggUCACcugugaccuaug gaauuggcaggGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 28) miR192-3p binding sites represented as lower case letter.

[0682] On the other hand, globin 3’UTR with miR27b-5p binding sites showed a surprising effect of prolonging mRNA expression of the mRNA encoded polypeptide, where the mRNA was constructed with the engineered globin 3’UTR with miR27b-5p binding sites. (FIG. 5D, UTR sequence of SEQ ID NO: 29). The construct contained a globin 3’UTR with miR27b-5p binding sites having the sequence:

[0683] CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCuucaccaaucagcuaagcuc uCGAUuucaccaaucagcuaagcucuUCUAGAuucaccaaucagcuaagcucuUCACuucaccaaucagcuaagcu cuGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO: 29). miR27b-5p binding sites represented as lower case letter.

[0684] mRNA having engineered globin 3’UTR with four miR27b-3p binding sites did not exhibit any cell-specific advantage for monocyte cells. (FIG. 5E). The respective engineered globin 3’UTR has the sequence:WSGR Docket No. 56371-768.601

[0685] CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCgcagaacuuagccacuguga aCGAUgcagaacuuagccacugugaaUCUAGAgcagaacuuagccacugugaaUCACgcagaacuuagccacugug aaGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO:30), where miR27b-3p miRNA binding sites are marked with lower case letters.

[0686] mRNA having engineered globin 3’UTR with four miR10a-5p binding sites and mRNA having engineered globin 3’UTR with four miR10a-3p binding sites also did not exhibit any cellspecific advantage for monocyte cells. (FIG. 5F and FIG. 5G respectively). The respective engineered globin 3’UTR with four miR10a-5p binding sites has the sequence:CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCcacaaauucggaucuacaggguaCG AUcacaaauucggaucuacaggguaUCUAGAcacaaauucggaucuacaggguaUCACcacaaauucggaucuacag gguaGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO:31); and the engineered globin 3’UTR with four miR10a-3p binding sites has the sequence: CUCGAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCuauuccccuagauacgaauuugCG AUuauuccccuagauacgaauuugUCUAGAuauuccccuagauacgaauuugUCACuauuccccuagauacgaauu ugGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAAGACAAUAGC (SEQ ID NO:32), miR10a-5p and miR10a-3p binding sites in each case are represented by lower case letters.

[0687] Taken together, there is a strong possibility of further optimizing mRNA sequences including the coding sequences and the UTRs, for providing improved therapeutic mRNA molecules encoding a polypeptides of interest and selectively express in myeloid cells and at the same time suppress expression of the encoded polypeptides in other cells, principally in liver cells when the mRNA molecule is administered systemically in vivo. Most nucleic acid materials delivered systemically accumulate and express in the liver cells and are not transported to other tissues and organs, and the above strategies may provide remedy for that.Example 7. Al based mRNA sequence optimization for stability, prolonged expression

[0688] An anti-TROP2 binding CFP mRNA was optimized by Artificial Intelligence based tools (LinearDesign). An exemplary optimized lambda 10 anti-TROP2 binding CFP mRNA coding sequence is shown in SEQ ID NO: 5. The anti-TROP2 binding CFP mRNA was then designed to have a 3’ UTR from agiobin (SEQ ID NO: 15). The anti-TROP2 binding CFP mRNA was further designed to have a modified 3’ UTR having four copies of miR27b binding sites, the sequence shown in SEQ ID NO: 16. Next, Fragment Analyzer was used to measure the purity and poly(A) tail for the mRNAs. As shown in FIG. 6, all the mRNAs, including the optimized lambda 10 anti-TROP2 binding CFP mRNA with agiobin 3’ UTR (“agiobin UTR”, SEQ IDWSGR Docket No. 56371-768.601NO: 15) and the optimized lambda 10 anti-TROP2 binding CFP mRNA with agiobin 3’ UTR as well as miR27b binding sites (“miR27b UTR”, SEQ ID NO: 16), along with the original codon optimized sequence with (i) bGH 3’ UTR, (ii) agiobin 3’ UTR, or (iii) agiobin 3’ UTR with miR27b binding sites all showed high purity and similar poly(A) length.

[0689] Next, the expression level and duration of the optimized lambda 10 anti-TROP2 binding CFP mRNAs were measured by flow cytometry. As shown in FIG. 7A, the optimized lambda 10 anti-TROP2 binding CFP mRNA having agiobin 3’ UTR with miR27b binding sites had significantly increased expression level and duration, as compared to other groups. The high expression was maintained after 4 days post electroporation of the mRNA to the cells. The result was further illustrated in FIG. 7B which shows the MFI of TROP2 binder expressed from the CFP mRNA. In FIG. 7B, the optimized lambda 10 anti-TROP2 binding CFP mRNA having agiobin 3’ UTR with miR27b binding sites showed the highest MFI as compared to other groups, suggesting significant increased expression level of CFP in the target cells.Example 8. Codon Optimized anti-TROP2 CFP Constructs

[0690] The coding sequences of the anti-TROP2 CFPs were further engineered for different UTRs and / or codon optimized. As shown in FIGs. 8A-8E, four additional constructs were designed. The construct shown in FIG. 8A (Construct #10) has an ARC A 5 ’Cap and BGH 3 ’UTR (SEQ ID NO: 14). The construct shown in FIG. 8B (Construct #11) comprises a codon optimized sequence of SEQ ID NO: 5 and further comprises a 1AG 5’Cap and cr-globin 3’UTR, with a full length sequence of SEQ ID NO: 15. Construct #12, shown in FIG. 8C comprises a codon optimized sequence of SEQ ID NO: 5 and further comprises a 1 AG 5’Cap and cr-globin 3’UTR with four miR27b binding sites (SEQ ID NO: 12), with a full length sequence of SEQ ID NO: 16. Construct #13, shown in FIG. 8D, comprises a codon optimized sequence of SEQ ID NO: 34 and comprises a 1 AG 5’Cap and cr-globin 3’UTR with a full length sequence of SEQ ID NO: 33. Construct #14, shown in FIG. 8E, comprises a codon optimized sequence (SEQ ID NO: 34) and further comprises a 1AG 5’Cap and cr-globin 3’UTR with four miR27b binding sites (SEQ ID NO: 37), with a full length sequence of SEQ ID NO: 36. Sequences for the codon optimized constructs are provided in Table 3.WSGR Docket No. 56371-768.601Table 3. Codon Optimized Construct Nucleotide SequencesWSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601WSGR Docket No. 56371-768.601

[0691] The constructs were characterized via electrophoresis (FIG. 9). To test the expression capacity of each construct, primary monocytes were obtained and electroporated with each of the constructs. The starting concentration of monocytes used was 8.0 x 106cells in 80uL. The final mRNA concentration was 0.2uM. Following electroporation, cells were harvested for flow cytometry analysis detecting anti-TROP2 CFP expression at 2h, 24h, 48h, 72h, and 144h following electroporation.

[0692] As shown in FIGs. 10A-10C, Construct #13 and Construct #14 demonstrated robust expression of the CFP at each time point tested. Analysis of mean fluorescence intensity (MFI) demonstrated that the PC codon optimized construct with the miR27b binding sites resulted in highest expression of the CFP (FIG. 11).WSGR Docket No. 56371-768.601Example 9. Clinical Protocol to Assess Safety and Tolerability of Engineered Nucleic Acid Encoding Anti-TROP2 CEP Monotherapy in Human For Treating Advanced Epithelial Cancer

[0693] Described herein is a multicenter, open-label, Phase 1 first-in-human study to assess the safety, tolerability, and define the recommended phase 2 dose (RP2D) for the drug. The Drug Product (DP) is an mRNA encoding anti-TROP2 CFP designed for myeloid cell-specific expression in vivo, encapsulated in a lipid nanoparticle (LNP). The anti-TROP2 CFP comprises a CD89 transmembrane domain. A variation of the DP, DPI is designed with identical LNP composition and mRNA encoding the same product (herein anti-TROP2-CD89 CFP; or alternatively or synonymously anti-Trop2-Fca-fusion receptor mRNA LNP), and additionally incorporating mRNA sequence optimizations aimed at increasing yield and purity, as well as reducing potential mRNA reactogenicity and is further optimized to enhance stability and translation efficiency. The DP (or DPI) is formulated for injection. The DP injection product is supplied as a 2.0 ml single use vial containing the drug at a concentration of 1.0 ± 0.3 mg / mL.

[0694] Study Objective: The safety, tolerability, PK, and pharmacodynamics of DP / DP1 will be evaluated in a Single- Agent Dose-Escalation Stage. Participants will be enlisted with cancers known for high levels of TROP2 expression. Dose-escalation steps of DP / DP1 will be tailored by monitoring safety data following the Bayesian Optimal Interval (BOIN) design (Liu, 2015; Yuan, 2016; Zhou, 2018). A Safety Review Committee (SRC) will determine the RP2D / expansion dose of DP / DP1 based on the totality of the safety, PK, correlative, and efficacy assessments. Participants will continue to receive the treatment they started with in the setting of clinical benefit.

[0695] Patient Population: Adults 18 years of age inclusive or older who provide written informed consent and meet all inclusion and exclusion criteria will be offered enrollment into the trial. The initial expansion cohort will enroll participants with advanced or metastatic, HER2 Negative (includes HER2 low, HER2 ultra-low, and HER2 0) and hormone receptor positive (HR+) breast cancer.

[0696] Dosing and Duration: DP / DP1 will be administered by intravenous (IV) infusion over approximately 60 ± 10 minutes. Dosing of DP / DP1 will be based on actual body weight (mg / kg). For participants with a body weight > 100 kg, the maximum total dose will be calculated based on 100 kg body weight. Standard institutional dose rounding rules can be applied. If not available, rounding should be based on the nearest tenth of a milligram.

[0697] The study consists of 4 Cycles. For Cohorts 1-3, during Cycle 1 (56 days), all participants will receive infusions of DP every 14 days for 3 doses. For Cohort 4, during Cycle 1 (42 days), all participants will receive infusions of DP every 7 days for 3 doses. For Cohorts 5-7, duringWSGR Docket No. 56371-768.601Cycle 1 (28 days) all participants will receive infusions of DP / DP1 every 14 days (Q14D) for 2 doses. Participants will continue in the study in the absence of unacceptable toxicity through to the End of Cycle 4 Evaluation visit (Table 8) but the dose regimen will be modified to once every 28 days for Cycles 2-4 for Cohorts 1- 4 (FIGs. 12A-12C). For Cohorts 5-7, including the DPI dose evaluations and the Expansion Cohort, the dosing regimen will remain Q14D (FIGs. 12A- 12C). If a participant has stable disease (SD) or is responding to therapy, they may continue on their current dosing regimen through the End-of-Treatment (EOT) until progressive disease (PD). Once a participant discontinues dosing, follow-up will continue with evaluations performed every 3 months until the end of the study or until progression of disease. Implementation of split dosing or step-up dosing during a Run-in Period (Days -14 to Day -1) may be employed to mitigate risk of CRS and decrease the rate and severity or CRS adverse events.

[0698] Table 8 shows Summary of Dose Escalation and Expansion Study Cohorts:*Based on SRC recommended expansion dose.**Indicates the 5 doses and dose regimen for DPI.Additional dose levels and regimens may be explored after approval of SRC.

[0699] In some embodiments, implementation of split or step dosing may decrease the rate or severity of CRS or adverse events.

[0700] Prophylactic tocilizumab or anakinra may be implemented to mitigate risk of CRS.Tocilizumab can be administered 1 h before DP / DP1 administration. The standard dose is 8 mg / kg (max 800 mg) IV over 60 minutes or per institutional guidelines. Anakinra is administered as subcutaneous (SC) injection at a dose of 100 mg SC once daily starting before or at the time of DP / DP1 administration. Alternate regimen may include 100 mg SC once every 12 h for high riskWSGR Docket No. 56371-768.601 participants. Yet higher dose may include 2 mg / kg IV every 6 h. The duration of anakinra dosage is approximately 3-5 days, but may continue longer.

[0701] Table 9 shows Dose Schedules:Additional Splits and exact step up doses will be determined in discussion and approval of Study Review Committee. For example, additional split could allow at least 3 days between dosing and maximum of 1 week.

[0702] BOIN Implementation

[0703] The steps to implement the BOIN design are summarized in FIG. 13. Participants need to be recruited in a cohort size of 3. Participants treated with DP in the first cohort were treated at dose level 1 (0.005 mg / kg). Participants treated with DPI will start at one dose level below the safe dose of DP (currently 0.06 mg / kg).

[0704] To assign a dose to the next cohort of participants, conduct dose escalation / de-escalation according to the rule displayed in Table 10. The decision to escalate, de-escalate, or eliminate at the tested dosing level depends on the number of evaluable participants and the number of evaluable participants with DLTs at the dosing level. “Eliminate” in Table 10 indicates eliminating the current and higher doses from the trial to prevent treating any future participants at these doses because they are overly toxic.

[0705] When a dose is eliminated, de-escalation to the next lower dose level should occur. When the lowest dose is eliminated, the trial is stopped for safety. If none of the actions (e.g., escalation, de-escalation, or elimination) is triggered, the new participants are treated in a cohort size of 3 at the current dose. If the current dose is the lowest dose and the rule indicates dose de- escalation, the new participants are treated at the lowest dose unless the number of DLTs reaches the elimination boundary, at which point terminate the trial for safety. If the current dose is the highest dose and the rule indicates dose escalation, the new participants are treated at the highest dose.WSGR Docket No. 56371-768.601Table 10 Summary of Dose Escalation / De-escalation rule for the BOIN designTable 11 Patient outcome from DPI monotherapy (not completed yet)

[0706] Table 11 shows a glimpse of FIH initial safety study trial with DP alone in advanced metastatic epithelial cancers. As of 22-July-2024, a total of 15 participants received at least one DP infusion. Initial safety experience supports that DP was well-tolerated in the studied population. Three participants had treatment-related SAEs: 1 pyrexia (Grade 2), and 2 cytokine release syndrome events (Grades 1 and 2), all of which resolved with protocol recommended management, the occurrence of these events will be monitored closely in this clinical study.WSGR Docket No. 56371-768.601Adverse events of neutropenia were also observed in three participants which resolved without intervention. There were no Grade 4 or 5 treatment related events or DLTs reported. The current safety profile supports continued clinical evaluation of DP and potential for combination with standard oncologic therapy regimens. Cohorts 1 to 3 are dosed every 14 days in Cycle 1 for 3 doses and dosed every 28 days from Cycle 2 onwards, Cohort 4 is dosed every 7 days in Cycle 1 for 3 doses and dosed every 28 days from Cycle 2 onwards, and Cohorts 5 to 7 are dosed every 14 days in Cycle 1 for 2 doses and dosed every 14 days from Cycle 2 onwards. Percentages are based on number of participants enrolled. *Participant D- SAE Pneumothorax spontaneous considered not related to DP. See Narrative in Investigator’s Brochure. **As of the date of this protocol, an additional 8 participants have received at least one DP infusion (3 participants at 0.1 mg / kg bi-weekly, 2 participants at 0.06 mg / kg bi-weekly, and 3 participants at 0.03 mg / kg weekly dosing schedule [Cohort 4]). Three participants at 0.1 mg / kg have cleared the DLT period without event, and therefore 0.1 mg / kg is considered the current safe dose and escalation continues (0.15 mg / kg). No Grade 4 or 5 treatment-related events or DLTs have been reported.Example 10. Clinical Protocol to Assess Safety and Preliminary Clinical Efficacy of Engineered Nucleic Acid Encoding Anti-TROP2 CEP (DP or DPI) Combination therapy in Gastroesophageal Cancer in Human

[0707] Patients with metastatic gastroesophageal cancer face a significant unmet medical need, as current treatments, primarily chemotherapy, offer limited efficacy and are associated with severe side effects. Survival rates are poor, with median overall survival (OS) often under a year, and real- world data shows little improvement over time (van Kleef 2020; Ter Veer 2016; Wagner 2017). New treatment strategies are urgently needed.

[0708] Efficacy of treatment with systemic cytotoxic therapies such as CapOx based regimens in metastatic esophagogastric cancer is evaluated in this study. The proposed therapy, (DP), is an mRNA-based approach designed to engineer myeloid cells to express a Chimeric Antigen Receptor (CAR) targeting TROP2, a protein overexpressed in about 80% of esophageal and gastric cancers. TROP2 is associated with aggressive cancer features and poor prognosis (Dum 2022; Zhao 2016). DP is intended to be combined with standard chemotherapy (CapOx), trastuzumab (for Human Epidermal Growth Factor Receptor [HER2] positive cases), and / or checkpoint inhibitors to boost immune responses. These therapies work together by inducing immunogenic cell death, increasing tumor antigen presentation, and enhancing T-cell activation, ultimately improving the immune system's ability to fight the cancer (Pol 2015; Bianchini 2014; Alexandrov 2013; Monteiro 2003). This combination approach aims to provide a more durable and effective treatment for patients with metastatic gastroesophageal cancer, leveraging the unique strengths of myeloid-based immunotherapy alongside traditional and targeted therapies.WSGR Docket No. 56371-768.601

[0709] Objectives :

[0710] Primary Objective

[0711] Safety and Tolerability: To assess the safety profile and identify any adverse effects of the new combination treatment.

[0712] Secondary Objectives

[0713] Preliminary Efficacy:

[0714] To evaluate direct anti -tumor activity by the investigator

[0715] To evaluate the level of TROP expression by immunohistochemistry (IHC) in the cancer

[0716] To evaluate the on-treatment changes in the tumor microenvironment

[0717] Main trial endpoints

[0718] Safety and tolerability, measured by the incidence and severity of adverse events (AEs)

[0719] AEs as characterized by type...

Claims

WSGR Docket No. 56371-768.601CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding TROP2, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36.

2. The composition of claim 1, wherein the CFP comprises an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), wherein the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises a sequence of SEQ ID NO: 180, the HCDR2 comprises a sequence of SEQ ID NO: 181, and the HCDR3 comprises a sequence of SEQ ID NO: 182; and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises a sequence of SEQ ID NO: 189, the LCDR2 comprises a sequence of SEQ ID NO: 190, and the LCDR3 comprises a sequence of SEQ ID NO: 191.

3. The composition of claim 2, wherein the VH comprises a sequence of SEQ ID NO: 175, and the VL comprises a sequence of SEQ ID NO: 176.

4. The composition of claim 3, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 39 or 179.

5. The composition of any one of claims 1-4, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5, 33, or 34.

6. The composition of any one of claims 1-5, wherein the nucleic acid further comprises a 5’ UTR, and wherein the 5’ UTR comprises a sequence of SEQ ID NO: 7 or 8.

7. The composition of any one of claims 1-6, wherein the nucleic acid further comprises a 3’ UTR,(i) wherein the 3’ UTR is a bGH 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 9; or(ii) wherein the 3’ UTR is a a globin 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 10.

8. The composition of claim 7, wherein the 3’ UTR comprises one or more microRNA binding site.

9. The composition of claim 8, wherein the 3’ UTR comprises four microRNA binding sites.

10. The composition of claim 8 or 9, wherein the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p, and wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11.WSGR Docket No. 56371-768.60111. The composition of claim 10, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 12 or 24.

12. The composition of any one of claims 7-11, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 13.

13. The composition of claim 8 or 9, wherein the microRNA binding site comprise a sequence that specifically binds to miR122-5p, and wherein 3’ UTR comprises a sequence of SEQ ID NO: 26.

14. The composition of any one of claims 1-13, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16.

15. The composition of claim 14, wherein the nucleic acid comprises a sequence of SEQ ID NO:16.

16. The composition of any one of claims 1-15, wherein the nucleic acid is an RNA; optionally, wherein the RNA is an mRNA.

17. The composition of any one of claims 1-16, wherein the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 33; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 33.

18. The composition of any one of claims 1-16, wherein the nucleic acid comprises a sequence with at least 80% sequence identity to SEQ ID NO: 36; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 36.

19. The composition of any one of claims 1-18, wherein the composition further comprises(i) a nucleic sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38; or(ii) a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7.

20. The composition of any one of claims 1-19, wherein the CFP for specifically binding TROP2 comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16.

21. The composition of any one of claims 1-20, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17.

22. A composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) for specifically binding GPC3, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 41.

23. The composition of claim 22, wherein the CFP comprises an amino acid sequence of SEQ ID NO: 40.WSGR Docket No. 56371-768.60124. The composition of claim 22 or 23, wherein the nucleic acid comprises a sequence having at least 95% sequence identity to SEQ ID NO: 41; optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 41.

25. The composition of any one of claims 22-24, wherein the nucleic acid further comprises a 5’ UTR, and wherein the 5’ UTR comprises a sequence of SEQ ID NO: 42.

26. The composition of any one of claims 22-25, wherein the nucleic acid further comprises a 3’ UTR, wherein the 3’ UTR is a a globin 3’ UTR, and wherein the 3’ UTR comprises a sequence of SEQ ID NO: 43.

27. The composition of claim 26, wherein the 3’ UTR comprises one or more microRNA binding site.

28. The composition of claim 27, wherein the 3’ UTR comprises four microRNA binding sites.

29. The composition of claim 27 or 28, wherein the microRNA binding site comprise a sequence that specifically binds to miR-27b-5p, and wherein the miR-27b-5p comprises a sequence of SEQ ID NO: 11.

30. The composition of claim 29, wherein the microRNA binding site comprises a sequence of SEQ ID NO: 12.

31. The composition of any one of claims 26-30, wherein the 3’ UTR comprises a sequence of SEQ ID NO: 13.

32. The composition of claim 27 or 28, wherein the microRNA binding site comprise a sequence that specifically binds to miR122-5p, and wherein 3’ UTR comprises a sequence of SEQ ID NO: 26.

33. The composition of any one of claims 26-32, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 44, optionally, wherein the nucleic acid comprises a sequence of SEQ ID NO: 44.

34. The composition of any one of claims 22-33, wherein the nucleic acid is an RNA, optionally, wherein the RNA is an mRNA.

35. The composition of any one of claims 22-34, wherein the composition further comprises(i) a nucleic acid sequence encoding a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 38, or(ii) a nucleic acid sequence encoding a polypeptide having a sequence comprising one or more sequences selected from the sequences presented in Tables 4-7.

36. A pharmaceutical composition, comprising the composition of any one of claims 1-35, and a pharmaceutical acceptable excipient.WSGR Docket No. 56371-768.60137. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition further comprises a lipid nanoparticle, wherein the nucleic acid is encapsulated in the lipid nanoparticle.

38. A pharmaceutical composition, comprising:(I) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising:(a) an extracellular domain comprising an antigen binding domain that specifically binds to TROP2 (a TROP2 binding domain), the TROP2 binding domain comprising (i) a heavy chain variable domain (VH) having complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises a sequence of GGFGSSYWYFDV; and (ii) a light chain variable domain (VL) having CDRs of LCDR1, LCDR2, and LCDR3, wherein the LCDR3 comprises a sequence of QQHYITPLT;(b) a CD89 transmembrane domain operatively linked to the extracellular domain; and(II) a lipid nanoparticle delivery vehicle encapsulating the RNA in (I).

39. The pharmaceutical composition of claim 38, wherein the HCDR1 comprises a sequence of NYGMN, the HCDR2 comprises a sequence of WINTYTGEPTYTDDFKG; and wherein the LCDR1 comprises a sequence of KASQDVSIAVA, the LCDR2 comprises a sequence of SASYRYT.

40. The pharmaceutical composition of claim 38 or 39, wherein the VH comprises a sequence of SEQ ID NO: 175, and the VL comprises a sequence of SEQ ID NO: 176.

41. The pharmaceutical composition of any one of claims 37-40, wherein the lipid nanoparticle delivery vehicle comprises an ionizable lipid, wherein the ionizable lipid is ALC-0366, and wherein the ionizable lipid comprises the chemical formula (Formula I):

42. The pharmaceutical composition of any one of claim 37-41, wherein the lipid nanoparticle delivery vehicle comprises a PEG-lipid, wherein the PEG-lipid(i) is ALC-0159, orWSGR Docket No. 56371-768.601(ii) comprises the chemical formula (Formula II):wherein n is an integer between 46 and 52.

43. The pharmaceutical composition of any one of claims 37-42, wherein the lipid nanoparticle delivery vehicle comprises di stearoylphosphatidylcholine (DSPC), and wherein the DSPC comprises the chemical formula (Formula III):

44. The pharmaceutical composition of any one of claims 37-43, wherein the lipid nanoparticle delivery vehicle comprises cholesterol, and wherein the cholesterol comprises the chemical formula (Formula IV):

45. The pharmaceutical composition of any one of claims 37-44, wherein the ratio of mRNA to ionizable lipid (N / P) is 6.0.

46. The pharmaceutical composition of any one of claims 37-45, wherein the concentration of the RNA is between about 1.5 to about 0.5 mg / mL or between about 1.3 to about 0.7 mg / mL when stored in a container.

47. The pharmaceutical composition of any one of claims 37-46, wherein the LNP comprises(a) an ionizable lipid present at a mol% of from about 35% to about 55%;(b) distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%;(c) cholesterol present at a mol% from about 30% to about 50%; and(d) a PEG lipid present at a mol% of from about 1% to about 10%.

48. The pharmaceutical composition of any one of claims 37-46, wherein:WSGR Docket No. 56371-768.601(a) the CFP has an amino acid sequence of SEQ ID NO: 39 or 179, and(b) the LNP comprises:(i) an ionizable lipid, wherein the ionizable lipid is ALC-0366 or comprises the chemical formula:wherein the ionizable lipid is present in the LNP at a mol% of about 47.5%,(ii) a PEG-lipid, wherein the PEG-lipid is ALC-0159 or comprises the chemical formula:wherein n is an integer between 46 and 52; wherein the PEG-lipid is present in the LNP at a mol% of about 2.5%,(iii) distearoylphosphatidylcholine (DSPC), wherein the DSPC is present in the LNP at a mol% of about 10%; and(iv) cholesterol, wherein the cholesterol is present in the LNP at a mol% of about 40%.

49. A pharmaceutical composition comprising:(a) an RNA comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an amino acid sequence of SEQ ID NO: 39 or 179; and(b) a lipid nanoparticle (LNP) delivery vehicle encapsulating the RNA, wherein the LNP comprises:(i) an ionizable lipid, wherein the ionizable lipid is ALC-0366 or comprises the chemical formula:WSGR Docket No. 56371-768.601 wherein the ionizable lipid is present in the LNP at a mol% of about 47.5%(ii) a PEG-lipid, wherein the PEG-lipid is ALC-0159 or comprises the chemical formula:wherein n is an integer between 46 and 52; wherein the PEG-lipid is present in the LNP at a mol% of about 2.5%;(iii) distearoylphosphatidylcholine (DSPC), wherein the DSPC is present in the LNP at a mol% of about 10%; and(iv) cholesterol, wherein the cholesterol is present in the LNP at a mol% of about 40%.

50. The pharmaceutical composition of any one of claims 38-49, wherein the sequence of the RNA encoding the CFP comprises a sequence according to any one of SEQ ID NOs: 4, 5, 14-16, 33, or 36.

51. A pharmaceutical composition comprising: a nucleic acid, encapsulated in an LNP, wherein the nucleic acid comprises a sequence encoding an anti-TROP2 chimeric fusion protein (CFP) having an amino acid sequence of SEQ ID NO: 39 or 179; wherein the nucleic acid comprises a sequence of SEQ ID NO: 14, 16, 33, or 36; and wherein the LNP comprises(a) an ionizable lipid present at a mol% of from about 35% to about 55%;(b) distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%;(c) cholesterol present at a mol% from about 30% to about 50%; and(d) a PEG lipid present at a mol% of from about 1% to about 10%.

52. A pharmaceutical composition, comprising: a nucleic acid, encapsulated in an LNP, wherein the nucleic acid comprises a sequence encoding an anti-TROP2 chimeric fusion protein (CFP), wherein the nucleic acid comprises a sequence of any one of SEQ ID NOs: 4, 5, or 34; wherein the LNP comprises(a) an ionizable lipid present at a mol% of from about 35% to about 55%;(b) distearoylphosphatidylcholine (DSPC) present at a mol% of from about 5% to about 20%;(c) cholesterol present at a mol% from about 30% to about 50%; and(d) a PEG lipid present at a mol% of from about 1% to about 10%.WSGR Docket No. 56371-768.60153. A cell comprising the composition of any one of claims 1-35.

54. The cell of claim 53, wherein the cell is an immune cell; optionally, wherein the cell is a myeloid cell, a lymphoid cell, a precursor cell, a stem cell or an induced pluripotent cell; optionally, wherein the cell is CD14+ CD16- cell.

55. A method for treating a patient in need thereof, comprising administering the cell of claim 53 or 54 to the patient.

56. A method for treating a cancer in a subject in need thereof, comprising administering the pharmaceutical composition of any one of claims 36-52 to the patient.

57. A method of treating a cancer in a subject in need thereof, comprising administering to the subject (i) a composition comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain operatively linked to the extracellular domain, and a CD89 intracellular domain operatively linked to the transmembrane domain; and (ii) at least one prophylactic agent for preventing or treating cytokine release syndrome (CRS).

58. A method of treating a cancer in a human in need thereof, the method comprising administering to the subject a combination of:(a) a first component comprising a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2;(b) at least one prophylactic agent; and(c) at least one anti-cancer agent selected from:(i) a chemotherapeutic agent(ii) a tumor target drug or(iii) an immune checkpoint inhibitor.

59. The method of claim 57 or 58, wherein the prophylactic agent is administered before, after, or simultaneously with the composition comprising the nucleic acid.

60. The method of any one of claims 57-59, wherein the prophylactic agent is an antiinterleukin (IL) agent; optionally, wherein the anti-IL agent is an IL receptor inhibitor; optionally, wherein the IL receptor inhibitor comprises tocilizumab and / or anakinra.

61. The method of claim 60, wherein the tocilizumab is administered(i) at least one hour before the administration of the composition comprising the nucleic acid,(ii) at the time of the administration of the composition comprising the nucleic acid,WSGR Docket No. 56371-768.601(iii) at a dose of 8 mg / kg (max 800 mg), and / or(iv) via IV over 60 minutes.

62. The method of claim 60 or 61, wherein the anakinra is administered(i) before the administration of the composition comprising the nucleic acid,(ii) at the time of the administration of the composition comprising the nucleic acid,(iii) subcutaneously,(iv) once daily, and / or(v) every 12 hours.

63. The method of any one of claims 60-62, wherein the amount of the anakinra administered is 100 mg.

64. The method of any one of claims 60-63, wherein the anakinra is administered(i) for from about 3 to about 5 days; (ii) intravenously;(iii) in an amount of 2 mg / kg; and / or(iv) every 6 hours.

65. The method of any one of claims 57-64, wherein the CRS is reduced as compared to a control group lacking the administration of the prophylactic agent, and wherein the CRS comprises elevated liver enzymes, D-dimer, ferritin, or C-reactive protein (CRP), wherein the CRP is measured according to ASTCT CRS grading.

66. A method of treating a cancer in a subject in need thereof, the method comprising administering the subject:(a) a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to TROP2 and at least a portion of a CD89 extracellular domain, a CD89 transmembrane domain, and a CD89 intracellular domain; and(b) an anti-cancer agent comprising: a chemotherapeutic agent; and(i) a tumor target drug, or(ii) an immune checkpoint inhibitor.

67. The method of any one of claims 58-66, wherein(i) the nucleic acid and the anti-cancer agent are administered at separate times;(ii) the nucleic acid is administered after administering the anti-cancer agent;(iii) the nucleic acid is administered before administering the anti-cancer agent; or(iv) the nucleic acid and the anti-cancer agent overlaps for at least one administration.

68. The method of any one of claims 58-67, wherein the chemotherapeutic agent is selected from the group consisting of capecitabine, oxaliplatin, fluoropyrimidine, cisplatin, and carboplatin.WSGR Docket No. 56371-768.60169. The method of any one of claims 58-68, wherein the chemotherapeutic agent comprises capecitabine and oxaliplatin (Capox).

70. The method of any one of claims 58-69, wherein the anti-cancer agent comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises an antibody specifically binds to PD-1 or PD-L1, optionally, wherein the antibody is pembrolizumab or nivolumab.

71. The method of any one of claims 58-70, wherein the anti-cancer agent comprises a tumor target drug; optionally, wherein the tumor target drug is an antibody specifically binds to a tumor antigen.

72. The method of claim 71, wherein the tumor antigen is HER-2.

73. The method of claim 71, wherein the antibody is trastuzumab.

74. The method of any one of claims 58-73, wherein the chemotherapeutic agent is capecitabine and oxaliplatin (Capox), and wherein the oxaliplatin is administered(i) at a dose of 130 mg / mA2,(ii) intravenously (IV), and / or(iii) on Day 1 of each cycle.

75. The method of claim 74, wherein the capecitabine is administered(i) at a dose of 1000 mg / mA2,(ii) orally, and / or(iii) twice daily on Days 1-14 of each cycle, every three weeks.

76. The method of any one of claims 70-75, wherein the cancer is PD-L1 -positive, and the anti-cancer agent comprises the immune checkpoint inhibitor.

77. The method of any one of claims 58-76, wherein the tumor target drug is(i) administered IV; and / or(ii) administered every 3 weeks.

78. The method of any one of claims 58-77, wherein the nucleic acid is administered(i) on the same day of the anti-cancer agent and is administered every 3 weeks; and / or(ii) before the anti-cancer agent on the same day.

79. The method of any one of claims 58-78, wherein the method comprises at least 3 cycles; optionally, wherein each of the at least 3 cycles comprises 3 weeks.

80. The method of claim 79, wherein the nucleic acid is administered on Day 1 and Day 8 of Cycle 1, Day 1 of Cycle 2 and afterwards.

81. The method of claim 80, wherein the oxaliplatin is administered on Day 8 of Cycle 1, and on Day 1 of Cycle 2 and afterwards.WSGR Docket No. 56371-768.60182. The method of claim 80 or 81, wherein the capecitabine is administered on Day 8 and Day 15 of Cycle 1, and Day 1 and Day 8 of Cycle 2 and afterwards.

83. The method of claim 82, wherein the nivolumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards.

84. The method of claim 79, wherein the trastuzumab and oxaliplatin is administered on Day 8 of Cycle 1, and Day 1 of Cycle 2 and afterwards.

85. The method of any one of claims 58-84, further comprising administering an additional drug, wherein the additional drug comprises an anti-emetic, analgesic, anti-pyretic, anti- diarrheal, anti-inflammatory drug.

86. The method of claim 85, wherein the additional drug comprises an anti-emetic drug, and wherein the anti-emetic drug comprises olanzapine, 5-HT3 receptor antagonist, or NK-1 receptor antagonist.

87. The method of claim 86, wherein the additional drug further comprises a steroid, optionally wherein the steroid comprises methylprednisolone.

88. The method of claim 86, wherein the 5-HT3 receptor antagonist is ondansetron or palonosetron.

89. The method of claim 86, wherein the NK-1 receptor antagonist is aprepitant or fosaprepitant.

90. The method of any one of claims 55-89, wherein the CFP comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 16 or any combination of sequences set forth in Table 16.

91. The method of any one of claims 55-90, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to any one sequence set forth in Table 17 or any combination of sequences set forth in Table 17.

92. The method of any one of claims 55-91, wherein the RNA is expressed predominantly in myeloid cells in vivo.

93. The method of any one of claims 55-92, wherein the administration of the pharmaceutical composition does not generate a cytokine response.

94. The method of any one of claims 55-79, and 85-93, wherein the method comprises administering the pharmaceutical composition to the subject(i) for at least one treatment cycle;(ii) for at least 2, 3, 4, or 5 treatment cycles;(iii) about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about or once every 9 weeks in each of the treatment cycle;WSGR Docket No. 56371-768.601(iv) about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle;(v) between 1 to 12 times for each treatment cycle; and / or(vi) between about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times for each treatment cycle.

95. The method of claim 94, wherein a second cycle follows a first cycle.

96. The method of claim 94 or 95, wherein the method further comprises administering the pharmaceutical composition to the subject during the second cycle.

97. The method of claim 96, wherein the pharmaceutical composition is administered to the subject(i) about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, or about once every 9 weeks during the second cycle,(ii) from about 1 to about 3, about 3 to about 6, about 6 to about 9, or about 9 to about 12 times in the second cycle, and / or(iii) 3 times in the second cycle.

98. The method of any one of claims 56-97, wherein an effective amount of the pharmaceutical composition ranges from 0.01 mg / kg / dose to 3.0 mg / kg / dose, from 0.05 mg / kg / dose to 2.5 mg / kg / dose, or from 0.1 mg / kg / dose to 1.0 mg / kg / dose.

99. The method of any one of claims 56-98, wherein an effective amount of the pharmaceutical composition ranges from about 0.001 to about 0.0015, about 0.0015 to about 0.002, about 0.002 to about 0.0025, about 0.0025 to about 0.003, about 0.003 to about 0.0035, about 0.0035 to about 0.004, about 0.004 to about 0.0045, about 0.0045 to about 0.005, about 0.005 to about 0.0055, about 0.0055 to about 0.006, about 0.006 to about 0.0065, about 0.0065 to about 0.007, about 0.007 to about 0.0075, about 0.0075 to about 0.008, about 0.008 to about 0.0085, about 0.0085 to about 0.009, about 0.009 to about 0.0095, or about 0.0095 to about 0.01 mg / kg of the RNA.

100. The method of claim 98 or 99, wherein the effective amount of the pharmaceutical composition is administered in a 60-minute intravenous (IV) infusion per dose.

101. The method of any one of claims 56-100, wherein the effective amount of the pharmaceutical composition is administered(i) for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IV infusion doses,(ii) at an interval of once in a week,(iii) at an interval of once every 10 days,(iv) at an interval of once every 2 weeks, and / orWSGR Docket No. 56371-768.601(v) for at least 5 doses.

102. The method of any one of claims 56-101, wherein the cancer is selected from the group consisting of cervical cancer, colorectal cancer, esophageal, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, and urothelial cancer.

103. The method of any one of claims 57-102, wherein the nucleic acid comprising a sequence encoding a CFP comprises a sequence with at least 80% sequence identity of SEQ ID NO: 4, 5, or 34, and wherein the CFP comprises an amino acid sequence of SEQ ID NO: 39.

104. The method of claim 103, wherein the nucleic acid comprises a sequence encoding a CFP comprises a sequence of SEQ ID NO: 4, 5, or 34.

105. The method of claim 103 or 104, wherein the nucleic acid comprises a sequence encoding a CFP comprises a sequence with at least 80% sequence identity of SEQ ID NO: 14, 16, 33, or 36.

106. The method of claim 105, wherein the nucleic acid comprises a sequence encoding a CFP comprises a sequence of SEQ ID NO: 14, 16, 33, or 36.

107. A combination therapeutic kit for treating a cancer in a subject in need thereof, comprising a first therapeutic agent and at least one additional therapeutic agent, wherein the first therapeutic agent comprises a nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) having an extracellular domain comprising an antigen binding domain that binds to Trophoblast Cell Surface Antigen 2 (TROP2), and a CD89 transmembrane domain operatively linked to the extracellular domain; and wherein the at least one additional therapeutic agent comprises (i) a prophylactic agent or (ii) a chemotherapeutic agent and at least one of (a) a tumor target drug or (b) an immune checkpoint inhibitor.

108. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) capecitabine and oxaliplatin; and(iii) nivolumab.

109. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) capecitabine and oxaliplatin; and(iii) pembrolizumab.WSGR Docket No. 56371-768.601110. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) fluoropyrimidine and cisplatin, and(iii) pembrolizumab.

111. A combination therapeutic kit for treating a PD-L1 -positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) fluoropyrimidine and cisplatin, and(iii) nivolumab.

112. A combination therapeutic kit for treating a HER2-positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) capecitabine and oxaliplatin; and(iii) trastuzumab.

113. A combination therapeutic kit for treating a HER-2 positive cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) capecitabine and oxaliplatin; and(iii) trastuzumab.

114. A combination therapeutic kit for treating a cancer, comprising:(i) an mRNA encoding a CFP, wherein the mRNA comprises a sequence of SEQ ID NO: 14, 16, 33, or 36;(ii) tocilizumab and anakinra.